· transgenic horticultural crops edited by beiquan mou and ralph scorza challenges and...

Transgenic HorTiculTural

crops

EditEd by

Beiquan Mou and ralph scorza

Challenges and Opportunities

Agriculture

As the world debates the risks and benefits of plant biotechnology, the proportion of the global area of transgenic field crops has increased every year, and the safety and value continue to be demonstrated. Yet, despite the success of transgenic field crops, the commercialization of transgenic horticultural crops (vegetables, fruits, nuts, and ornamentals) has lagged far behind. Transgenic Horticultural crops: challenges and opportunities examines the challenges for the creation and commercialization of horticultural biotechnology and identifies opportunities, strategies, and priorities for future progress.

A “must read” for anyone working in the fields of genetic engineering or plant breeding, for policy makers, educators, students, and anyone interested in the issues of genetic engineering of fruits, vegetables and ornamentals, this book covers:

• Past achievements, newest developments, and current challenges in transgenic fruit, nut, vegetable, ornamental, and pharmaceutical crops • Reviews transgenic horticultural crops in the U.S., Europe, Africa, and Asia • Hurdles to the commercialization of transgenic technology in economics and the marketplace, consumer acceptance, intellectual property rights protection, public–private partnership, and regulation • Critical evaluation of the benefits and risks of genetically engineered horticultural crops, including risk assessment and transgene containment • Presents case studies and an industry perspective on transgenic horticultural crops

The production and commercialization of transgenic horticultural crops are enormous tasks—their progress and realization require an informed research community, horticultural industry, government, and body of consumers. To aid in this effort, this book provides facts, analyses and insights by leading experts in this field to inform a wide audience of students, agricultural and genetic professionals, and the interested public. Part of the global conversation on the pros and cons of transgenic foods, Transgenic Horticultural crops aims to stimulate more interest and discussion on the subject and to promote the development of safe and sustainable genetically modified horticultural crop varieties.

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v

ContentsPreface......................................................................................................................viiEditors........................................................................................................................ixContributors...............................................................................................................xi

Chapter 1 Transgenic.Fruit.and.Nut.Tree.Crops.Review.......................................1

Ana M. Ibáñez, Cecilia B. Agüero, Mathew A. Escobar, and Abhaya M. Dandekar

Chapter 2 Transgenic.Vegetables......................................................................... 31

Owen Wally, J. Jayaraj, and Zamir K. Punja

Chapter 3 Transgenic.Ornamental.Crops............................................................ 55

Beverly A. Underwood and David G. Clark

Chapter 4 Expression.and.Manufacture.of.Pharmaceutical.Proteins.in Genetically.Engineered.Horticultural.Plants.................................. 83

Qiang Chen

Chapter 5 Transgenic.Fruit.Crops.in.Europe..................................................... 125

Henryk Flachowsky and Magda-Viola Hanke

Chapter 6 Transgenic.Horticultural.Crops.on.the.African.Continent................ 147

Idah Sithole-Niang

Chapter 7 Transgenic.Horticultural.Crops.in.Asia............................................ 155

Desiree M. Hautea, Von Mark Cruz, Randy A. Hautea, and Vijay Vijayaraghavan

Chapter 8 The.Economic.and.Marketing.Challenges.of.Horticultural.Biotechnology.................................................................................... 175

Steven Sexton and David Zilberman

vi Contents

Chapter 9 Consumer.Acceptance.of.Genetically.Modified.Foods:.Traits, Labels,.and.Diverse.Information............................................ 193

Wallace E. Huffman

Chapter 10 Intellectual.Property.and.Development.of.Transgenic.Horticultural.Crops........................................................................... 219

Cecilia L. Chi-Ham and Alan B. Bennett

Chapter 11 Structuring.University–Private.Partnerships.for.Developing.and Commercializing.Transgenic.Horticultural.Crops..................... 233

Gordon Rausser and Reid Stevens

Chapter 12 Why.Are.Regulatory.Requirements.a.Major.Impediment.to Genetic.Engineering.of.Horticultural.Crops?...............................249

Steven H. Strauss

Chapter 13 Virus-Resistant.Transgenic.Horticultural.Crops:.Safety.Issues.and.Lessons.from.Risk.Assessment.Studies..................................... 263

Jonathan E. Oliver, Paula F. Tennant, and Marc Fuchs

Chapter 14 Molecular.Approaches.for.Transgene.Containment.and.Their.Potential.Applications.in.Horticultural.Crops................................... 289

Yi Li and Hui Duan

Chapter 15 Prospects.for.the.Commercialization.of.Transgenic.Ornamentals...... 305

Michael S. Dobres

Chapter 16 Genetic.Engineering.of.Grapevine.and.Progress.toward.Commercial.Deployment.................................................................. 317

Dennis J. Gray, Sadanand A. Dhekney, Zhijian T. Li,

and John M. Cordts

vii

PrefaceAt.the.dawn.of.the.twenty-first.century,.food.insecurity.and.malnutrition.continue.to.plague.humankind,.especially.in.developing.countries..It.has.been.estimated.that.world.food.supplies.must.increase.by.up.to.50%.over.the.next.20.years.due.to.popula-tion.growth,.even.while.farming.land.is.being.rapidly.lost.to.housing,.transportation,.and.industrial.uses..Global.warming.and.climate.change.also.pose.serious.threats.to.agricultural.production.and.place.unprecedented.pressures.on.the.sustainability.of.food.supplies..Transgenic.field.crop.production.is.a.major.component.of.modern.agriculture.and.promises. to.play.an. important.role.in.meeting. the.food.supply.challenges.that.we.face.today.and.in.the.future..The.global.planted.area.of.genetically.engineered.field.crops.soared.to.330.million.acres.in.25.countries.in.2009,.of.which.158.million.acres.(48%).were.in.the.United.States..Today,.more.than.three.quarters.of.the.soybean,.nearly.half.of.the.cotton,.and.more.than.a.quarter.of.the.global.maize.production. are. from. biotech. varieties,. primarily. with. herbicide. tolerance,. insect.resistance,.or.stacked.genes.for. the.two. traits..As.the.world.debates. the.risks.and.benefits.of.plant.biotechnology,.the.proportion.of.the.global.area.of.transgenic.field.crops.has.increased.every.year,.and.the.safety.and.benefits.continue.to.be.demon-strated..Yet,.despite.the.success.of.transgenic.field.crops,.the.commercialization.of.transgenic.horticultural.crops.(vegetables,.fruits,.nuts,.and.ornamentals).has.lagged.far.behind..Transgenic Horticultural Crops: Challenges and Opportunities.examines.the.challenges,.advances,.and.opportunities.for.the.creation.and.commercialization.of.transgenic.horticultural.crops.

The.consumption.and.production.of.horticultural.products.continue.to.increase,.and.now.horticultural.crops.account.for.50%.of.the.value.for.all.agricultural.crops.in.the.United.States..With.the.rising.demand.for.fruits.and.vegetables.by.health-conscious. consumers,. there. are. ever-increasing. interests. in. horticultural. crops..Although. horticultural. crops. were. the. first. biotech. crops. commercialized. in. the.United.States,.beginning.with.the.Flavr.Savr.tomato.in.1994,.they.have.not.made.an.impact.on.production.due.to.factors.that.include.consumer.concern.over.genetically.modified. (GM). food,.which. results. in. the. reluctance.of.processors.and.marketers.to. accept. the. biotech. products. already. developed;. complex. and. costly. regulatory.processes;.the.limited.acreage.of.most.horticultural.crops,.which.makes.it.difficult.to. recover. the. costs. of. research. and. development;. and. costly. segregation. of. GM.and. non-GM. commodities.. Current. practices. in. patenting. and. intellectual. prop-erty.protection.have.added.barriers.to.the.use.of.biotechnology.for.the.creation.and.commercialization.of.new.horticultural.crop.varieties..Additional.challenges.to.the.development.and.commercialization.of.GM.horticultural.crops.include.technical.dif-ficulties.in.the.transformation.of.certain.horticultural.crops,.barriers.to.regulatory.approval.in.many.countries,.and.the.uncertainties.of.post-commercialization.stew-ardship.. Although. there. are. many. volumes. dealing. with. plant. biotechnology. and.transgenic.plants,.those.focusing.on.horticultural.crops.are.rare..In.this.book,.inter-nationally.acclaimed.experts.from.different.disciplines.assess.the.current.status.of.

viii Preface

transgenic.horticultural.crops,.examine.the.challenges.for.the.creation.and.com-mercialization.of.horticultural.biotechnology,.and.identify.opportunities,.strategies,.and.priorities.for.future.progress.in.this.important.field..The.authors.of.the.chapters.are.leading.experts.who.were.asked.not.to.conform.to.a.set.outline.but.to.approach.their.topics.in.their.own.unique.ways.

Chapters.1.through.4.describe.the.past.achievements,.newest.developments,.and.current.challenges.in.transgenic.fruit,.nut,.vegetable,.ornamental,.and.pharmaceuti-cal. crops.. Chapters. 5. through. 7. provide. reviews. of. transgenic. horticultural. crops.in.other.parts.of. the.world. such.as.Europe,.Africa,. and.Asia..Chapters.8. through.12. discuss. in. detail. hurdles. to. the. commercialization. of. transgenic. technology. in.economics.and.the.marketplace,.consumer.acceptance,. intellectual.property.rights.protection,.public–private.partnership,.and.regulation..Chapters.13.and.14.describe.the.risk.assessment.of.transgenic.crops.and.transgene.containment..Finally,.Chapters.15.and.16.present.some.case.studies.and.an.industry.perspective.on.transgenic.hor-ticultural.crops.

The. production. and. commercialization. of. transgenic. horticultural. crops. are.enormous. tasks—their. progress. and. realization. need. the. efforts. of. the. research.community,.horticultural.industry,.government,.and.consumers..Although.this.book.covers.many. topics,. it.by.no.means.has.exhausted.all. issues. related. to. transgenic.technology.in.horticulture..We.have.sought.to.provide.facts,.analyses,.and.insights.by.leading.experts.in.this.field.that.can.inform.a.wide.audience,.including.graduate.and.advanced. undergraduate. students. of. agriculture—and. horticulture. in. particular,.educators. at. all. levels,. breeders. of. horticultural. crops,. plant. geneticists,. biotech-nologists,.biologists,.molecular.biologists,.cell/tissue.culture.specialists,.horticultur-ists,.agronomists,.entomologists,.plant.pathologists,.physiologists,.nutrition.and.food.technologists,. food. safety. specialists,. economists,. environmentalists,. agricultural.extension.personnel,.growers,.farm.managers,.pest.control.agents,.government.regu-lators,.and.the.interested.public..We.hope.that.this.book.will.stimulate.more.interest.and.discussion.on.the.subject.and.promote.the.advancement.of. research.on.trans-genic.horticultural.crops.and.the.development.of.safe,.sustainable.GM.horticultural.crop.varieties.

ix

EditorsDr. Beiquan Mou. is. currently. a. research. plant. geneticist. with. the. Agricultural.Research.Service,.U.S..Department.of.Agriculture,.Salinas,.California..He.obtained.his. PhD. degree. in. plant. breeding. and. genetics. from. Oregon. State. University. in.1993..He.then.worked.on.the.mechanism.and.inheritance.of.self-.and.interspecific.incompatibility. in.Nicotiana. at. the.University.of.Missouri–Columbia. and.carried.out.postdoctoral.research.on.the.transgenic.modification.of.cornstarch.structure.and.functionality. at. Iowa. State.University.. Since. 2001,. Dr..Mou. has. been. conducting.research.on.the.genetics.and.breeding.of.lettuce.and.spinach.for.disease.and.insect.resistance,. nutritional. improvement,. and. horticultural. traits.. He. has. released. 15.lettuce.and.spinach.varieties.possessing.unique.traits,.improved.quality,.new.genes,.and/or.disease.or.insect.resistance..He.currently.serves.as.chair.of.the.USDA.Leafy.Vegetable.Crop.Germplasm.Committee.and.the.Vegetable.Breeding.Working.Group.of.the.American.Society.for.Horticultural.Science..He.is.sought.out.for.consultation.nationally.and.internationally.by.other.researchers,.government.agencies,.industry,.and.the.media.

Dr. Ralph Scorza. is. a. research. horticulturist. and. lead. scientist. for. the. Genetic.Improvement. of. Fruit. Crops. Research.Unit. at. the. USDA-ARS.Appalachian.Fruit.Research.Station,.Kearneysville,.West.Virginia..He. received.his.BS. in.agronomy.and.MS.in.fruit.crops,.both.from.the.University.of.Florida,.and.his.PhD.in.genetics.and.plant.breeding.from.Purdue.University.in.1979..The.broad.objectives.of.his.research.program.at.the.USDA.are.to.develop.stone.fruit.(Prunus).germplasm.with.improved.fruit.quality,.resistance.to.biotic.and.abiotic.stress,.and.improved.tree.growth.habits.for.high-yielding,.mechanically.integrated.orchard.systems..His.breeding.program.combines.classical.and.molecular.approaches.

Dr..Scorza.has.released.nine.stone.fruit.varieties.developed.through.conventional.breeding..His.genetic.engineering.work.has.included.the.successful.development.of.disease-resistant.grapes,.pears,.and.plums..The.plum.pox.virus–resistant.plum.cultivar.‘HoneySweet’. developed. by.Dr.. Scorza. and.his. colleagues. is. the.first. genetically.engineered.temperate.fruit.crop.to.be.deregulated.and.approved.by.the.APHIS,.FDA,.and.EPA.in.the.United.States.

Dr..Scorza.is.a.recipient.of.the.Arthur.S..Flemming.Award.and.was.selected.as.an.ARS-NAA.Senior.Research.Scientist.of.the.Year..He.is.also.a.corecipient.of.three.Secretary.of.Agriculture.Honor.Awards..He.has.authored.over.190.research.publica-tions.and.is.a.fellow.of.the.American.Society.for.Horticultural.Science.

xi

Contributors

Cecilia B. AgüeroViticulture.and.Enology.DepartmentUniversity.of.California,.DavisDavis,.California

Alan B. BennettDepartment.of.Plant.SciencesUniversity.of.California,.DavisDavis,.California

Qiang ChenLaboratory.of.Plant.Pharmaceutical.

ResearchBiodesign.Institute.and.College.

of Technology.and.InnovationArizona.State.UniversityTempe,.Arizona

Cecilia L. Chi-HamDepartment.of.Plant.SciencesUniversity.of.California,.DavisDavis,.California

David G. ClarkDepartment.of.Environmental.HorticultureUniversity.of.FloridaGainesville,.Florida

John M. CordtsBiotechnology.Regulatory.ServicesAnimal.and.Plant.Health.Inspection.

ServiceUnited.States.Department.

of AgricultureRiverdale,.Maryland

Von Mark CruzAgricultural.Research.ServiceUnited.States.Department.

of AgricultureNational.Center.for.Genetic.Resources.

PreservationFort.Collins,.Colorado

Abhaya M. DandekarPlant.Sciences.DepartmentUniversity.of.California,.DavisDavis,.California

Sadanand A. DhekneyMid-Florida.Research.and.Education.

CenterInstitute.of.Food.and.Agricultural.

SciencesUniversity.of.FloridaApopka,.Florida

Michael S. DobresNovaFlora.Inc.West.Grove,.Pennsylvania

Hui DuanJ..R..Simplot.CompanyBoise,.Idaho

Mathew A. EscobarDepartment.of.Biological.SciencesCalifornia.State.University,.San.MarcosSan.Marcos,.California

Henryk FlachowskyInstitute.for.Breeding.Research.on.

Horticultural.and.Fruit.CropsJulius.Kühn-InstituteDresden,.Germany

Marc FuchsDepartment.of.Plant.Pathology.

and Plant-Microbe.BiologyCornell.UniversityGeneva,.New.York

xii Contributors

Dennis J. GrayMid-Florida.Research.and.Education.



Magda-Viola HankeInstitute.for.Breeding.Research.on.

Horticultural.and.Fruit.CropsJulius.Kühn-InstituteDresden,.Germany

Desiree M. HauteaInstitute.of.Plant.BreedingUniversity.of.the.PhilippinesLos.Baños,.Philippines

Randy A. HauteaInternational.Service.for.the.Acquisition.

of.Agri-Biotech.ApplicationsSEAsia.CenterManila,.Philippines

Wallace E. HuffmanDepartment.of.EconomicsIowa.State.UniversityAmes,.Iowa

Ana M. IbáñezPlant.Sciences.DepartmentUniversity.of.California,.DavisDavis,.California

J. JayarajDepartment.of.Biological.SciencesSimon.Fraser.UniversityBurnaby,.British.Columbia,.Canada

Yi LiDepartment.of.Plant.Science.

and Landscape.ArchitectureUniversity.of.ConnecticutStorrs,.Connecticut

Zhijian T. LiMid-Florida.Research.and.Education.



Jonathan E. OliverDepartment.of.Plant.Pathology.

and Plant-Microbe.BiologyCornell.UniversityGeneva,.New.York

Zamir K. PunjaDepartment.of.Biological.SciencesSimon.Fraser.UniversityBurnaby,.British.Columbia,.Canada

Gordon RausserDepartment.of.Agricultural.

and Resource.EconomicsUniversity.of.California,.BerkeleyBerkeley,.California

Steven SextonDepartment.of.Agricultural.


Idah Sithole-NiangDepartment.of.BiochemistryUniversity.of.ZimbabweHarare,.Zimbabwe

Reid StevensDepartment.of.Agricultural.


xiiiContributors

Steven H. StraussDepartment.of.Forest.Ecosystems.

and SocietyOregon.State.UniversityCorvallis,.Oregon

Paula F. TennantDepartment.of.Life.SciencesThe.University.of.the.West.IndiesKingston,.Jamaica

Beverly A. UnderwoodDepartment.of.Environmental.

HorticultureUniversity.of.FloridaGainesville,.Florida

Vijay VijayaraghavanSathguru.Management.Consultants.

Pvt. Ltd.Hyderabad,.India

Owen WallyDepartment.of.Biological.SciencesSimon.Fraser.UniversityBurnaby,.British.Columbia,.Canada

David ZilbermanDepartment.of.Agricultural.


1

1 Transgenic Fruit and Nut Tree Crops Review

Ana M. Ibáñez, Cecilia B. Agüero, Mathew A. Escobar, and Abhaya M. Dandekar

CONTENTS

Introduction.................................................................................................................2Genetic.Transformation.of.Fruit.and.Nut.Tree.Crops.................................................3

Importance.of.Genetic.Engineering.......................................................................3Current.Status.of.Genetic.Transformation.of.Perennial.Crops..............................3

Genetic.Transformation.via.Organogenesis......................................................4Genetic.Transformation.via.Somatic.Embryogenesis.......................................4

Methods.of.Transformation....................................................................................5Agrobacterium-Mediated.Transformation.........................................................5Biolistic.Bombardment.....................................................................................6

Transient.Expression..............................................................................................6Rootstock.Transformation......................................................................................6

Input.Traits..................................................................................................................6Pathogen.Resistance...............................................................................................7

Viral.Resistance.................................................................................................7Bacterial.Resistance..........................................................................................9Fungal.Resistance............................................................................................ 12

Insect.Resistance.................................................................................................. 13Cydia pomonella............................................................................................. 13Epiphyas postvittana.(Light.Brown.Apple.Moth)........................................... 14

Output.Traits............................................................................................................. 14Delayed.Fruit.Ripening........................................................................................ 16Improving.Fruit.Quality.and.Nutritive.Value....................................................... 16Removal.of.Undesirable.Phytochemicals............................................................ 18Antigen.Production.............................................................................................. 18

Conclusions............................................................................................................... 19References................................................................................................................. 19

2 Transgenic Horticultural Crops: Challenges and Opportunities

INTRODUCTION

Fruit.crop.plants.constitute.an.important.group.among.agricultural.crops,..adding.tens. of. billions. of. dollars. per. year. to. the. global. economy.. Worldwide,. over.45 million.hectares.of.land.have.been.devoted.to.their.production,.and.millions.of. growers. depend. on. continued. global. trade.1. Among. fruit. crops,. citrus. pro-duction.is.most.significant,.with.more.than.134.million.tons.produced.from.an.area.of.8.7.million.hectares. in.2008..This. is. followed.by.bananas.with.world-wide.production. at. just.under.100.million. tons,.apples. at. 76.million. tons,.and.grapes.at.74.million. tons.2. In.California,. the.production.of. fruit.and.nut.crops.contributes. to. more. than. one-third. of. the. total. farm. gate. value. of. the. state’s.agricultural.commodities..The.fruit.and.nut.crops.are.cultivated.using.a.highly.sophisticated.production.system.that.uses.vegetatively.propagated.clonal.cultivar.materials.grafted. to.clonal.or.seedling.rootstocks..The.plantings.are.perennial.and.orchard.systems.represent.several.decades.of.investment.to.a.grower..These.crop.plants.have.a.juvenile.phase,.which.can.last.from.2.to.8.years,.during.which.they.grow.vegetatively.without.any.fruit.production..Thus,.traditional.fruit.tree.breeding.takes.a.very.long.time—20.to.40.years.to.develop.new.varieties..This.is.primarily.due.to.the.time.it.takes.to.backcross.promising.selections.with.current.commercial.cultivars,.which.is.required.to.create.a.new.commercial.cultivar.with.optimal.fruit.characteristics..Transgenic.plants.offer.a.more.direct.and.quicker.strategy.to.provide.genetic.solutions.

Fruit.and.nut.crops.face.many.challenges,.including.the.need.to.be.grown.on.less.land.with.less.resources.and.the.need.to.yield.consistently.high.quality.fruit.that.can.sustain. the.economics.of.production..Disease.and.pests.pose. the.great-est.challenge.to.fruit.and.nut.crop.production,.and.we.discuss.below.the.different.diseases. and. pests. that. are. important. to. these. crops. and. the. specific. solutions.that.can.be.provided.using.transgenic.technologies..Many.of.these.are.production-eliminating. diseases,. so. finding. genetic. solutions. is. critical.. In. some. cases,. the.disease.resistance.traits.are.present.in.wild.germplasm,.so.introgression.of.disease.resistance.by. traditional.breeding. takes.place. at. the. cost. of. fruit.quality,.which.means. that. many. backcrosses. have. to. be. conducted. to. restore. fruit. quality.. In.some.cases,.there.is.no.resistance.even.in.the.wild.germplasm,.so.novel.sources.of. resistance.have. to.be.developed. (e.g.,. pathogen-derived. resistance)..For. these.strategies,.transgenic.technologies.need.to.be.employed..These.technologies.have.been.developed.in.many.of.the.tree.crops.(as.outlined.in.next.section);.however,.a.major.challenge.is.the.recalcitrance.that.these.plants.display.in.tissue.culture..The.percentage.of. tissue-cultured.explants. that.give. rise. to.a. transgenic.plant. is. low.and.thus.it.is.difficult.to.transform.these.plants.using.Agrobacterium tumefaciens..Successful.plant.transformation.requires.high.frequencies.of.both.transformation.and.regeneration,.such.that.a.successful.overlap.of.such.events.can.take.place.at.a.tissue.level.to.obtain.transgenic.plants..While.there.are.many.examples.of.success,.there.are.also.many.fruit.tree.species.that.have.proved.very.difficult.to.transform.due.to.this.problem.

Two.general.classes.of.traits.that.are.of.commercial.significance.are.discussed.in.this.review:.input.traits.and.output.traits..Input.traits.are.specifically.important.

3Transgenic Fruit and Nut Tree Crops Review

for. growers,. producers,. and. handlers,. because. they. are. focused. on. sustaining.yields. and. productivity.. Disease. and. pest. resistance. are. examples. of. important.input.traits..Output.traits,.which.preserve.the.quality.and.nutritional.value.of.the.fruit,.are.of.primary.importance.to.the.consumer,.though.they.ultimately.benefit.the.grower.as.well..Increasingly,.fruit.consumption.has.been.linked.with.improved.nutrition.and.disease.prevention,.and.thus.USDA.guidelines.suggest.that.fruit.and.nuts.are.an.important.component.of.the.daily.diet..Many.fruit.quality.traits,.like.flavor,.are.important.for.consumption,.as.a.good.flavor.makes.the.fruit.more.desir-able..Fruit.quality.is.also.important.for.downstream.products.like.fruit.juice.and.wine..In.this.chapter,.we.outline.the.progress.that.has.been.made.in.different.fruit.crops.to.manipulate.both.input.and.output.traits.

GENETIC TRANSFORMATION OF FRUIT AND NUT TREE CROPS

Importance of GenetIc enGIneerInG

A. major. breeding. objective. is. to. combine. fruit. quality. with. other. horticulturally.important.traits.such.as.plant.architecture,.adaptation.to.extreme.environmental.con-ditions,.and.decreasing.the.amount.of.chemical.input.required.to.control.pests.and.diseases..The.production.of.new.varieties.by.conventional.breeding.is.a.complex.and.time-consuming.process,.especially.for.fruit.trees,.due.to.the.high.level.of.heterozy-gosity,.pronounced.inbreeding.depression,.long.juvenile.phase,.and.complex.repro-ductive.biology..The.plant.breeder.must.wait.many.years.to.be.able.to.evaluate.fruit.quality,.and,.because.fruit.trees.are.large.perennial.plants,.a.relatively.small.number.of.seedling.progeny.can.be.evaluated.3,4.In.addition,.the.characters.that.constitute.a.good.cultivar.in.most.instances.are.polygenic.in.their.inheritance;.thus,.the.probability.of.recombining.sets.of.genes.that.determine.the.essential.properties.of.a.given.cultivar.is.low.5.In.wine.grape.breeding,.legal.and.economic.hurdles.can.be.even.larger.than.the.biological.obstacles.described.above..New.wine.grape.varieties.are.not.welcome.because.the.wine.industry.relies.predominantly.on.a.few.select.and.ancient.cultivars;.also,.vintners.are.constrained.by. tradition,. regulation,.and.economics,. so. they.use.classical.varieties,.such.as,.‘Cabernet.Sauvignon’,.‘Merlot’,.and.‘Chardonnay’.6

Direct.genetic.modification.by.the.introduction.of.single.genes.offers.the.opportu-nity.to.direct.very.specific.changes.in.existing.cultivars,.to.increase.resistance.to.abi-otic.stress,.diseases,.and.pests.and.to.modify.fruit.composition.in.very.precise.ways..Vegetative.propagation.means.that.modifications.to.traditional.cultivars.by.genetic.transformation.leave.the.essential.characters.and.identity.of.the.cultivars.unaltered,.a.result.that.would.be.very.difficult.to.achieve.by.conventional.breeding.where.two.genomes.are.intermingled.7.Last.but.not.least,.genetic.transformation.also.offers.the.opportunity.to.study.how.genes.control.the.growth.and.development.of.plants.

current StatuS of GenetIc tranSformatIon of perennIal cropS

A. wide. variety. of. transformation. and. regeneration. protocols. have. been. used. to.produce.transgenic.fruit.trees..However,.in.most.species,.transformation.and.regen-eration. of. commercial. cultivars. are. not. routine,. generally. being. limited. to. a. few.


genotypes..Most.of.the.basic.protocols.were.developed.several.years.ago.and.subse-quent.efforts.have.been.oriented.toward.optimization.or.adaptation.of.the.technique.to.new.genotypes..Unfortunately,.the.time.required.to.produce.transgenic.fruit.trees.is.too.long,.especially.for.fruit.character.analysis,.and.there.is.a.need.for.faster.sta-ble.transformation.systems..Transient.expression.systems.are.less.time-consuming.methods.that.are.receiving.more.attention.as.a.tool.for.gene.functional.analysis..The.production.of.marker-free.transgenic.plants.is.another.growing.area.of.interest.due.mainly.to.consumer.concerns.

Genetic Transformation via OrganogenesisAlthough.a.common.factor.in.many.transformation.protocols.is.the.use.of.embryo-genic. cultures. as. starting.material. for. transformation,. some. species.have. been.successfully. transformed. through. the.use.of.organogenesis..This. system.offers.an.alternative.approach.for.genotypes.that.are.recalcitrant.to.plant.regeneration.by.somatic.embryogenesis,.although.the.probability.of.obtaining.chimeric.plants.is.higher.

Current.protocols. for. the.production.of. transgenic.citrus.involve.the. transfor-mation.of.cells.in.seedling.stem.pieces.and.regeneration.of.shoots.from.organo-genic.cultures..The.first.reliable.protocol.reported.the.transformation.of.the.citrus.relative. Poncirus trifoliata. by. cocultivation. of. etiolated. epicotyl. segments. with.Agrobacterium tumefaciens.8.A.similar.transformation.procedure.with.modifica-tions.has.been.used.to.produce.transgenic.plants.of.other.citrus.genotypes.9.In.general,.rooting. of. transgenic. shoots. in. citrus,. except. for. P. trifoliata. and. grapefruit,. is.rather.inefficient.but.can.be.alleviated.by.the.use.of.shoot.tip.grafting.9,10.Mature.tissues.have.also.been.used.as.explant.material.in.order.to.maintain.genotype.iden-tity.and.overcome.the.long.juvenile.growth.phase..Buds.collected.from.trees.are.grafted.onto.seedlings.of.a.vigorous.rootstock.grown.under.glasshouse.conditions;.new.shoots.elongated.from.them.are.then.used.as.starting.material.9

Transformation.via.organogenesis.has.also.been.applied.in.several.other.fruit.tree.species..For.example,.apple.transformation.was.first.reported.by.James.et.al.,.who.used.Agrobacterium-mediated.transformation.of.leaf.disks.from.the.apple.cultivar.‘Greensleeves’.11,12.Although.somatic.embryogenesis.is.the.most.prevalent.regenera-tion.method.used.in.grape.genetic.transformation,.transgenic.plants.of.table.grape.cultivars.‘Silcora’.and.‘Thompson.Seedless’.have.also.been.produced.via.organo-genesis..The.method.is.based.on.the.formation.of.meristematic.bulk.tissue.with.a.high. regenerative.capacity,.using.adventitious. shoots.as.a.starting.material..Shoot.regeneration.is.obtained.30.days.after.meristematic.bulk.slices.are.inoculated.with.Agrobacterium.13.Alternatively,. shoot. tips. subjected. to.wounding. and. then.cocul-tivated. with. Agrobacterium. have. been. shown. to. rebuild. complete. meristems. and.produce.non-chimeric.transgenic.‘Thompson.Seedless’.plants.14

Genetic Transformation via Somatic EmbryogenesisEmbryogenic.cultures.are. the.preferred.starting.materials. for. transformation.with.either.Agrobacterium.inoculation.or.microprojectile.bombardment.because.they.are.the.most.responsive.tissues,.with.competence.for.in.vitro.regeneration.and.genetic.transformation.15.The.entire.process. involves. induction.of. somatic.embryogenesis,.


maintenance.of.the.embryogenic.callus,.and.transformation,.selection,.and.regenera-tion.of.plants..The.large.number.of.cultivars.and.rootstocks.increases.the.complexity.due.to.genotype.differences.observed.at.all.tissue.culture.steps.

In.walnut,.somatic.embryogenesis.techniques.were.developed.for.Juglans regia.but.have.been.applied.to.other.Juglans. species.. In. this.system,. immature.walnuts.are.cultured.on.a.basal.medium.and.secondary.embryos. that.originate. from.cells.in. the.epidermal. layer.of. the.embryo.display.high.susceptibility. to. infection.with.Agrobacterium.16. A. major. challenge. today. in. walnut. tissue. culture. is. to. obtain.embryogenesis. from. maternal. tissue.. This. is. important. because. embryos. from.zygotic.tissue.do.not.allow.the.exact.genotype.to.be.predicted..Repetitively,.embryo-genic.cultures.have.been.obtained.from.immature.anther.tissue,.but.only.from.the.“Chandler”.cultivar..Recently,.a.modified.protocol.allowed.a.somatic.embryo.line.to.be.generated.from.immature.anthers.of.a.“Paradox”.hybrid.that.may.provide.a.source.of.elite.rootstock.tissue.for.genetic.transformation.17

In.grapevine,. anthers.collected.during.first. pollen.mitosis. are. the.most.widely.used.explant.for.culture.initiation,.with.the.embryogenic.callus.arising.from.diploid.tissue.18.Embryogenic.callus.maintenance.has.been.accomplished.in.liquid,.semisolid.or.solid.media.supplemented.with.a.variety.of.growth.regulator.combinations.19–23.The.variety.of.protocols.developed.for.maintenance.of.embryogenic.cultures.is.due.to.the.fact.that.the.production.of.suitable.embryogenic.cultures.for.transformation.with.Agrobacterium.has.represented.a.greater.challenge.than.the.initiation.step,.with.higher.transformation.efficiencies.being.obtained.with.embryogenic.lines.composed.of.fine.cells,.arrested.in.a.very.early.pre-embryogenic.state.7

methodS of tranSformatIon

Agrobacterium-Mediated TransformationThe. vast. majority. of. transformation. protocols. use. A. tumefaciens. (biovar. 1). as. a.vector.. Agrobacterium. strains. that. work. efficiently. for. walnut,. citrus,. and. apple.transformation.include.disarmed.derivatives.of. the.tumorigenic.strains.A281.(e.g.,.EHA101. and. EHA105). and. C58. (e.g.,. C58C1).3,12,16. The.disarmed. strains. possess.the.Ti.plasmid-based.vir.genes.required.for.plant.transformation,.but.lack.the.native.T-DNA.(which.is.associated.with.pathogenesis)..These.strains.are.transformed.with.broad. host. range. binary. plasmids. that. contain. the. desired. T-DNA. region,. carry-ing.the.gene.of.interest.and.selectable.marker.genes,.mainly.conferring.resistance.to. antibiotics,. such. as. kanamycin. and. hygromycin.24. However,. given. the. public.concern.with. the. introduction.of.antibiotic.resistance.genes. into.food,.methods. to.eliminate.these.selectable.marker.genes.from.the.transformed.plants.and.strategies.that. avoid. selection.with.antibiotics.are.being.developed.. In.citrus,.workable. trans-formation.efficiencies.have.been.achieved.using.a.multi-auto-transformation.(MAT).vector.combined.with.an.inducible.recombinase/recombination.sites.(R/RS)–specific.recombination.system.and.the.phosphomannose.isomerase/mannose.conditional.posi-tive.selection.system.25.In.addition,.a.cotransformation.system.using.a.mixture.of.two.Agrobacterium. strains. (one.harboring. the.gene.of. interest.and. the.other.containing.both.positive.and.negative.selectable.markers.genes).has.been.used.to.obtain.transgenic.grapevines.free.of.selectable.marker.genes.26


Biolistic BombardmentThe.biolistic.process.has.been.successfully.applied.to.grape;.however,.the.equipment.and.expertise.required.together.with.the.degree.of.optimization.needed.to.establish.a.suitable.bombardment.protocol.for.specific.cultivars.have.limited.the.value.and.wide.scale.application.of. this. technology..It.was.initially.tested.using.embryogenic.cell.suspensions.of.‘Chancellor’,.a.Vitis.complex.interspecific.hybrid,.and.then.success-fully.extended.to.Vitis vinifera.cultivars.‘Chardonnay’.and.‘Merlot’.27,28

tranSIent expreSSIon

Agrobacterium-mediated.transient.assays.have.become.an.important.tool.for.gene.function. analysis.. Two. high. quality. draft. genome. sequences. have. been. reported.for. grapevine.29,30.They. are. the.first. genome. sequences. produced. for. a. fruit. crop.and. will. stimulate. the. search. for. rapid. functional. genomics. screening. systems..Agroinfiltration.has.been.used. to. transform. leaves.of. in. vitro. cultured.grapevine.plants.31.Conversely,.transient.transformation.in.grape.berry.skin.has.been.achieved.by. treating. half-cut. berries. with. an. A. tumefaciens. suspension.32. In. intact. fruit.of. citrus,. efficient. transgene. expression. was. accomplished. through. injection. of.Agrobacterium.into.the.fruit.33

Viral.vectors.represent.an.alternative.transient.expression.system.that.has.been.shown.to.be.a.useful.strategy.for.overexpression.or.silencing.of.plant.genes.in.annu-als.. RNA. virus–based. vectors,. carrying. a. green. fluorescent. protein. coding. gene,.have.been.developed.for.citrus. trees..The.most.successful.vectors.were.unusually.stable.and.continued.producing.fluorescence.more.than.4.years.after.inoculation.of.Citrus macrophylla.seedlings.34

rootStock tranSformatIon

Nearly. all. commercial. fruit. trees. are. propagated. vegetatively.. Generally,. cuttings.or.buds.from.desired.varieties.are.grafted.onto.rootstocks.selected.for.adaptation.to.specific.soil.conditions.and.resistance.to.root-destroying.diseases.and.insects..For.this.reason,.genetic.transformation.of.rootstocks.has.been.oriented.to.improve.these.characteristics..However,.rootstock.genetic.engineering.can.be.directed.to.modify.the. properties. of. the. scion.. Currently,. the. signal. peptide. sequences. derived. from.mRNAs.found.in.grape.xylem.exudates.are.being.evaluated.for.delivery.of. thera-peutic.proteins.into.the.xylem.35.Such.transgenic.products,.if.synthesized.in.a.root-stock,.could.move.through.the.graft.union.and.confer.resistance.to.xylem-specific.infections.such.as.Xylella fastidiosa,.a.Gram-negative.bacterium.that.causes.Pierce’s.disease.(PD).in.grapevines.

INPUT TRAITS

Genetic. transformation. is.a. tool. for.creating.new.fruit. and.nut. tree.crop.varieties.with.improved.input.traits,.which.enhance.agronomic.characteristics.and.crop.per-formance,.conferring.great.benefit.to.growers..Traditional.breeding.to.improve.input.traits.like.resistance.to.pathogens.and.insects.is.very.inefficient,.labor-intensive,.and.


time-consuming.. However,. genetic. engineering. offers. a. real. possibility. to. create.fruit.and.nut.tree.crop.varieties.with.desirable.agronomic.traits.that.exhibit.increased.resistance.to.pathogens.and.insects..Here,.we.discuss.recent.progress.in.increasing.resistance.to.viruses,.bacteria,.fungi,.and.insects.through.the.genetic.transformation.of.fruit.and.nut.tree.crops.

pathoGen reSIStance

Viral ResistanceViruses. are. the.main. cause. of. economic. losses. to. agricultural. crops. worldwide..For.example,.Citrus tristeza virus.(CTV).causes.huge.economic.losses.for.the.cit-rus. industry.and.Plum pox virus. (PPV).causes.one.of. the.most.devastating.viral.diseases. of. stone. fruit. (Prunus. spp.).. Several. transgenic. approaches. have. been.successfully.applied.for.virus.resistance.in.fruit.crops.based.on.pathogen-derived.resistance.(PDR).strategies..Virus.resistant.transgenic.crops.have.been.developed.during.the.last.two.decades.through.both.the.expression.of.transgenic.virus.RNA.in.host.plants.and.protein-mediated.resistance.36–39.Described.as.a.potent.genetic.vaccination. against. viral. sequences,. RNA-mediated. virus. resistance. (RMVR). is.less. susceptible. to. variation. in. virus. sequence. and. is. highly. sequence-specific,.often.providing.complete. immunity. to. the. inoculated.virus.or.RNA.40,41.Protein-mediated.resistance.has.the.potential.for.broader.protection.but.generally.results.in.mild.resistance,.which.only.delays.symptoms.and.decreases.viral.titers..However,.the. combination. of. RNA-. and. protein-mediated. resistance. in. transgenic. plants.could.prove. to.be.potent,.broad,.and.durable.41.Gene.silencing. is.another.power-ful.biotechnological.tool.used.to.gain.virus.resistance.for.fruit.crops..This.method.has.produced.transgenic.plants.able.to.switch.off.endogenous.genes.and.invading.nucleic.acids.42–47

Papaya Ringspot VirusPapaya ringspot virus. (PRSV). causes. a. destructive. disease. in. papaya. (Carica papaya. L.),. an.economically. important. fruit. crop. in. tropical.and. subtropical. areas,.resulting.in.drastic.reduction.of.fruit.quality..PRSV.is.naturally.transmitted.by.aphids.in. a. nonpersistent. manner. and. induces. stunting. in. growth,. symptoms. of. mosaic.and.distortion.on. leaves,.and.streaks.on.petiole.and.stem..Resistance.against.PRSV.in.papaya.has.not.been.obtained.by.conventional.breeding.. In. the. last. two.decades,.effective.strategies.based.on.PDR.have.been.widely.used..The.coat.protein.(CP).gene.of.PRSV,.HA.5-1,.was.transferred.into.papaya.via.micro-projectile.bombardment.48–50.Transgenic.papaya.lines.highly.resistant.to.the.severe.Hawaiian.PRSV.strain.PRSPV.HA.were.selected.under.greenhouse.and.field.conditions.49,50.Rainbow.and.SunUp.cul-tivars.have.been.successfully.commercialized.in.Hawaii.since.1998,.representing.the.first. practical. application. of. transgenic. fruit. crop.51,52. However,. the.CP-hemizygous.line.Rainbow.is.susceptible.to.non-Hawaiian.PRSV.isolates.and.the.CP-homozygous.line.SunUp,.while.resistant.to.a.wider.range.of.isolates.from.Jamaica.and.Brazil,.is.still.susceptible.to.isolates.from.Thailand.and.Taiwan.51,53,54

In.Taiwan.transgenic.papaya.lines.carrying.the.CP.gene.of.a.Taiwan,.severe.strain,.PRSV.YK,.have.also.been.successfully.generated.55.Several.transgenic.lines.provide.


broad.spectrum.resistance.against.homologous.and.geographically.distinct.strains.of.PRSV.under.greenhouse.conditions56.and.a.high.degree.of.resistance.under.field.trials.57.During.the.field.trial,.some.transgenic.papaya.lines.showed.susceptibility.to. the.new.emerging.papaya leaf distortion mosaic virus. (PLDMV).which. indi-cates.that.the.virus.is.a.threat.for.PRSV-resistant.transgenic.papaya.in.Taiwan.and.elsewhere.58

Recently,.transgenic.papaya.lines.with.double.resistance.to.PRSV.and.PLDMV.were.generated.using.a.chimeric.construct.strategy.containing.a.fused.cDNA.frag-ment.composed.of.the.truncated.PLDMV.P-TW-WF.CP.and.PRSV.YK.CP.coding.regions..Three.of.nine.resistant.transgenic.papaya.lines.carrying.the.chimeric.con-struct.showed.high.levels.of.resistance.to.heterologous.PRSV.strains.from.Hawaii,.Thailand,. and. Mexico.. Transgenic. papaya. lines. look. promising. for. resistance. to.PRSV.strains.and.PLDMV.in.Taiwan.and.elsewhere.59

Citrus Tristeza VirusCitrus tristeza virus. (CTV). (Closterovirus). is. considered. the. most. economically.important. virus. affecting. citrus. production. worldwide.. CTV. is. spread. by. several.species. of. aphids. with. piercing-sucking. mouthparts,. Toxoptera citricidus. being.the. most. efficient. vector.. This. viral. disease. is. now. endemic. in. several. economi-cally.important.production.regions.around.the.world..Different.strains.of.CTV.can.cause.diverse.disease.syndromes. that.vary. in.severity..Symptoms.may.be.as.mild.as.weak.and.scattered.vein.clearing,.whereas.quick.decline,.a.syndrome.in.which.a.tree.with.normal.appearance.starts.showing.wilt.symptoms.and.completely.collapses.in. a. few. weeks,. is. the. most. dramatic. manifestation. of. disease.. The. development.and.identification.of.CTV.tolerant.rootstocks.was.one.of.the.main.strategies.for.the.control. of. quick-decline. disease.60. Recently,. biotechnology. tools. like.gene. silenc-ing. (GS).have. been. successfully. applied.. Specifically,. targeting. CTV.using. RNA.interference.(RNAi).strategies.is.the.major.strategy.in.plant.transformation.for.CTV.resistance..Citrus.lines.expressing.p23.CTV,.a.silencing.suppressor,.were.reported.as.resistant.to.CTV,.and.transgenic.viral.protein.accumulation.was.negatively.cor-related. to. resistance.. In. this.study,.posttranscriptional.gene.silencing.(PTGS).was.proposed.as.the.mechanism.for.resistance.to.CTV,.since.resistant.citrus.plants.had.multiple.copies.of.the.transgene,.low.levels.of.the.corresponding.mRNA,.methyla-tion.of.the.silenced.transgene,.and.accumulation.of.the.p23-specific.small.interfer-ing.RNAs. (siRNAs).61.RNA-mediated. resistance. for.CTV.has. also.been.obtained.in. transgenic. grapefruit.62,63. To. successfully. obtain. CTV. resistant. plants. through.gene.silencing,.it.is.important.to.understand.all.the.viral.counterdefense.strategies,.like.the.virus’s.RNAi.suppression.strategies.developed.during.their.coevolution.with.plants.43,45,64. Protein-mediated. resistance. strategies. have. also. been. described. for.CTV..CTV-resistant.citrus.plants.that.express.the.CP.of.the.virus.have.been.obtained.and. in. these.plants.virus. resistance.was. related. to. the. accumulation.of. the. trans-genic.viral.protein.in.plant.tissue,.with.PDR.as.the.proposed.mechanism.of.resis-tance.65–69.Protoplast.fusion.is.another.promising.biotech.tool.because.it.can.produce.somatic.hybrids,.which.would.be.very.valuable.for.the.development.of.CTV-resistant..rootstocks..Citrus.somatic.hybrids.with.resistance.to.CTV.infection.have.been.suc-cessfully.obtained.by.protoplast.fusion.70


Plum Pox VirusSharka,.or.plum.pox,.caused.by.the.plum pox virus.(PPV,.genus.Potyvirus),.is.one.of. the. most. serious. diseases. for. Prunus. stone. fruit. and. nut. species.. This. virus.has.caused.serious.economic.losses.to.the.stone.fruit.industry.in.Europe,71,72.and.may.eventually.spread.widely.throughout.North.America.and.South.America.73–75.Prune dwarf virus.(PDV),.prunus necrotic ringspot virus.(PNRSV),.and.tomato ringspot virus.(ToRSV).are.other.viruses.widespread.in.production.areas.world-wide. that. are. very. destructive. for. peach,. plum,. cherry,. and. apricot. production..Viruses. endemic. to. North. America,. including. peach mosaic virus. (PMV). and.American plum line pattern virus. (APLPV),.are.also.known. to.cause. important.diseases.of.stone.fruit.76,77

PPV.mainly.infects.plum,.apricot,.and.peach.trees.72,78,79.Sharka.disease.symptoms.range.from.weak.necrosis.on.leaf.blades.and.stems,.to.death.of.young.shoots..PPV.is.transmitted.by.aphids.in.a.nonpersistent.manner,.its.main.vectors.being.Myzus persicae.and.Aphis spiraecola.80–83.Due.to.the.severe.economic.losses.in.crop.production.for.the.stone. fruit. industry,.efforts.have.focused.on. the.development.of.PPV-resistant.Prunus.cultivars.either.by.conventional.breeding.or.by.biotechnology.approaches..PPV-resistant. plant. selection. by. traditional. breeding. has. not. been. successful,. but.the. use. of. biotechnology. approaches. to. obtain. resistant. plants. looks. promising..Pathogen-derived.resistance.to.PPV.has.been.achieved.by.the.introduction.of.either.wild.type.or.mutated.structural.and.nonstructural.genes.into.host.plants.38.A.gene.silencing. approach46. was. used. to. obtain. transgenic. European. plums. carrying. the.CP.gene.of.PPV..The.diversity.and.dynamics.of.PPV.and.aphid.population.in.trans-genic. European. plums. has. also. been. evaluated. in. the. field. during. 7. years. under.Mediterranean.conditions.84.After.7.years.of.experimental.trials,.the.transgenic.line.C5.(cv..Honey.Sweet).was.reported.to.be.free.from.PPV.84–86.The.resistance.mecha-nism.of.line.C5.was.confirmed.to.be.based.on.PTGS.46,87

Because.PPV.is.only.one.of.the.multiple.viruses.affecting.Prunus.stone.fruit.pro-duction,.it.is.highly.desirable.to.engineer.multivirus.resistance.in.plums..Accordingly,.PTGS-based.approaches.have.been.used.to.provide.multiple.resistances.to.important.viruses.affecting.Prunus.stone.fruit.and.nut.species..A.single.chimeric.transgene,.PTRAP6,.was.created.by.the.fusion.of.400–500.bp.long.fragments.from.six.major.Prunus. fruit.viruses. (APLPV,.PMV,.PPV,.PDV,.PNRSV,.and.ToRSV)..Nicotiana benthamiana.plants.transformed.with.PTRAP6.displayed.resistance.to.PDV,.PPV,.and.ToRSV.88

Bacterial ResistanceErwinia amylovoraFire. blight,. caused. by. the. Gram-negative. bacterium. Erwinia amylovora,. is. an.incredibly.destructive.bacterial.disease.of.pear,.apple,.and.other.members.of. the.Rosaceae.family,.as.well.as.grape..Most.commercially.available.apple.scion.cul-tivars.and.rootstocks.are.particularly.sensitive.to.E. amylovora..Resistance.to.fire.blight. in.apple.and.pear.has.been.engineered.using.several. transgenic. strategies..Expression.of. the.lytic.peptide.attacin.E.in.transgenic.apples.and.pears.provided.good. resistance. to. the. pathogenic. bacterium.89–96. Other. genes. used. to. improve.


fire.blight. resistance. are.epo,. a. gene. encoding. EPS-depolymerase,97,98. SB-37,91,99.T4. lysozyme,91,95,99,100. hrpN. (harpin),101. cecropin. MB. 39. (modified. SB-37),102,103.and. Shiva-1.91. Transgenic. apples. resistant. to. fire. blight. have. also. been. obtained.by.partially.silencing.certain.genes.encoding.pathogen-protein.receptors.104.There.are.also.opportunities.for.breeding.fire.blight-resistant.apple.and.pear.cultivars.by.exploiting.genetic.variation.in.germplasm.and.by.developing.quantitative.trait.loci.(QTL).markers.105,106

Agrobacterium tumefaciensCrown.gall,.caused.by.the.soil.bacterium.Agrobacterium tumefaciens,.greatly.dimin-ishes.tree.productivity.and.is.a.serious.disease.for.many.fruit,.nut,.and.ornamental.crops..Walnuts.in.particular.are.very.susceptible.to.this.disease..Losses.are.incurred.from.both.contaminated.nursery.stock.and.infected.orchard.trees..Current.prophy-lactic.measures.and.the.mechanical.removal.of.galls.have.not.adequately.controlled.the.problem..Pathogenic.A. tumefaciens. transforms.infected.plants.with.the.genes.iaaM. (tryptophan. monooxygenase),. iaaH. (indole-3-acetamide. hydrolase),. and. ipt.(isopentenyl. transferase)..The. iaaM. and. iaaH.gene.products.convert. tryptophan.into. indole-3-acetic. acid,. an. auxin,. while. the. ipt. gene. product. catalyzes. the. pro-duction.of.adenosine.monophosphate.(AMP),.which.is.converted.to.cytokinins.by.endogenous.plant.enzymes..The.resulting.overproduction.of.auxin.and.cytokinins.induces.proliferation.of.callus.tissue.at.the.wound.site,.resulting.in.the.development.of.large.galls.17,107

To.engineer.resistance.to.this.pathogen,.a.binary.vector.plasmid.containing.inverted.repeats.of.portions.of.the.iaaM.and.ipt.genes.was.constructed.and.transformed.into.walnut.108.Constitutive.expression.of.this.construct.induces.RNAi-mediated.degrada-tion.of.the.iaaM.and.ipt.transcripts,.demonstrating.the.use.of.RNAi.to.generate.resis-tance.to.a.major.bacterial.disease.109.Because.the.construct.and.the.oncogenes.do.not.need.to.have.perfect.homology.for.silencing.to.be.effective,.the.resulting.transgenic.plants.are.resistant.to.a.very.wide.range.of.A. tumefaciens.strains,.displaying.a.broad.spectrum.durable.resistance.108.Transgenic.walnut.plants.containing.the.iaaM-.and.ipt-silencing.constructs.are.currently.in.field.trials..RNAi has.also.been.used.to.gen-erate.crown.gall.resistance.for.tomato,110.apple,111,112.and.grape.113

Xylella fastidiosaXylella fastidiosa.(Xf ).is.a.xylem-limited,.Gram-negative.bacterium,114..transmitted.by. insect. vectors. (especially. the. glassy. winged. sharpshooter,. Homalodisca coagulate).. Different. Xf. strains. cause. devastating. diseases. in. economically.important.plants,.for.example.PD.in.grape,.citrus.variegated.chlorosis.(CVC).in.citrus,115. and.almond. leaf. scorch.disease. (ALSD). in.almond..Grape.PD.symp-toms.include.yellowing.and.gradual.necrosis.of.the.petiole.attached.to.the.cane.after. leaf. fall.. The. disease. progresses. rapidly,. resulting. in.occlusion.of. xylem.vessels.and.consequent.water.stress..Vine.death.often.occurs.within.2.years.116.Citrus.CVC.symptoms. include. leaf.variegated. chlorosis,. defoliation,. twig.die-back,.size.reduction,.and.hardening.of.fruits.115.The.disease.management.strat-egy.for.PD.in.grape.focuses.on.containing.vector.transmission.using.insecticides,.with.limited.success.


Recently,. transgenic. grapevines. containing. xylem-targeted. effector. proteins.like. polygalacturonase. inhibiting. protein. (PGIP). and/or. chimeric. antimicrobial.proteins.have.been.obtained.117–121.The.use.of.PGIP.to.transform.grapevines.was.based.on.the.knowledge.that.Xf.has.genes.that.encode.plant.cell.wall–degrading.enzymes,. including.polygalacturonases.117.Five.PGIP.expression.constructs.were.designed,.each.containing.different.signal.peptide.sequences.in.order.to.identify.which. most. efficiently. localizes. PGIP. to. xylem. tissues,. as. well. as. which. pro-vides. the. best. distribution. of. PGIP. through. the. graft. union. into. untransformed.scion.tissues.120.Chimeric.antimicrobial.protein.strategy.provides.a.protein-based..therapeutic.that.targets.the.causative.agent.specifically.resulting.in.its.clearance,.which. results. in. resistance. to. the. causative. agent.. Chimeric. proteins. have. two.domains,.a.surface.binding.domain.and.a.clearance.domain. linked.by.a.flexible.linker.such.that.both.components.can.act.independently.120,121.A.chimeric.antimi-crobial.protein.was.designed.to.obtain.resistance.to.PD.that.contained.a.surface.recognition.domain.(SRD).and.a.clearance.domain.(CD)..The.SRD.targeted. the.Xf surface.protein.mopB,.highly.conserved.among.all.Xf.strains.and.believed.to.be. unique. to. Xf118. linked. to. the. CD,. a. lytic. peptide. with. antimicrobial. activity.against. Gram-negative. bacteria.. Transgenic. grapevines. expressing. PGIP. or. the.chimeric. antimicrobial. protein. have. been. greenhouse. propagated. and. mechani-cally.inoculated.with.Xf.to.validate.their.efficiency.against.PD;.the.results.obtained.look.promising.120.Transgenic.grapevines.field.trials.in.multiple.locations.started.in.2010.

XanthomonasCitrus. canker. (CC). is. a. serious. endemic. disease. caused. by. Xanthomonas citri. (syn. Xanthomonas campestris. pv..citri. or. related.Xanthomonas campestris.pv. aurantifolii).. X. citri. affects. various. citrus. species. and. is. dispersed. by. wind-blown. rain,. contaminated. equipment,. and. human. activity.. Canker. threatens. the..existence. of. citrus. industry,. affecting. leading. citrus. producers. from. Brazil. and.Florida..Canker.symptoms.are.characterized.by.pustule-like.lesions.that.can.cover.the.surfaces.of.leaves,.stems,.and.fruits..Such.lesions.later.become.corky.and.sur-rounded.by.water-soaked.margins.with.a.yellow.halo.122.It.has.been.observed.that.soon.after..infection, X. citri.simultaneously.suppresses.the.host.basal.defense.and.induces.remarkable.changes.in.the.transcriptional.profiles.of.genes.associated.with.cell.wall.remodeling,.cell.division.and.expansion,.vesicle.trafficking.and.response.to.the.hormones.auxin.and.gibberellin.123–125

Genetic.transformation.has.been.used.in.citrus.cultivars.to.improve.CC.disease.resistance.by.the.expression.of.the.Sarcophaga peregrine.sarcotoxin,126.the.atta-cin E.antimicrobial.peptide.from.Tricloplusia ni,127,128.the.Xa21.gene.from.Oryza sativa,129.and.the.harpin.N.gene.(hrpN).from.Erwinia amylovora.130.For.example,.transgenic. Citrus sinensis. plants. expressing. hrpN. under. transcriptional. control.of.a.pathogen. inducible.promoter. (gst1).showed.up. to.79%.reduction. in.suscep-tibility.to.CC.compared.with.non-transgenic.plants.130.In.addition,.researchers.at.Integrated.Plant.Genetics.have.developed.the.Disease.Block™.technology,.which.consists. of. the. expression. of. a. recombinant. antibody. fragment. directed. against.PthA,.an.effector.protein.associated.with.division,.enlargement,.and.death.of.the.


host.cell.during.the.first.stages.of.canker.formation.131,132.The.cytoplasm-expressed.antibody.fragment.should.bind.to.PthA.upon.delivery.into.the.host.cell.and.prevent.its.nuclear.localization..This.strategy.did.not.produce.fully.resistant.plants.but.was.efficient.in.slowing.the.disease.process,.resulting.in.a.2000-fold.reduction.in.the.number.of.X. citri.cells.released.after.one.cycle.of.infection..Complete.immunity.is.considered.possible.by.improving.the.stability.and.expression.level.of.the.recom-binant.antibody.133

Fungal ResistanceVenturia inaequalisApple.scab,.caused.by.the.fungal.pathogen.Venturia inaequalis,.is.a.serious.dis-ease.in.almost.all.apple.cultivars.grown.commercially.around.the.world.and.causes.significant. losses. worldwide.. A. scab. resistance. locus,. Vf,. has. been. identified. in.the.crabapple.species.Malus floribunda.134.The.Vf.locus.confers.resistance.to.five.races. of. V. inaequalis135. but. not. to. races. 6. and. 7,. identified. in. Europe.105,136,137.A number.of.other.scab.resistance.genes.have.been.mapped.onto.the.apple.genome,.but.only.the.Vf.locus.has.been.analyzed.in.detail..Vf.is.a.complex.locus.contain-ing. four. paralogs,. Vfa1,. Vfa2,. Vfa3,. and Vfa4.138,139. HcrVf2,. a. homolog. to. the.Cladosporium fulvum.resistance.gene.of.tomato,.was.transformed.into.the.‘Gala’.cultivar. of. apple,. conferring. scab. resistance.140. In. addition,. the. Vfa1,. Vfa2,. and.Vfa4.genes.were.introduced.into.a.plant.cloning.vector,.pCAMBIA2301,.and.used.for.Agrobacterium-mediated.transformation.of.‘Galaxy’.and.‘McIntosh’.apple.cul-tivars.. Transformed. lines. expressing. Vfa1. and. Vfa2. exhibited. partial. resistance.to.apple.scab,.while.transformed.lines.expressing.Vfa4.were.found.to.be.suscep-tible.to.apple.scab.141.The.stilbene.synthase.(Vst)gene.from.Vitis vinifera.L..is.also.being.used.to.transform.apple.with.apple.scab.resistance.142.Alternative.approaches.to. engineer. resistance. include. expression. of. an. antimicrobial. peptide,143,144. chi-tinases,96,145,146. exochitinases,147–149. and. endochitinases.147,150,151. Transgenic. apple.plants.expressing.high. levels.of.endochitinase.are. resistant. to.V. inaequalis,.but.are.also. stunted.150.One.promising.strategy. for.breeding.durable. scab. resistance.is.to.combine.several.functionally.different.resistance.genes.into.a.single.cultivar.(pyramiding.of.resistance.genes).105,152,153

Botrytis cinereaGray.mold,.caused.by.Botrytis cinerea,.attacks.the.shoots,.leaves,.flowers,.and.fruits.of.grape,.tomato,.and.strawberry,.as.well.as.other.crops..It.is.one.of.the.most.destruc-tive.fruit.diseases,.resulting.in.significant.economic.losses.pre-.and.postharvest..The.most.common.strategy.to.control.B. cinerea.is.the.regular.application.of.fungicides.throughout.flowering..However,. new. strategies.utilizing.PGIPs,. cell.wall. proteins.that.can.inhibit.fungal.polygalacturonases,.have.been.shown.to.be.effective.in.con-trolling.B. cinerea.154.Transgenic.expression.of.pear.PGIP.in.tomato155.and.grape117.limits.fungal.colonization.by.inhibiting.fungal.polygalacturonases,.reducing.suscep-tibility.to.B. cinerea..Strategies.based.upon.altering.the.expression.of.native.plant.cell.wall–modifying.enzymes.have.also.proved.effective.in.controlling.gray.mold..For. example,. suppression. of. the. endo-beta-1,4-glucanases. Cel1. and. Cel2. reduces.gray. mold. in. tomato.156. Likewise,. simultaneous. suppression. of. genes. encoding.


polygalacturonase.(LePG).and.expansin.(LeExp1).in.transgenic.tomato.plants.dra-matically.reduced.the.susceptibility.of.ripening.fruit.to.B. cinerea..Pathogen.hydro-lases.targeting.the.plant.cell.wall.are.well-known.components.of.virulence,.and.it.has.been.experimentally.established. that.wall.disassembly.by. the.plant. itself.also.contributes.to.susceptibility.157.Strawberry.has.a.lack.of.natural.genetic.resistance.to.gray.mold,.but.transgenic.expression.of.genes.like.ch5B.encoding.a.chitinase158.and.thau II.encoding.thaumatin.II159.from.other.plant.species.have.been.used.to.obtain.transgenic. strawberry. lines. with. resistance. to. B. cinerea.. Transgenic. wild. straw-berry.(Fragaria vesca).fruits.overexpressing.the.Fragaria × ananassa.pectin.methyl.esterase.gene.(FaPE1).showed.increased.resistance.to.B. cinerea..This.resistance.is.related.to.the.increase.in.pectin-derived.oligogalacturonides,.essential.components.for.elicitation.of.defense.responses.to.B. cinerea.160

Phytophthora nicotianaePhytophthora.species.are.considered.one.of.the.most.important.soil.borne.problems.for. citrus,. leading. to. considerable. losses. worldwide.161,162. Gumosis,. encompassing.both.trunk.rot.and.root.rot,.is.the.most.relevant.disease.caused.by.this.pathogen.and.is.characterized.by.necrosis.and.gum.exudation.at.the.ground.level..Phytophthora nicotianae.has.been.most.frequently.associated.with.the.disease..Transgenic.orange.plants.have.been.engineered.using. the.coding.region.of. the. tomato.pathogenesis–related.protein.PR-5,.a.chitinase.with.antifungal.activity..Transgenic.plants.as.well.as.their.detached.bark.were.challenged.with.oomycete.cultures.and.P. citrophthora,.respectively..A.significant.reduction.in.lesion.development.was.observed.in.one.of.the.transgenic.lines.when.compared.with.the.control.plants.163.Somatic.hybridization.has.also.been.used.to.produce.citrus.somatic.hybrids.tolerant.to.trunk.and.root.rot.caused.by.Phytophthora nicotianae..In.Florida,.more.than.70.somatic.hybrids.have.already.entered.into.commercial.field.trials.as.potential.rootstocks.70

Uncinula necatorPowdery.mildew,.caused.by.Uncinula necator,.is.one.of.the.most.threatening.fun-gal. problems. for. grape.. Transgenic. plants. harboring. a. constitutively. expressing.rice. class. I. chitinase. gene. showed. enhanced. disease. resistance. to. powdery. mil-dew. and. anthracnose. when. compared. with. the. control. lines.164. Also,. transgenic.‘Chardonnay’. grapevines. containing. the. magainin. gene. (mag2),. a. peptide. with.broad.spectrum.antimicrobial.activity,.showed.measurable.symptom.reduction. in.response.to U. necator.under.greenhouse.conditions.165

InSect reSIStance

Cydia pomonellaThe.larvae.of.the.codling.moth.(CM).are.a.severe.threat.to.apple.and.walnut.pro-duction. worldwide.. The. CM. lays. its. eggs. on. fruit. or. leaf. clusters. near. fruit,. and.when.they.hatch,.the.larvae.feed.on.the.fruit,.causing.considerable.economic.dam-age..Application.of.chemical.pesticides,.the.main.method.of.controlling.this.insect,.has.failed.to.control.CM.larvae..Biotechnology.is.an.alternative.to.create.resistance.


to. insect. pests. and. to. avoid. chemical. pesticide. use.. The. predominant. strategy. to.engineer. CM. resistance. in. tree. crops. has. been. through. expression. of. Bacillus thuringiensis.(Bt).genes.that.encode.insecticidal.crystal.proteins.(ICP).166–169.In.vitro.studies.have.shown.that.ICPs.bind.with.high.specificity.and.affinity.to.specific.cell.receptors.on.the.insect.brush.border.membrane.of.midgut.epithelial.cells.170–172.This.binding.correlates.with. the. formation.of.pores.and.membrane. lesions. in. the.mid-gut. that. lead. to. swelling,. leakage,. and. lysis.of. the. epithelium,. ultimately. causing.death.of.the.insect.through.starvation.and.septicemia.172–174.ICP.genes.from.differ-ent. Bacillus. species. are. categorized. according. to. the. host. range. of. their. activity.and.DNA.sequence.homology.174,175.The.crylA(c).protein.was.found.to.be.the.most.toxic.to.CM.larvae,176.but.the.transformation.of.apple166.and.walnut167.with.cry1A(c).produced.very.low.levels.of.gene.expression..This.was.a.result.of.codon.bias.in.the.bacterial.gene.sequence,.which.resulted.in.very.low.levels.of.translation.167,169.Codon-optimized.synthetic.versions.of.cryIAc.have.been.introduced.into.apple,.where.they.confer.high.levels.of.mortality.to.CM.larvae.both.under.greenhouse.and.field.con-ditions.. Similar. experiments. have. also. been. done. in. walnut177. and. persimmon,178.where. chemically. synthesized. versions. of. cryIAc. provided. excellent. protection.against.target.insect.larvae..Transgenic.tissues.expressing.cryIAc.protein.at.as.low.as.0.02%.of.total.cellular.protein.produced.100%.mortality.in.CM.larvae.177.Recent.studies.have.shown.evidence.that.RNA.silencing.pathways.also.play.a.role.in.plant.defense.against.insects,.which.can.provide.an.alternative.biotechnological.strategy.to.develop.pest.control.in.plants.179

Epiphyas postvittana (Light Brown Apple Moth)Epiphyas postvittana,.the.light.brown.apple.moth,.is.a.native.pest.of.horticultural.crops. in. Australia. and. New. Zealand,180. and. is. now. present. in. New. Caledonia,.North-Western.Europe,.Hawaii,.and.California..The.larvae.of.the.light.brown.apple.moth.cause.significant.damage.to.apple.foliage.and.fruit..To.confer.resistance.to.E. postvittana,. the. apple. cultivar. ‘Royal. Gala’. was. transformed,. incorporating.genes.encoding.the.potato.(Solanum tuberosum).biotin.binding.proteins.avidin.and.strepavidin.. In. the.absence.of.biotin,.an.essential.vitamin.for. insects,.80%–90%.of.larvae.feeding.on.the.transgenic.apple.lines.died.in.a.period.of.3.weeks,.com-pared.to.14%.on.the.control.plants.181.The.recent. identification.of.genes.involved.in. E. postvittana. digestion182. has. allowed. proof-of-concept. research. focused. on.the. development. of. an. alternative,. RNAi-based. strategy. to. combat. this. pest.. In.controlled.feeding.experiments.with.E. postvittana.larvae,.oral.delivery.of.dsRNA.homologous.to.the.EposCXE1.led.to.silencing.of.the.native.carboxylesterase.gene.EposCXE1,.which.encodes.a.larval.midgut.enzyme.involved.in.digestion.183

OUTPUT TRAITS

The.genetic.modification.of.input.traits.enhances.the.agricultural.properties.and.per-formance.of.a.crop,.with.the.grower.as.the.primary.beneficiary..In.contrast,.trans-genic.crops.with.altered.output.traits,.such.as.increased.shelf.life,.enhanced.nutritive.value,.or.reduced.allergenicity,.primarily.benefit.the.consumer..In.perennial.orchard.


crops,. the. modification. of. output. traits. necessarily. involves. characteristics. mani-fested.in.fruit.or.seed.tissue..Because.most.fruit.and.nut.tree.crops.have.an.extended.juvenile.period,.initial.characterization.of.modified.output.traits.in.transgenic.trees.often.cannot.occur.until.5–10.years.after.the.initial.transformation..Thus,.with.some.notable.exceptions,.most.work.outlining.the.genetic.modification.of.output.traits.in.fruit.and.nut.tree.crops.is.preliminary,.with.many.transgenic.lines.currently.described.only.in.USDA.field.trial.listings.(Table.1.1)..Below,.we.discuss.current.progress.in.the.modification.of.ripening,.the.improvement.of.fruit.quality.and.nutritive.value,.the.removal.of.undesirable.phytochemicals,.and.antigen.production.in.transgenic.fruit.and.nut.tree.crops.

TABLE 1.1U.S. Field Trials of Transgenic Fruit and Nut Tree Crops with Enhanced Output Traits

Crop Trait Gene Institution(s)Related

Publication

Apple Reduced.postharvest.browning

Polyphenol.oxidase.(antisense)

Cornell.Univ.;.Okanagan.Specialty.Fruits,.Inc.

Murata.et.al.198,199

Delayed.ripening ACC.synthase.(antisense);.ACC.oxidase.(antisense)

Cornell.Univ.;.UC Davis;.UC Berkeley

Dandekar.et al.187

Delayed.ripening SAM.transferase Excelcis;.Agritope —

Altered.sugar.profile Sorbitol-6-phosphate.dehydrogenase.(antisense)

UC.Davis;.Cornell.Univ.;.Oregon.State.Univ.

Teo.et.al.195

Coffee Delayed.ripening ACC.oxidase.(antisense)

Univ..of.Hawaii —

Reduced.caffeine Xanthosine.methyltransferase.(antisense)

Univ..of.Hawaii Ogita.et.al.201

Grapefruit Altered.carotenoid.profile

Chalcone.synthase,.chalcone.isomerase,.rhamnosyl.transferase

Univ..of.Hawaii Costa.et.al.193

Papaya Delayed.ripening ACC.synthase.(antisense)

Univ..of.Hawaii —

Pear Delayed.ripening SAM.transferase Excelcis;.Agritope Gao.et.al.191

Persimmon Delayed.softening Polygalacturonase.inhibitor.protein

UC.Davis —

Pineapple Delayed.ripening ACC.synthase.(antisense)

Univ..of.Hawaii Botella.et.al.188

Increased.sweetness Mabinlin Univ..of.Hawaii —

Plum Delayed.ripening ACC.oxidase.(antisense)

United.States.Dept..of.Agriculture

—


delayed fruIt rIpenInG

Many. tree. fruits. are. transported. long. distances. from. their. point. of. cultivation. to.their.point.of.sale.to.the.consumer..As.demonstrated.in.several.annual.plant.systems,.genetically.manipulating.the.biosynthesis.of.the.hormone.ethylene.can.substantially.slow.fruit.ripening,.allowing.increased.flexibility.in.transport.time.and.fruit.maturity.at.harvest.184,185.In.climacteric.fruits,.ethylene.triggers.a.rapid.increase.in.respiratory.rate.and.initiates.a.cascade.of.biochemical.and.physiological.changes.associated.with.ripening.88.Beginning.with.the.precursor.S-adenosyl.methionine.(SAM),.ethylene.is.synthesized.in.two.enzymatic.steps.in.plants..First,.SAM.is.converted.to.1-amino-cyclopropane-1-carboxylic.acid. (ACC),. a. reaction. catalyzed.by. the.ACC.synthase.enzyme..Second,.the.ACC.oxidase.enzyme.converts.ACC.to.ethylene.186.The.strat-egies. applied. to. minimize. ethylene. production. in. transgenic. fruit. tree. crops,. and.thereby. slow.ethylene-associated. fruit. ripening.processes,. have.primarily. focused.on.posttranscriptional.silencing.of.the.genes.encoding.the.ACC.synthase.and.ACC.oxidase.enzymes.

Delayed.ripening.in.transgenic.fruit.trees.has.been.demonstrated.most.convinc-ingly.in.apple.and.papaya.88,187.Silencing.of.ACC.oxidase.in.papaya.resulted.in.a.40%.reduction.in.fruit.ethylene.production,.with.a.corresponding.delay.in.fruit.softening.and.the.retention.of.green.peel.color..However,.the.fruit.appears.to.have.limited.com-mercial.application,.as.the.unripe.fruit.retains.high.susceptibility.to.pathogen.infection/decay,. leading. to. a. direct. transition. from. unripe. fruit. to. rotten. fruit.88. Dandekar.et. al.. pursued. a. similar. approach. in. apple,. silencing. both. the. ACC. synthase. and.ACC..oxidase.genes.in.separate.transgenic.lines..Apples.collected.from.both.ACC.synthase-. and. ACC. oxidase-silenced. lines. displayed. >90%. reduction. in. ethylene.production..The.transgenic.fruits.were.firmer.and.had.a.longer.shelf.life.than.compa-rable.controls.(Figure.1.1),.but.their.sugar/acid.balance.(a.key.aspect.of.fruit.flavor).was.unaffected..Fruit.aroma.was.likely.altered.in.these.lines,.however,.as.total.vola-tile.ester.production.was.reduced.by.65%–70%.compared.to.controls.187

Several.additional.studies.have.described.comparable.delayed.ripening.strategies.in.pineapple,188.mango,189.and.avocado,190.though.no.phenotypic.characterization.of.the.transgenic.plants.has.been.reported..Similarly,.the.silencing.of.ACC.oxidase.in.transgenic.pear.reduced.ethylene.production.by.85%.in.in.vitro.shoots,.but.effects.on.pear.fruit.have.not.yet.been.determined.191

ImprovInG fruIt QualIty and nutrItIve value

Several.recently.published.studies.have.focused.on.the.generation.of.transgenic.fruit.and.nut.tree.crops.with.enhanced.nutritive.value.or.increased.levels.of.specific.phyto-chemicals.beneficial.to.human.health..For.example,.the.grape.stilbene.synthase.gene,.which.is.responsible.for.the.synthesis.of.the.phytoalexin.resveratrol,.has.been.intro-duced.into.apple.and.kiwifruit.143,192.Resveratrol.is.an.antifungal.compound.involved.in.pathogen-induced.plant.defense.responses,.but.it.also.has.a.variety.of.beneficial.effects.on.human.health,.including.anti-inflammatory.and.anticarcinogenic.proper-ties.143.The.accumulation.of.glycosylated.resveratrol.derivatives.was.demonstrated.in.the.leaves.of.both.transgenic.kiwifruit.and.apple.plants,.though.it.is.currently.unclear.


whether.the.fruit.from.these.plants.would.accumulate.these.compounds.at.levels.that.could.potentially.benefit.human.health.142,192.Preliminary.reports.have.also.described.attempts. to. increase. vitamin. A. content. in. grapefruit. (through. expression. of. the..carotenoid.biosynthetic.genes.phytoene.synthase,..phytoene.desaturase,.and.lycopene.β-cyclase).and.attempts.to.increase.the.content.of.“heart.healthy”.oleic.acid.in.palm.oil.(through.expression.of.the.fatty.acid.modifying.genes.β-ketoacyl-ACP.synthase II,.Δ9-stearoyl-ACP-desaturase,. and. palmitoyl-ACP-thioesterase. .[antisense]).193,194.However,.no.phenotypic.characterization.of.the.transgenic.grapefruit.and.oil.palm.plants.has.yet.been.reported.

A.unique.balance.of.soluble.sugars,.starch,.acids,.and.volatiles.underlies.the.flavor.and.quality.of.each.type.of.fruit..The.recent.work.of.Teo.et.al..has.demonstrated.that.fruit.quality/flavor.can.be.dramatically.altered.using.transgenic.approaches.195.Unlike.most. plant. species,. apples. accumulate. and. transport. photosynthate. largely. in. the.form.of.the.sugar.alcohol.sorbitol,.rather.than.sucrose..Sorbitol.is.synthesized.from.glucose-6-phosphate.through.the.action.of. the.enzyme.sorbitol-6-phosphate.dehy-drogenase.(S6PDH),.and.Teo.et.al..generated.transgenic.apple.plants.expressing.an.antisense.copy.of.the.S6PDH.gene.195.Leaves.from.several.resultant.transgenic.lines.

(a) (b)

(c)

(d)

(e)

FIGURE 1.1 (See color insert.). Delayed. fruit. ripening. in. transgenic. apples.. (a). An.apple.tree.expressing.an.antisense.ACC.oxidase.(ACO).transgene..Ethylene.production.is.reduced.>90%.in.this.transgenic.line.compared.to.wild-type.controls..(b, c).ACO-silenced.(b) and.wild-type.(c).apples.stored.at.room.temperature.for.1.month..(d, e).ACO-silenced.(d). and. wild-type. (e). apples. stored. at. room. temperature. for. 3. months.. (Reprinted. with.kind.permission.from.Springer.Science+Business.Media:.Transgenic Res.,.Effect.of.down-regulation.of.ethylene.biosynthesis.on.fruit.flavor.complex.in.apple.fruit,.13,.2004,.373,.Dandekar,.A..M..et.al.)


displayed.80%–90%.reductions. in.S6PDH.enzyme.activity.and.70%–80%.reduc-tions.in.sorbitol. levels..Dramatic.changes.in.fruit.composition.were.also.observed.in.the.transgenic.lines,.including.significant.increases.in.glucose.levels,.flesh.firm-ness,.and.soluble.solids.content,.and.corresponding.significant.decreases.in.fructose.levels,.sorbitol.levels,.starch.levels,.malic.acid.levels,.and.titratable.acidity..Overall,.these.results.demonstrate.that.fruit.quality.characteristics.(and.likely.flavor).are.real-istic.targets.for.manipulation/improvement.via.biotechnology.

removal of undeSIrable phytochemIcalS

Postharvest.enzymatic.browning,.caused.primarily.by.the.enzyme.polyphenol.oxi-dase,. is.a. substantial.quality.problem. in.a.variety.of. fruit. crops,. including.apple,.pear,. grape,. and.pineapple.. Polyphenol.oxidase. comes. in. contact.with. its. pheno-lic.substrates.during.fruit.ripening.or.tissue.damage,.generating.reactive.quinones.that.crosslink.to.form.dark-colored.phytomelanins.196.Following.the.lead.of.studies.performed. in. annual. crops,197. several. groups. have. attempted. to. silence. the. poly-phenol.oxidase.gene. in.order. to. reduce.postharvest.browning. in. tree. fruits..Most.notable. is.work.by.Murata.et.al.,. in.which.expression.of.an.antisense.polyphenol.oxidase.gene.in.transgenic.apple.lines.reduced.polyphenol.oxidase.activity.and.tis-sue.browning.by ~50%.compared.to.wild-type.controls.198,199.However,.only.callus.and.shoot.tissues.were.examined;.it.remains.to.be.seen.whether.reductions.in.post-harvest.browning.will.also.be.observed.in.apple.fruit.198,199.Similar.approaches.have.been. pursued. to. reduce. blackheart. in. pineapple200. and. grape. berry. darkening,196.though.no.characterization.of.the.transgenic.polyphenol.oxidase-silenced.plants.has.yet.been.reported.

Posttranscriptional. gene. silencing. has. also. been. utilized. to. reduce. caffeine.content. in.coffee.plants..Caffeine. is.generated.from.xanthosine.by. two.sequential.methylation.reactions.catalyzed.by.the.enzymes.theobromine.synthase.and.caffeine.synthase..Ogita.et.al.. transformed.coffee.plants.with.an.RNAi.vector.designed. to.initiate.posttranscriptional.gene.silencing.of.the.theobromine.synthase.gene.201.The.resultant. transgenic. lines. were. morphologically. normal,. but. displayed. 50%–70%.less.caffeine.in.leaf.tissues.than.controls..Effects.on.caffeine.content.in.coffee.fruit.were.not.described..Although.not.yet.reported.in.the.literature,.a.similar.gene.silenc-ing.strategy.could.be.used.to.reduce.the.levels.of.allergenic.proteins.in.tree.nuts.(e.g.,.walnuts.and.chestnuts)..This.approach.was.recently.applied.to.reduce.the.levels.of.the.immunodominant.Ara.h.2.protein.in.transgenic.peanut.plants.202

antIGen productIon

Several. studies. have. shown. that. transgenic. plants. that. produce. viral. or. bacterial.antigen.proteins.can.induce.a.protective.immune.response.(as.measured.by.the.pro-duction. of. antigen-specific. serum. antibodies). when. fed. to. mice. and. humans.203,204.These.plants,.which.act.as.“edible.vaccines,”.have.several.significant.advantages.over.traditionally.purified.and.administered.vaccines,.including.ease.of.human.delivery,.increased.stability,.and.decreased.production.and.transport.costs.205.While.most.stud-ies. on. edible. vaccines. have. focused. on. annual. crops,. recently. the. “s”. gene. of. the.


Hepatitis.B.surface.antigen.(HBsAg).was.expressed.in.banana.plants.206.Hepatitis.B.is.the.major.cause.of.persistent.viremia.in.humans,.and.banana.is.an.ideal.host.for.expression.of.HBsAg.due.to.its.palatability.to.infants.and.year-round.availability.in.the.tropics..By.expressing.an.HBsAg.“s”.gene-ER.retention.sequence.fusion.under.the.control.of.the.banana.ethylene.forming.enzyme.promoter,.Sunil-Kumar.et.al..success-fully.produced.antibody-reactive.antigen.in.banana.leaves.and.fruits.206.Unfortunately,.antigen.levels.in.banana.fruit.were.very.low.compared.to.antigen.levels.achieved.in.several.previously.described.annual.plant.systems.(1.ng/g.vs..16.μg/g.fresh.weight.in.potato.tuber),.precluding.immunogenicity.testing.and.the.use.of.these.bananas.for.oral.vaccination.

CONCLUSIONS

The.high.value.of.fruit.and.nut.crops.and.their.increasing.dietary.importance.due.to.their.unique.nutritional.and.disease-preventing.attributes.has. focused.attention.on.genetic.strategies.to.solve.production.limitations..The.technology.to.produce.trans-genic.trees.is.relatively.mature,.especially.the.use.of.the.Agrobacterium-mediated.transformation.process..However,.regeneration.of.transgenic.plants.continues.to.pose.the.greatest.challenge.for.many.of.these.crops,.especially.Prunus..In.most.fruit.and.nut.tree.species,.only.a.few.of.the.commercially.significant.phenotypes.have.been.exten-sively.tested.in.the.field..Viral.resistance.in.plum.and.papaya.has.been.deregulated,.and.commercialized.in.the.case.of.papaya..Many.tree.crops.are.promising.candidates.for.the.use.of.transgenic.technologies.to.prevent.disease.and.pest.infestation,.espe-cially.through.the.use.of.Bt.insecticidal.proteins.that.target.lepidopteran.insect.pests..Resistance.to.bacterial.pathogens.poses.the.greatest.challenge,.as. they.present. the.greatest.threat.to.commercial.viability.in.many.perennial.crop.species..Substantial.progress.has.been.made. toward.gaining.resistance. to.PD. in.grapevine.and.crown.gall.disease.in.walnut..Many.fruit.quality.traits.have.also.been.manipulated,.primar-ily.through.the.RNAi-mediated.regulation.of.ethylene. .biosynthesis.and/or.sorbitol.biosynthesis,.where.the.phenotypes.have.been.documented.in.field-grown.transgenic.fruit..Many.interesting.transgenic.approaches.have.been.developed.for.other.quality.traits.that.are.of.importance.to.consumers,.including.postharvest.storage.disorders,.fruit. texture,. appearance,. sugar. and. lipid. composition,. and. allergenicity;. how-ever,.these.phenotypes.remain.to.be.established.in.the.field..With.the.exception.of.papaya,.no.other.transgenic.fruit.variety.has.been.commercialized.and.the.technol-ogy.development.toward.commercialization.for.scion.varieties.in.particular.has.been.“soft-pedaled”.due.to.the.public.acceptance.issue..Perhaps.the.greatest. immediate.promise.lies.in.the.potential.for.commercialization.of.transgenic.rootstocks,.as.they.can.provide.resistance.to.disease,.pest,.and.environmental.issues.while.sustaining.the.production.of.a.wild-type.fruit.crop.from.the.grafted.(non-transgenic).scion.

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.185.. Ayub,.R..et.al.,.Expression.of.ACC.oxidase.antisense.gene.inhibits.ripening.of.canta-loupe.melon.fruits,.Nat. Biotechnol.,.14,.862,.1996.

.186.. Adams,.D..O..and.Yang,.S..F.,.Ethylene.biosynthesis—Identification.of.1-aminocyclopropane-1-carboxylic.acid.as.an.intermediate.in.the.conversion.of.methionine.to.ethylene,.Proc. Natl. Acad. Sci.,.76,.170,.1979.

.187.. Dandekar,.A..M..et.al.,.Effect.of.down-regulation.of.ethylene.biosynthesis.on.fruit.flavor.complex.in.apple.fruit,.Transgenic Res.,.13,.373,.2004.

.188.. Botella,.J..R.,.Cavallaro,.A..S.,.and.Cazzonelli,.C..I.,.Towards.the.production.of.trans-genic.pineapple.to.control.flowering.and.ripening,.Acta Hort.,.529,.115,.2000.

.189.. Cruz-Hernandez,.A.,. Gomez-Lim,. M..A.,. and. Litz,. R.. E.,. Transformation. of. mango.somatic.embryos,.Acta Hort.,.455,.292,.1997.

.190.. Litz,.R..E..et.al.,.Plant.recovery.following.transformation.of.avocado.with.anti-fungal.protein.and.SAM.hydrolase.genes,.Acta Hort.,.738,.447,.2007.

.191.. Gao,. M.. et. al.,. Gene. expression. and. ethylene. production. in. transgenic. pear. (Pyrus communis.cv..‘La.France’).with.sense.or.antisense.cDNA.encoding.ACC.oxidase,.Plant Sci.,.173,.32,.2007.

.192.. Kobayashi,. S.. et. al.,. Kiwifruits. (Actinidia deliciosa). transformed. with. a. Vitis.stilbene. synthase. gene. produce. piceid. (resveratrol-glucoside),. Plant Cell Rep.,.19, 904,.2000.

.193.. Costa,.M..G..C.,.Otoni,.W..C.,.and.Moore,.G..A.,.An.evaluation.of.factors.affecting.the.efficiency. of. Agrobacterium-mediated. transformation. of. Citrus paradisi. (Macf.). and.production. of. transgenic. plants. containing. carotenoid. biosynthetic. genes,. Plant Cell Rep.,.21,.365,.2002.

.194.. Yunus,.A..M..M..and.Kadir,.A..P..G.,.Development.of.transformation.vectors.for.the.production.of.potentially.high.oleate.transgenic.oil.palm,.Electron. J. Biotechnol.,.11,.1,.2008.

.195.. Teo,.G..et.al.,.Silencing.leaf.sorbitol.synthesis.alters.long-distance.partitioning.and.apple.fruit.quality,.Proc. Natl. Acad. Sci.,.103,.18842,.2006.

.196.. Thipyapong,.P.,.Stout,.M..J.,.and.Attajarusit,.J.,.Functional.analysis.of.polyphenol.oxi-dases.by.antisense/sense.technology,.Molecules,.12,.1569,.2007.

.197.. Rommens,. C.. M.. et. al.,. Crop. improvement. through. modification. of. the. plant’s. own.genome,.Plant Physiol.,.135,.421,.2004.

.198.. Murata,.M..et.al.,.A.transgenic.apple.callus.showing.reduced.polyphenol.oxidase.activ-ity.and.lower.browning.potential,.Biosci. Biotechnol. Biochem.,.65,.383,.2001.

.199.. Murata,.M..et.al.,.Transgenic.apple.(Malus.x.domestica).shoot.showing.low.browning.potential,.J. Agric. Food Chem.,.48,.5243,.2000.

.200.. Ko,. H.. L.. et. al.,. The. introduction. of. transgenes. to. control. blackheart. in. pineapple.(Ananas comosus.L.).cv..Smooth.Cayenne.by.microprojectile.bombardment,.Euphytica,.150,.387,.2006.

.201.. Ogita,.S..et.al.,.Producing.decaffeinated.coffee.plants,.Nature,.423,.823,.2003.


.202.. Dodo,.H..W..et.al.,.Alleviating.peanut.allergy.using.genetic.engineering:.The. silenc-ing.of. the. immunodominant.allergen.Ara.H.2. leads. to. its. significant. reduction.and.a.decrease.in.peanut.allergenicity,.Plant Biotechnol. J.,.6,.135,.2008.

.203.. Thanavala,.Y.. et. al.,. Immunogenicity. of. transgenic. plant-derived. hepatitis-b. surface-antigen,.Proc. Natl. Acad. Sci.,.92,.3358,.1995.

.204.. Kong,.Q..X.. et. al.,.Oral. immunization. with.hepatitis.B. surface. antigen. expressed. in.transgenic.plants,.Proc. Natl. Acad. Sci.,.98,.11539,.2001.

.205.. Mor,. T.. S.,. Gomez-Lim,. M.. A.,. and. Palmer,. K.. E.,. Perspective:. Edible. vaccines—A.concept.coming.of.age,.Trends Microbiol.,.6,.449,.1998.

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31

2 Transgenic Vegetables

Owen Wally, J. Jayaraj, and Zamir K. Punja

INTRODUCTION

There. are. a. range. of. vegetable. crop. species. that. are. grown. worldwide. to. provide.humans.with.a.source.of.nutrients,.vitamins,.and.fiber..These.vegetables.are.usually.consumed.fresh,.e.g.,.carrot,.cucumber,.lettuce,.and.tomato,.while.others.may.be.eaten.after.cooking,.e.g.,.broccoli,.cabbage,.peas,.and.squash..Vegetable.crops.are.usually.grown.under.field.conditions,.and.certain.high-value.crops.can.be.grown.in.the.green-house,.e.g.,.tomato.and.cucumber..These.crops.have.been.bred.to.produce.cultivars.with.high.yield,.optimal.flavor.and.appearance,.and.disease.and.pest.resistance.

The.tools.of.biotechnology.have.been.applied.to.vegetable.crops.to.achieve.for-eign.gene. insertion.and.expression.of.additional. and,. in. some.cases,. novel. traits..The. work. conducted. during. the. period. 1995–2002. on. 23. vegetable. crop. species.belonging.to.9.botanical.families.has.been.summarized.by.Punja.and.Feeney.1.In.this.chapter,.recent.work.over.the.period.2002–2008.on.genetic.transformation.of.vegetable.crops.for.the.purpose.of.introducing.potentially.useful.traits.is.discussed..The.traits.that.have.been.engineered.are.diverse.and.include.insect.and.nematode.resistance,.disease.resistance.(to.viruses,.fungi,.and.bacteria),. tolerance. to.abiotic.

CONTENTS

Introduction............................................................................................................... 31Insect.Resistance....................................................................................................... 39Nematode.Resistance................................................................................................40Pathogen.Resistance..................................................................................................40

Virus.Resistance...................................................................................................40Fungal.and.Bacterial.Resistance.......................................................................... 41

Abiotic.Stress.Tolerance........................................................................................... 42Medical.Applications................................................................................................ 43

Edible.Vaccines.................................................................................................... 43Therapeutic.Products............................................................................................44

Nutritional.Modifications..........................................................................................44Enhanced.Nutritional.Quality..............................................................................44Enhanced.Taste..................................................................................................... 45Enhanced.Antioxidant.Activities..........................................................................46Fruit.Ripening...................................................................................................... 47

Discussion................................................................................................................. 47References.................................................................................................................48


TAB

LE 2

.1V

eget

able

Cro

p Sp

ecie

s Th

at H

ave

Bee

n En

gine

ered

to

Expr

ess

Spec

ific

Tran

sgen

es, a

nd t

heir

Res

ulti

ng E

ffec

ts

Cro

pTr

aits

Gen

e Ex

pres

sed

Res

ult

Ref

eren

ces

Car

rot

Path

ogen

.re

sist

ance

Whe

at.c

hitin

ase/

gluc

anas

e,.r

ice.

pero

xida

sePe

roxi

dase

.exp

ress

ion.

enha

nced

.res

ista

nce.

to.f

olia

r.fu

ngal

.pa

thog

ens

[31]

Tric

hode

rma.

chiti

nase

Enh

ance

d.to

lera

nce.

to.f

unga

l.pat

hoge

ns[3

0]

Pse

udom

onas

fluo

resc

ens .

mic

robi

al.f

acto

r.3

Enh

ance

d.to

lera

nce.

to.f

olia

r.fu

ngal

.pat

hoge

ns[1

04]

Bar

ley.

chiti

nase

,.whe

at.li

pid.

tran

sfer

.pro

tein

Low

er.f

olia

r.di

seas

e.du

e.to

.fun

gal.p

atho

gens

[33]

Ric

e.th

aum

atin

-lik

e.pr

otei

nL

ower

.fol

iar.

dise

ase.

sym

ptom

s.to

.a.r

ange

.of.

fung

al.p

atho

gens

[34]

Abi

otic

.str

ess.

resi

stan

ceC

arro

t.bet

aine

.ald

ehyd

e.de

hydr

ogen

ase,

.chl

orop

last

.ex

pres

sion

Incr

ease

d.re

sist

ance

.to.s

alin

ity.u

p.to

.400

.mM

.NaC

l[5

1]

Met

abol

ic.

engi

neer

ing

Hae

mat

ococ

cus

pluv

iali

s.β-

caro

tene

.ket

olas

eE

nhan

ced.

prod

uctio

n.of

.sev

eral

.ket

o-ca

rote

noid

s[9

8]

Med

ical

.ap

plic

atio

nsM

easl

es.p

olyp

eptid

es.in

.sus

pens

ion.

cultu

reFu

nctio

nal.m

easl

es.v

acci

ne.p

rodu

ctio

n[6

9,70

]

[74]

Shal

lot

Inse

ct.

resi

stan

ceB

acil

lus

thur

ingi

ensi

s.(B

T).

Cry

1Ca.

or.H

04.to

xin.

gene

sC

ompl

ete.

resi

stan

ce.to

war

d.ar

myw

orm

.(S.

exi

gua)

[8]

Cab

bage

,.C

hine

se.

cabb

age.

and.

colla

rds

Path

ogen

.re

sist

ance

Ant

i-se

nse.

Tur

nip.

Mos

aic.

Vir

us.N

ib.g

ene

Hig

h.le

vels

.of.

resi

stan

ce.to

.Tur

nip.

Mos

aic.

Vir

us[2

4]

Asp

ergi

llus

nig

er.g

luco

se.o

xida

seD

ecre

ased

.lesi

ons.

by.X

anth

omon

as c

ampe

stri

s[1

05]

Met

abol

ic.

engi

neer

ing

Thr

ee.A

rabi

dops

is.g

enes

.(M

AM

1,.C

YP7

9F1,

.C

YP8

3A1)

.invo

lved

.in.g

ener

atio

n.of

.alip

hatic

.gl

ucos

inol

ates

Alte

red.

gluc

osin

olat

e.co

mpo

sitio

n.to

war

ds.p

harm

acol

ogic

al.

impo

rtan

t.met

hyls

ulph

inyl

alky

l.glu

cosi

nola

tes

[101

]

33Transgenic Vegetables

Thr

ee.A

rabi

dops

is.g

enes

.(C

YP7

9B3,

.CY

P79B

2,.

CY

P83B

1).in

volv

ed.in

.indo

le.g

luco

sino

late

.pr

oduc

tion

Alte

red.

gluc

osin

olat

e.ac

cum

ulat

ion,

.dri

ving

.acc

umul

atio

n.of

.in

dole

.glu

cosi

nola

tes

[102

]

Inse

ct.

resi

stan

ceB

T.C

ry1a

b.pr

otei

n,.c

hlor

opla

st.e

xpre

ssio

nH

igh.

leve

ls.o

f.re

sist

ance

.to.P

lute

lla

xylo

stel

la[9

]

BT

.Cry

1AC

.or.

Cry

1CC

olla

rds.

expr

essi

ng.e

ither

.gen

e.w

ere.

high

ly.r

esis

tant

.to.P

. xy

lost

ella

[10]

Abi

otic

.str

ess

E. c

oli.s

uper

oxid

e.di

smut

ase.

(SO

D).

and.

cata

lase

,.bot

h.co

nstit

utiv

e.an

d.ch

loro

plas

ticC

o-ex

pres

sion

.of.

both

.gen

es.e

nhan

ced.

tole

ranc

e.to

.SO

2.an

d.ot

her.

reac

tive.

oxyg

en.s

peci

es.(

RO

S)[5

9,58

]

Bra

ssic

a na

pus,

.LE

A.p

rote

inE

nhan

ced.

resi

stan

ce.to

.sal

t.and

.dro

ught

.str

esse

s[4

5]

E. c

oli .t

reha

lose

-6-p

hosp

hate

.syn

thas

e.ge

ne.o

r.ho

t.pe

pper

.LE

A.p

rote

in.g

ene

Res

ista

nce.

to.lo

w.le

vels

.of.

NaC

l.(20

0.m

M).

and.

mod

erat

e.le

vels

.of.

heat

.str

ess.

(45°

C)

[47]

Bro

ccol

i/ca

ulifl

ower

Nut

ritio

nal.

mod

ifica

tion

Bro

ccol

i.chl

orop

hylla

se,.a

ntis

ense

.exp

ress

ion

Slow

ing.

of.c

hlor

ophy

ll.b

reak

dow

n.an

d.1–

2.da

y.de

lay.

in.

post

harv

est.y

ello

win

g[1

06]

Bro

ccol

i.sol

uble

.aci

d.in

vert

ase,

.ant

isen

se.e

xpre

ssio

nH

ighe

r.le

vels

.of.

suga

rs.a

nd.p

rote

in.p

osth

arve

st.a

nd.s

low

ed.

flore

t.yel

low

ing

[107

]

Bro

ccol

i.AC

C.s

ynth

ase.

1.an

d.A

CC

.oxi

dase

,.ant

isen

se.

expr

essi

onD

ecre

ased

.leve

l.of.

post

harv

est.e

thyl

ene.

and.

dela

yed.

chlo

roph

yll.l

oss.

by.2

.day

s[1

08,1

09]

Inse

ct.

resi

stan

ceB

T.C

ry1a

b,.in

duci

ble.

expr

essi

onPr

otec

tion.

to.P

. xyl

oste

lla.

for.

up.to

.8.w

eeks

.aft

er.in

duct

ion

[110

]

Synt

hetic

.BT

.Cry

1Ab

Enh

ance

d.re

sist

ance

.to.P

. xyl

oste

lla

[12]

BT

.Cry

1AC

.and

.Cry

1C.o

r.C

ry1A

.and

.Cry

1CR

educ

ed.s

prea

d.of

.P. x

ylos

tell

a[1

3,11

1]

BT

.Cry

1Ab

Inhi

bite

d.gr

owth

.of.

P. x

ylos

tell

a[1

4]

Indi

an.

mus

tard

Inse

ct.

resi

stan

ceB

T.C

ry1A

c,.C

ry1C

.and

.co-

expr

esse

d.C

ry1A

c.an

d.C

ry1C

Res

ista

nce.

to.P

. xyl

oste

lla.

and.

BT-

resi

stan

t.mot

h.va

riet

ies

[15]

Nut

ritio

nal.

mod

ifica

tion

Ara

bido

psis

.γ-to

coph

erol

.met

hyltr

ansf

eras

eSh

ift.f

rom

.γ-t

ocop

hero

l.poo

ls.to

.the.

mor

e.ac

tive.

vita

min

.E.

(α-t

ocop

hero

l)

[112

]

(con

tinu

ed)


TAB

LE 2

.1 (

cont

inue

d)V

eget

able

Cro

p Sp

ecie

s Th

at H

ave

Bee

n En

gine

ered

to

Expr

ess

Spec

ific

Tran

sgen

es, a

nd t

heir

Res

ulti

ng E

ffec

ts

Cro

pTr

aits

Gen

e Ex

pres

sed

Res

ult

Ref

eren

ces

Pea/

bean

Dis

ease

.re

sist

ance

Bea

n.go

lden

.mos

aic.

viru

s.(B

GM

V).

AC

1.ge

ne,.R

NA

i.ex

pres

sion

Hig

h.re

sist

ance

.tow

ards

.BG

MV

[26]

Mut

ated

.rep

.gen

e.of

.BG

MV

Incr

ease

d.nu

mbe

r.of

.sym

ptom

less

.pla

nts.

com

pare

d.to

.no

ntra

nsfo

rmed

[25]

Abi

otic

.str

ess

B. n

apus

.LE

AIm

prov

ed.r

esis

tanc

e.to

.bot

h.N

aCl.a

nd.d

roug

ht.s

tres

ses

[46]

Nut

ritio

nal.

mod

ifica

tion

Mod

ified

.fee

dbac

k.in

sens

itive

.ric

e.an

thra

nila

te.

synt

hase

Hig

her.

leve

ls.o

f.fr

ee.tr

ypto

phan

.acc

umul

atio

n.an

d.to

tal.

tryp

toph

an.le

vels

[113

]

Inse

ct.

resi

stan

ceB

ean.

alph

a.am

ylas

e.in

hibi

tor.

1D

elay

ed.la

rval

.dev

elop

men

t.of.

the.

pea.

wee

vil

[6]

Com

mon

.bea

n.α-

amyl

ase.

inhi

bito

r.2

Incr

ease

d.re

sist

ance

.to.M

exic

an.b

ean.

wee

vil

[114

]

Cuc

umbe

rD

isea

se.

resi

stan

ceR

ice.

clas

s.I.

chiti

nase

Hig

h.re

sist

ance

.tow

ards

.B. c

iner

ea[2

9]

Abi

otic

.str

ess

Wild

.pot

ato.

dehy

drin

sE

nhan

ced.

resi

stan

ce.to

.chi

lling

.str

ess.

and.

incr

ease

d.fr

eezi

ng.

tole

ranc

e[1

15,1

16]

Nut

ritio

nal.

mod

ifica

tion

Cas

sava

.sup

erox

ide.

dism

utas

e,.f

ruit.

expr

essi

onH

ighe

r.ac

cum

ulat

ion.

of.S

OD

[117

]

Tha

umat

ococ

cus

dani

elli

i .tha

umat

in.I

ISw

eet.t

astin

g.ph

enot

ype,

.with

.min

or.r

esis

tanc

e.to

.P

seud

oper

onos

pora

cub

ensi

s[1

18]

Let

tuce

/sp

inac

hD

isea

se.

resi

stan

ceC

oat.p

rote

in.(

CP)

.gen

e.of

.lettu

ce.b

ig-v

ein.

asso

ciat

ed.

viru

s.(L

BV

aV).

in.s

ense

.or.

antis

ense

.ori

enta

tion

Sens

e.C

P.pr

ovid

ed.r

esis

tanc

e.to

.LB

VaV

,.whi

le.a

nti-

sens

e.C

P.ex

pres

sion

.gav

e.re

sist

ance

.to.L

BV

aV.a

nd.m

irafi

ori.l

ettu

ce.

viru

s

[20]

Fla

mm

ulin

a .sp

..oxa

late

.dec

arbo

xyla

se.g

ene

Red

uced

.Scl

erot

inia

scl

erot

ioru

m.d

isea

se.s

ympt

oms

[37]

Abi

otic

.str

ess

Ara

bido

psis

.AB

F3,.A

BA

.res

pons

ive.

gene

.tran

scri

ptio

n.fa

ctor

Hig

her.

tole

ranc

e.to

.bot

h.dr

ough

t.and

.col

d.st

ress

es[1

19]

B. n

apus

.LE

AIn

crea

sed.

tole

ranc

e.to

.sal

t.and

.dro

ught

.str

ess

[48]


Nut

ritio

nal.

mod

ifica

tion

E. c

oli.a

spar

agin

e.sy

nthe

tase

.AIn

crea

sed.

aspa

ragi

ne,.a

spar

tate

.and

.glu

tam

ine.

in.a

dditi

on.to

.en

hanc

ed.v

eget

ativ

e.gr

owth

[120

]

Ara

bido

psis

.R2R

3-M

YB

.tran

scri

ptio

n.fa

ctor

Inhi

bite

d.pr

oduc

tion.

of.a

ntho

cyan

in.p

igm

ents

.and

.less

.red

.co

lora

tion

[121

]

E. c

oli .a

spar

agin

e.sy

nthe

tase

.AD

ecre

ased

.suc

rose

,.glu

cose

.and

.fru

ctos

e.an

d.in

crea

sed.

inul

in.

leve

ls[9

2]

Ara

bido

psis

.hom

ogen

tisat

e.ph

ytyl

tran

sfer

ase.

(hpt

).or

.to

coph

erol

.cyc

lase

.(T

C)

Ele

vate

d.vi

tam

in.E

.leve

ls[9

4]

Part

heno

ciss

us h

enry

ana.

stilb

ene.

synt

hase

Incr

ease

d.pr

oduc

tion.

of.r

esve

ratr

ol[1

22]

Tast

e.m

odif

ying

.mir

acul

in.f

rom

.Ric

hade

lla

dulc

ifica

Enh

ance

d.sw

eetn

ess.

and.

tast

e[8

9]

Ara

bido

psis

.gam

ma-

toco

pher

ol.m

ethy

ltran

sfer

ase

Alte

red.

pool

.of.

γ-to

coph

erol

.to.p

rodu

ce.m

ore.

activ

e.α-

toco

pher

ols

[93]

Med

ical

.ap

plic

atio

nsC

hick

en.α

-int

erfe

ron.

(ChI

FN)

Rec

ombi

nant

.ChI

FN.w

as.c

orre

ctly

.fol

ded.

and.

activ

e.in

.le

aves

,.with

.hig

h.le

vels

.of.

antiv

iral

.act

ivity

[80]

Cho

lera

.toxi

n.B

-pro

insu

lin.f

usio

n.pr

otei

nH

igh.

leve

ls.o

f.pr

oins

ulin

,.and

.red

uced

.sym

ptom

s.of

.pa

ncre

atic

.insu

litis

.in.m

ice

[83]

Synt

hetic

.E. c

oli.e

nter

otox

in.B

.(sL

TB

),.f

or.a

djuv

ant.

for.

co-a

dmin

iste

red.

antig

ens

Bio

logi

cally

.act

ive.

sLT

B.w

ere.

form

ed.in

.the.

leav

es[8

2]

Synt

hetic

.cho

lera

.toxi

n.B

.sub

unit.

(sC

TB

)H

igh.

leve

ls.o

f.sC

TB

,.pot

entia

lly.f

or.a

n.ed

ible

.vac

cine

[71]

HIV

-1.T

at.p

rote

inSp

inac

h.w

as.o

rally

.con

sum

ed.b

y.m

ice.

and.

wer

e.pr

imed

.upo

n.fu

rthe

r.di

rect

.DN

A.v

acci

natio

n.fo

r.hi

gh.ti

tre.

prod

uctio

n.of

.Ta

t.ant

ibod

ies

[72]

Rab

ies.

viru

s.gl

ycop

rote

inSp

inac

h.w

as.o

rally

.con

sum

ed.a

nd.d

eliv

ered

.Rab

ies.

viru

s.pr

otec

tion.

to.m

ice.

and.

hum

an.v

olun

teer

s[7

3]

Mea

sles

.hem

aggl

utin

in.p

rote

inL

ettu

ce.o

rally

.con

sum

ed.b

y.m

ice.

resu

lted.

in.h

igh.

leve

l.of.

mea

sle.

antib

ody.

prod

uctio

n[7

5]

Swee

t.po

tato

Nut

ritio

nal.

mod

ifica

tions

RN

Ai.o

f.gr

anul

e-bo

und.

star

ch.s

ynth

ase.

IR

emov

ed.o

ver.

90%

.of.

the.

amyl

ase.

in.s

tarc

h[8

7]

(con

tinu

ed)


TAB

LE 2

.1 (

cont

inue

d)V

eget

able

Cro

p Sp

ecie

s Th

at H

ave

Bee

n En

gine

ered

to

Expr

ess

Spec

ific

Tran

sgen

es, a

nd t

heir

Res

ulti

ng E

ffec

ts

Cro

pTr

aits

Gen

e Ex

pres

sed

Res

ult

Ref

eren

ces

Med

ical

.ap

plic

atio

nsH

uman

.lact

ofer

rin

Hig

h.le

vels

.of.

viab

le.p

oten

tially

.ther

apeu

tic.p

rote

in[1

23]

Tom

ato

Dis

ease

.re

sist

ance

Whe

at.o

xala

te.o

xida

seR

educ

tion.

in.f

olia

r.le

sion

s.by

.B. c

iner

ea.a

nd.S

. scl

erot

ioru

m[3

6]

Swee

t.pep

per.

ferr

edox

in.I

.(PF

LP)

Res

ista

nce.

to.R

. sol

anac

earu

m.a

nd.E

. car

otov

ora

[39]

Tom

ato.

β-1,

3.gl

ucan

ase.

(GL

U),

.alf

alfa

.def

ensi

n.ge

ne.

(AFP

).an

d.bi

vale

nt.G

LU

-AFP

Hig

h.le

vels

.of.

the.

GL

U-A

FP.r

esul

ted.

in.e

nhan

ced.

resi

stan

ce.

to.R

alst

onia

sol

anac

earu

m[3

8]

Pepp

er.b

asic

.PR

-1.a

nd.a

scor

bate

.per

oxid

ase-

like.

1E

nhan

ced.

tole

ranc

e.to

.Phy

toph

thor

a ca

psic

i[3

5]

Toba

cco.

osm

otin

.and

.bea

n.ch

itina

seR

esis

tanc

e.to

.Fus

ariu

m o

xysp

orum

f. s

p. L

ycop

ersi

ci[3

2]

Tom

ato.

leaf

.cur

l.vir

us.(

TL

CV

).re

plic

ase.

1,an

ti-se

nse.

expr

essi

onH

igh.

leve

l.of.

resi

stan

ce.to

.TL

CV

[22]

RN

Ai.f

or.T

LC

V.r

ep1.

and.

rep4

Res

ista

nce.

to.T

LC

V.w

as.o

bser

ved,

.the.

addi

tion.

of.th

e.se

cond

.R

NA

i.pre

vent

ed.e

volu

tion.

of.r

esis

tanc

e[2

3]

Mir

abil

is ja

lapa

.ant

imic

robi

al.p

eptid

e.an

d.m

aize

.gl

ucan

ase

Enh

ance

d.re

sist

ance

.to.A

lter

nari

a so

lani

[124

]

Tom

ato.

spot

ted.

wilt

.vir

us.(

TSW

V).

nucl

eopr

otei

nC

ompl

ete.

resi

stan

ce.in

.the.

field

.with

.no.

outc

ross

ing

[27,

28]

TL

CV

.coa

t.pro

tein

.(C

P)Sy

mpt

omle

ss.w

hen.

inoc

ulat

ed.w

ith.T

LC

V.a

fter

.15.

days

[19]

Ara

bido

psis

.thio

nin.

(thi

2.1)

,.fru

it.in

activ

e.ex

pres

sion

Lea

ves.

and.

root

s.w

ere.

sign

ifica

ntly

.mor

e.re

sist

ant.t

o.F

usar

ium

.and

.bac

teri

al.w

ilt[1

25]

Abi

otic

.str

ess

Tom

ato.

hydr

oxyc

inna

moy

l.CoA

.qui

nate

.tran

sfer

ase

Incr

ease

d.ph

enol

ic.le

vels

.and

.incr

ease

d.to

lera

nce.

tow

ard.

UV

.ra

diat

ion

[60]

Tom

ato.

omeg

a-3.

fatty

.aci

d.de

satu

rase

Res

ista

nce.

to.c

hilli

ng.s

tres

s.at

.low

.irra

dian

ce[6

1]

Chi

li.pe

pper

.ank

yrin

.rep

eat.d

omai

n.zi

nc.fi

nger

Dec

reas

ed.a

ccum

ulat

ion.

of.f

ree.

oxyg

en.r

adic

als.

and.

enha

nced

.re

sist

ance

.to.s

alin

ity.a

nd.o

xida

tive.

stre

ss[6

2]


Tom

ato.

glyc

erol

-3-p

hosp

hate

.acy

ltran

sfer

ase.

gene

Incr

ease

d.le

vels

.of.

cis-

unsa

tura

ted.

fatty

.aci

ds.in

.the.

thyl

akoi

d.m

embr

ane,

.incr

easi

ng.to

lera

nce.

tow

ard.

chill

ing

[63]

Popl

ar.b

oilin

g.st

able

.pro

tein

.A.(

bspA

)In

crea

se.in

.wat

er.s

tres

s.to

lera

nce

[126

]

Art

hrob

acte

r gl

obif

orm

is.c

holin

e.ox

idas

eA

ccum

ulat

ion.

of.g

lyci

ne.b

etai

ne.a

nd.in

crea

se.in

.chi

lling

.to

lera

nce

[50]

E. c

oli .c

atal

ase,

.fol

iar.

expr

essi

onE

nhan

ced.

resi

stan

ce.to

.the.

herb

icid

e.pa

raqu

at.a

nd.in

crea

sed.

tole

ranc

e.to

.chi

lling

.and

.dro

ught

.str

esse

s[5

7]

Atr

iple

x ho

rten

sis.

beta

ine.

alde

hyde

.deh

ydro

gena

seE

nhan

ced.

tole

ranc

e.to

.mod

erat

e.le

vels

.of.

NaC

l[4

9]

Nut

ritio

nal.

mod

ifica

tion

Soyb

ean.

isofl

avon

e.sy

ntha

seM

ouse

.met

allo

thio

nein

s.(M

T-1)

Hig

h.le

vel.o

f.is

oflav

one.

accu

mul

atio

n.in

.the.

frui

t.ski

nE

leva

ted.

leve

ls.o

f.Z

n.an

d.SO

D[1

27]

[128

]

Ric

hade

lla

dulc

ifica

.mir

acul

inH

igh.

leve

ls.o

f.m

irac

ulin

.acc

umul

atio

n,.in

crea

sing

.tom

ato.

swee

tnes

s[9

0]

Oci

mum

bas

ilic

um.g

eran

iol.s

ynth

ase,

.rip

enin

g-sp

ecifi

c.ex

pres

sion

Alte

red.

terp

enoi

d.po

ol.a

nd.e

nhan

ced.

flavo

r[9

1]

Synt

hetic

.mam

mal

ian.

GT

P.cy

cloh

ydro

lase

,.fr

uit-

spec

ific.

expr

essi

onH

eigh

tene

d.ac

cum

ulat

ion.

of.p

teri

dine

.and

.fol

ates

[84]

[85]

Bac

teri

al.ly

cope

ne.b

eta-

cycl

ase.

(LB

c),.p

last

id.

expr

essi

onC

onve

rted

.hig

h.le

vels

.of.

lyco

pene

.to.β

-car

oten

e[9

7]

RN

Ai.o

f.en

doge

nous

.pho

tom

orph

ogen

esis

.reg

ulat

ory.

gene

,.det

iola

ted.

1.(D

ET

1),.f

ruit-

spec

ific.

expr

essi

onR

educ

tion.

in.D

ET

1.ac

tivity

,.with

.bot

h.ca

rote

noid

.and

.fla

vono

id.c

onte

nts.

incr

ease

d.si

gnifi

cant

ly[9

6]

Ara

bido

psis

.3-h

ydro

xym

ethy

lglu

tary

l.CoA

.and

.ba

cter

ial.1

-deo

xy-d

-xyl

ulos

e-5-

phos

phat

e.sy

ntha

seE

leva

ted.

tota

l.phy

tost

erol

s.an

d.ph

ytoe

ne.a

nd.β

-car

oten

e[9

5]

RN

A.s

ilenc

ing.

of.p

olyg

alac

turo

nase

.(PG

).an

d.ex

pans

in.f

ruit-

spec

ific.

expr

essi

onSi

gnifi

cant

ly.fi

rmer

.fru

it.th

roug

hout

.rip

enin

g.an

d.re

duce

d.su

scep

tibili

ty.to

.det

erio

ratio

n.in

.long

-ter

m.s

tora

ge[1

03]

[129

]

T. d

anie

llii

.thau

mat

inFr

uit.n

otic

eabl

y.sw

eete

r[8

8]

(con

tinu

ed)


TAB

LE 2

.1 (

cont

inue

d)V

eget

able

Cro

p Sp

ecie

s Th

at H

ave

Bee

n En

gine

ered

to

Expr

ess

Spec

ific

Tran

sgen

es, a

nd t

heir

Res

ulti

ng E

ffec

ts

Cro

pTr

aits

Gen

e Ex

pres

sed

Res

ult

Ref

eren

ces

Vac

cine

/m

edic

alH

uman

.inte

rleu

kin-

12.(

IL12

)R

educ

ed.b

acte

rial

.load

.whe

n.fe

d.to

.mic

e.in

fect

ed.w

ith.

myc

obac

teri

a[7

9]

Mod

ified

.hum

an.a

lpha

-1-a

ntitr

ypsi

nA

ccum

ulat

ion.

of.h

igh.

leve

ls.o

f.bi

olog

ical

ly.a

ctiv

e,.th

erap

eutic

.pr

otei

n[1

30]

Hum

an.b

eta-

amyl

oid

Acc

umul

atio

n.of

.hig

h.le

vels

.of.

the.

prot

ein,

.whe

n.fe

d.to

.mic

e.ex

hibi

ted.

an.im

mun

e.re

spon

se[1

31]

HIV

-1.T

at.p

rote

in,.f

ruit-

spec

ific.

expr

essi

onH

igh.

leve

ls.o

f.pr

otei

n.ac

cum

ulat

ion,

.that

.pro

duce

d.an

.im

mun

e.re

spon

se.in

.mic

e.w

hen.

cons

umed

[65]

Hep

atiti

s.B

.vir

us.la

rge.

surf

ace.

antig

en,.f

ruit-

spec

ific.

expr

essi

onH

igh.

leve

ls.o

f.pr

otei

n.ac

cum

ulat

ion.

in.m

atur

e.fr

uits

[66]

Hum

an.c

oagu

latio

n.Fa

ctor

.IX

,.fru

it-sp

ecifi

c.ex

pres

sion

Hig

h.le

vels

.of.

func

tiona

l.pro

tein

[81]

Hep

atiti

s.B

.sur

face

.ant

igen

Imm

une-

prim

ed.m

ice.

whe

n.fr

uit.w

as.o

rally

.con

sum

ed[6

7]

Part

ial.O

RF2

.of.

Hep

atiti

s.E

.vir

usPr

oduc

ed.im

mun

oact

ive.

Hep

atiti

s.E

.pro

tein

[68]

Inse

ct.

resi

stan

cePo

tato

.pol

yphe

nol.o

xida

seB

acil

lus

thur

ingi

ensi

s.C

ry6A

Incr

ease

d.re

sist

ance

.to.c

omm

on.c

utw

orm

sIn

crea

sed.

resi

stan

ce.to

.roo

t.kno

t.nem

atod

e[2

][1

6]

Popl

ar.c

hitin

ase,

.leaf

.exp

ress

ion

Del

ayed

.dev

elop

men

t.of.

Col

orad

o.po

tato

.bee

tle.la

rvae

[3]

Pota

to.P

I-II

.and

.car

boxy

pept

idas

e.in

hibi

tors

,.pr

otei

nase

.inhi

bito

rs,.l

eaf-

spec

ific.

expr

essi

onIn

crea

sed.

resi

stan

ce.to

.Hel

ioth

is o

bsol

eta.

and.

Lir

iom

yza

trif

olii

.larv

ae[5

]

Not

e:.

Gen

e.ex

pres

sion

.was

.con

stitu

tive.

unle

ss.o

ther

wis

e.st

ated

.


stresses.(salinity.and.drought),.production.of.edible.vaccines.and.therapeutic.prod-ucts,.nutritional.enhancement,.and.metabolic.engineering..The.published.results.from.these.studies.show.some.very.interesting.trends.toward.better.stress.tolerance,.pest.and.disease.resistance,.and.more.nutritious.vegetable.crops.(Table.2.1)..For.the.most.part,.these.transgenic.vegetable.crop.species.have.been.evaluated.under.laboratory.and.greenhouse.conditions,.with.limited.field.trials..Several.studies,.however,.have.evaluated.these.materials.in.trials.with.animal.systems.for.the.assessment.of.vaccines.and.therapeutics..Collectively,.these.studies.clearly.show.the.potential.of.transgenic.vegetable.crops.with.beneficial.traits,.and.they.are.discussed.in.more.detail.below.

INSECT RESISTANCE

Resistance.of.plants.to.insects.is.a.much.desired.attribute,.potentially.reducing.the.number. of. insecticide. sprays. required.. The. following. are. examples. of. transgenic.strategies.used.to.reduce.insect.damage.on.vegetable.crops.

. 1..Manipulation.of.polyphenol.oxidase.(PPO).activity.could.provide.resistance.simultaneously.to.both.diseases.and.insect.pests.2.Transgenic.tomato.plants.constitutively.expressing.potato.PPO.gene.were.tested.for.insect.resistance..These.transgenic.plants.exhibited.up.to.nearly.sixfold.higher.PPO.activity.levels.compared.to.controls.2.The.PPO.transgenic.plants.clearly.showed.an.increase.in.resistance;.simple.growth.rates.of.common.cutworms.on.these.plants.were.up.to.three.times.lower.than.on.controls.and.larvae.consumed.less.foliage..In.addition,.increased.PPO.activity.led.to.higher.larval.mortality.2.These.results.suggest.a.critical.role.for.PPO-mediated.phenolic.oxidation.in.resistance.to.this.insect.2

. 2.. Insect.cytoskeletons.and.digestive.systems.contain.large.amounts.of.chitin,.which.can.be.broken.down.by.certain.plant.chitinases..Transgenic.tomato.seedlings. expressing. a. poplar. chitinase. gene. were. tested. for. resistance.toward.the.Colorado.potato.beetle,.which.can.cause.severe.losses.in.tomato.and.other.crops.3.The.plants.expressing.the.poplar.chitinase.gene.reduced.the. larval.development. allowing.approximately.50%. to. reach. the. second.larval.instar,.as.compared.to.over.90%.in.the.control.plants,3.suggesting.the.possibility.of.using.a.plant.chitinase.gene.for.promoting.insect.resistance.3

. 3..Plant.proteinase.inhibitors.(PIs).have.been.well.established.to.play.a.potent.defensive. role. against. predators. and. pathogens. (reviewed.by. Mosolov.and.Valueva4)..The.defensive.capacities.of.plant.PIs.rely.on.inhibition.of.proteases.present.in.insect.guts.or.secreted.by.microorganisms,.causing.a.reduction.in.the.availability.of.amino.acids.necessary.for.their.growth.and.development..Unlike. other. insecticidal. proteins,. for. PIs. to. be. active,. very. high. levels.of.recombinant.PI.protein.are.required.to.achieve.pest.resistance.4.Tissue.specific.or. inducible.expression.would.yield.higher. levels.at. the.required.site. and. time..Leaf-specific.overexpression.of. the.potato.protease. inhibi-tor.and.carboxypeptidase.inhibitors.in.tomato.resulted.in.increased.resis-tance.to.Heliothis obsoleta.and.Liriomyza trifolii.larvae.in.high-expressing.


homozygous. transgenic. lines. compared. to. controls.5. Seeds. of. transgenic.peas.expressing.a.α-amylase.inhibitor.reduced.pea.weevil.(Bruchus piso-rum.L.).survival.by.nearly.100%,.with.the.larval.mortality.occurring.at.an.early.instar..Conversely,.in.nontransgenic.cultivars,.essentially.100%.of.the.pea.weevils.emerged.as.adults.6

. 4..Bacillus thuringiensis. (Bt).crystalline. toxin.genes.(cry).have.been.exten-sively.used.to.develop.transgenic.insect.resistant.crop.plants.over.the.past.two.decades..Several.reports.are.available.on.different.synthetic.Bt.genes.expressed.both.individually.and.in.a.combinatorial.way.and.their.efficiency.in.pest. control. (reviewed. in.Christou. et. al.7)..A.cry1Ca. or. a.H04. hybrid.gene. was. expressed. in. leaf. tissues. of. beet. and. shallot.8. When. either. of.these.genes.were.expressed.at.high.levels.in.the.leaves,.it.resulted.in.nearly.complete.resistance.against.beet.armyworm.in.shallot.or.beet.8.Similarly,.transgenic. garlic. plants. expressing. cry1Ca. were. completely. resistant. to.beet.armyworm.in.a.number.of.in.vitro.bioassays.8.There.are.several.other.successful. examples. of. increased. insect. resistance. in. a. variety. of. trans-genic.vegetables.using.Bt.cry.genes.alone.or.in.combination..These.include.cabbage,9. collards,10. broccoli,11–13. and. cauliflower.14. The. success. of. Bt. in.controlling.insects.has.been.very.promising,.with.the.only.major.concern.being. the.development. of. pest. resistance. to. particular. Cry. proteins.. The.use. of. trap. plants. and. pyramiding. different. Cry. proteins. at. high. levels.through. chloroplast. expression. can.help. alleviate. the.potential. for. future.pest.resistance.13,15

NEMATODE RESISTANCE

Some.of.the.Bt.toxin.genes.also.have.known.nematicidal.activity..Expression.of.nema-ticidal.Cry6A.in.transgenic.tomato.plants.provided.protection.against.plant-parasitic.nematodes.16. When. Cry6A-expressing. roots. were. challenged. with. Meloidogyne incognita,.the.nematode.was.able.to.ingest.the.toxin,.which.resulted.in.a.significant.decrease.in.gall.production.by.up.to.fourfold..This.report.suggests. that.Bt.protein.can.confer.resistance.to.endoparasitic.nematodes,.and.therefore.Bt.toxin.proteins.are.potential.candidates.for.developing.nematode-resistant.transgenic.plants.17

PATHOGEN RESISTANCE

vIruS reSIStance

The.earliest.successful.development.of.virus-resistant.transgenic.vegetable.crops.uti-lized.the.viral.coat.protein-mediated.resistance.(CPMR).strategy.to.engineer.patho-gen.resistance.18.The.use.of.CPMR.continues.to.be.one.of.the.most.useful.mechanisms.for.viral.resistance,.having.been.used.for.tomato.leaf.curl.virus.resistance.in.tomato19.and.big.vein-associated.virus.resistance.in.lettuce.20.CPMR.requires.expression.of.the. viral. coat. (or. capsid). protein. gene. from. the. virus. in. transgenic.plants..When.exposed.to.the.viral. inoculum,.the.transgenic.plants.displayed.reduced.number.of.


lesions,.reduced.rate.of.systemic.disease.development,.and.very.low.levels.of.virus.accumulation.compared.to.control.plants..In.some.instances,.the.transgenic.plant.was.protected.not.only.against.the.virus.from.which.the.coat.protein.gene.was.derived.but.also.against.other.serologically.unrelated.viruses.(reviewed.in.Prins.et.al.18)..Despite.the.widespread.use.of.CPMR,.it.is.believed.that.multiple.molecular.mechanisms.of.resistance.exist,.which.are.not.fully.understood.21.Currently,.CPMR.is.registered.for.use. in. transgenic.squash.and.zucchini,.with.the.potential.for.many.new.vegetable.crops.in.the.future.

Expression.of.viral.RNA-dependant.RNA-polymerases.(replicase).using.antisense.or.sense.constructs.has.also.been.effective.at.limiting.infection.and.spread.of.RNA.viruses..Replicase-mediated.resistance.appears. to.be.strain-specific.and. functions.through.lowering.the.overall.level.of.viral.replicases,.thus.reducing.the.viral.concen-tration..Replicase-mediated.viral.resistance.has.been.successfully.used.in.tomato.to.increase.resistance.to.tomato.leaf.curl.virus.22,23

Plant.DNA.viruses. do.not. encode. polymerases;. instead,. they. require. an. inter-action.between.viral.replication-associated.protein.(rep).and.the.host.plant’s.DNA.polymerase..Expression.of.mutated.viral.rep.gene.or.rep-gene.silencing.through.anti-sense.technologies.can.reduce.viral.titer.and.has.been.used.to.improve.viral.resis-tance.in.cabbage24.and.bean.25,26

Other.mechanisms.of.viral.resistance.include.expression.of.viral.nucleoproteins,.which.serve.to.disrupt.normal.movement.of.viral.proteins.and.nucleic.acid.packing.leading. to. resistance,. reducing. viral. symptoms.. Expression. of. nucleoproteins. has.been.used.in.tomato.to.reduce.symptoms.in.response.to.tomato-spotted.wilt.virus.27,28

funGal and bacterIal reSIStance

Enhanced.resistance.to.fungi.and.bacteria.has.been.demonstrated.using.a.number.of.different.strategies.for.several.vegetable.crop.species.(Table.2.1)..These.include.carrot,. cucumber,. lettuce,. and. tomato.. The. genes. and. corresponding. enzymes..produced. include. hydrolytic. enzymes. (chitinases. and. glucanases),. peroxidase,..pathogenesis-related.(PR).proteins.(thaumatin),.and.peptides..Expression.of.chitin-ases.or.β-1,3-glucanases,.which.are.PR-proteins.that.can.hydrolyze.the.major.struc-tural.polysaccharides.in.the.fungal.cell.wall.(chitin.and.laminarin),.provided.varying.levels. of. resistance. to. Botrytis cinerea. in. cucumber29. and. to. a. number. of. fungal.pathogens.infecting.carrot.30,31.Chitinase.expression.has.been.achieved.in.combina-tion.with.other.genes. including. osmotin. in. tomato32. to. reduce.Fusarium.wilt. and.with. lipid-transfer.protein. in. carrot,33.which. resulted. in. reduced. fungal. infection..Expression.of.another.PR-protein,. thaumatin,.also. enhanced. fungal.disease. resis-tance.in.carrot.34.Peroxidase.expression.reduced.Botrytis.and.Sclerotinia.infection.of.carrot.leaves31.and,.when.expressed.in.combination.with.PR-1,.reduced.Phytophthora.development.in.tomato.35

A.secondary.approach.has.been.to.engineer.expression.of.enzymes.that.degrade.pathogen. virulence. products,. e.g.,. oxalic. acid. oxidase. or. decarboxylase. to. break.down.oxalic.acid..For.example,.expression.of.a.wheat.oxalate.oxidase.gene.in.tomato.reduced.the.development.of.Botrytis.and.Sclerotinia36.while.an.oxalate.decarboxyl-ase.gene.from.the.fungus.Flammulina.also.led.to.reduced.Sclerotinia.development.


in. transgenic. lettuce.37.Both.of. these.genes.exhibited.the.ability.to.degrade.oxalic.acid,.an.important.pathogenicity.factor.for.this.fungus.36,37

For.bacterial.resistance,.gene.products.such.as.peroxidase,.ferredoxin,.and.defen-sins.reduced.disease.development.on.tomato.(Table.2.1).caused.by.several.pathogens..In.tomato,.bacterial.wilt.development.was.reduced.through.combined.expression.of.a.β-1,3-glucanase.and.defensin.gene38.while.expression.of.a.sweet.pepper.ferredoxin.gene.also.reduced.the.development.of.bacterial.blight.39

ABIOTIC STRESS TOLERANCE

Abiotic.stresses,.including.salinity,.temperature,.and.water.stresses,.are.among.the.most. limiting.stresses,. lowering.crop.yield.and.quality.worldwide..Growing.areas.that.experience.extreme.salt.and.water.stress.typically.grow.limited.amounts.of.veg-etable.crops,.which.can. lead. to. local.human.nutritional.deficiencies..Transgenic.vegetable. crops. capable.of.growing. on. saline. soil. or.with.minimum. irrigation. in.these.areas.could.help.stabilize.food.and.nutritional.production.in.these.regions.40

Osmotic.adjustment.at.the.cellular.level.is.a.vital.response.in.dealing.with.water,.salinity,. and. freezing. stresses.. Natural. plant. defenses. to. these. stresses. include.the. synthesis. and. accumulation. of. plant. compatible. solutes,. including. glycine.betaine,. sugars,. proline,. and. compatible. proteins. (late-embryogenesis. abundant.protein.[LEA],.dehydrins,.and.heat.shock.proteins).(reviewed.in.Cheong.and.Yun41)..Compatible. osmolytes. protect. the. plant. through. osmotic. adjustment,. which. helps.maintain. turgor,. scavenging. of. reactive. oxygen. species. (ROS),. and. the. stabiliza-tion.of.protein.structure.40.The.ability.to.regulate.osmotic.potential.using.compatible.solutes.has.increased.yield.under.drought.conditions.in.many.crop.plants.42,43.There.are. reports. of. transgenic. vegetables.with. increased. production. of. compatible. sol-utes,.resulting.in.increased.resistance.to.abiotic.stresses.(Table.2.1)..Two.of.the.more.commonly.used.strategies.in.transgenic.vegetables.are.the.overexpression.of.LEA.genes.and.overproduction.of.glycine.betaine..Normally,.LEA.genes.are.expressed.during. the. late. stages. of. seed. development;. however,. many. LEA. genes. are. also.expressed.in.vegetative.tissue.when.the.plants.undergo.an.abiotic.stress.44.Many.of.the.LEA.proteins.have.been.shown.to.confer.tolerance.to.drought,.salinity,.and.freez-ing.stresses.45.Overexpression.of.heterologous.LEA.proteins.has.been.successful.in.increasing.abiotic.stress.resistance.in.kidney.bean,46.Chinese.cabbage,45,47.and.let-tuce.48.Glycine.betaine.is.naturally.produced.in.many.plant.species,.and.high.accu-mulation.levels.are.associated.with.drought.and.salt.tolerance..Tomato.plants.do.not.produce.endogenous.glycine.betaine;.therefore,.plants.were.transformed.to.express.heterologous.betaine.aldehyde.dehydrogenase.(BADH)49.or.choline.oxidase.50.These.tomato.plants.were.able.to.withstand.moderately.high.levels.of.salinity.and.produced.viable.fruit.and.seeds..Carrot.accumulates.low.levels.of.glycine.betaine.naturally,.but.chloroplastic.overexpression.of.endogenous.BADH.resulted.in.enhanced.glycine.betaine.levels.51.These.transgenic.carrots.were.able.to.grow.on.soil.containing.physi-ologically.very.high.salt.levels.

Another. strategy. used. by. drought. and. salinity-resistant. plants. is. to. compart-mentalize. the.abundant.but. toxic.Na+. ions. to.utilize. them.for.osmotic.adjustment.


by. transporting. the. ions. into. the. vacuole. using. tonoplastic. Na+/H+. antiporters.52.Transgenic.tomato.plants,.overexpressing.the.Arabidopsis.Na+/H+.antiporter.(Atnhx).gene,.were.able.to.grow.and.produce.fruit.at.relatively.high.levels.of.Na+,.without.accumulating.excess.Na+.in.the.fruit.52.The.Atnhx.gene.has.also.been.overexpressed.in.sugar.beet,.which.became.more.resistant.to.drought.stress,.in.addition.to.increas-ing.the.natural.level.of.sucrose.in.the.taproot.53,54

Abiotic.stresses.disrupt.normal.plant.metabolism.by.affecting.reaction.rate,.protein. stability,. and. membrane. integrity.. This. alteration. in. metabolism. leads.to.an. increase. in.accumulation.of.ROS. to.potentially. toxic. levels. (reviewed. in.Ashraf55).. Detoxification. of. ROS. occurs. naturally. to. some. extent. in. all. plant.species.through.the.production.of.detoxifying.enzymes.and.antioxidants.56.The.recently.employed.strategies.for.detoxifying.ROS.in.vegetable.crops.include.the.overexpression. of. the. detoxifying. enzymes. superoxide. dismutase. and. catalase.in. tomato57. and. Chinese. cabbage,58,59. resulting. in. enhanced. tolerance. to. abi-otic.stresses..Additionally,.more.complex.metabolic.engineering.to.increase.the.production.of.antioxidants.has.been.made.to.enhance.resistance.toward.abiotic.stresses.in.tomato.60–63

MEDICAL APPLICATIONS

edIble vaccIneS

Transgenic.vegetables.offer.an.attractive.option.for.vaccine.production,.since.nearly.any.antigenic.protein.can.be.expressed.in.plants.at.a.large.scale,.and.the.crops.can.be.grown.locally.64.Vaccines.can.also.be.produced.in.edible.parts.of.plants,.which.aids.in.delivery..Edible.plant-based.vaccines.can.be.made.against.an.entire.protein.or.just.the.desired.antigenic.peptides.from.viruses,.parasites,.or.bacterial.pathogens.64.Development.of. edible.vaccines.has.been.primarily.directed. to.pathogens,. which.infect.the.host.via.the.mucosal.surfaces.lining.the.digestive,.respiratory,.and.urinor-eproductive.tracts.of.the.body,.since.these.vaccines.need.to.be.absorbed.in.a.similar.fashion..Much.of.the.research.on.edible.vaccines.has.focused.on.tomato.fruit,.car-rot.taproots,.and.leafy.greens,65–73.since.they.can.be.consumed.raw,.eliminating.the.possibility. of. degradation. of. the. vaccine. during. the. cooking. process.. Expression.of. these. polypeptides. has. been. mainly. with. constitutive. promoters. while. some.research.in.tomatoes.used.fruit-specific.E8.promoter.65,66.In.recent.years,.successful..vegetable-based.edible.vaccines.have.been.produced.against.common.viruses.such.as.measles,69,70,74,75.rabies,73.and.hepatitis.B,66,68.common.bacterial.diseases.such.as.cholera,71.and.HIV,65,72,76.which.are.extremely.difficult.to.treat.

While.there.are.many.benefits.associated.with.the.production.of.vegetable-based.edible. vaccines,. there. are. a.number. of. limitations. to. this. technology.. One.of. the.major.limitations.is.obtaining.adequate.protein.concentrations.that.initiate.sufficient.immune.response.to.result.in.complete.immunity..Additionally.timing.of.doses.and.application.of.multiple.doses.is.crucial.and.will.need.to.be.addressed.before.these.edible.vaccines.are.used.for.larger.scale.applications..Despite.the.promise.in.allevi-ating.important.global.diseases,.there.are.currently.no.commercial.transgenic.veg-etable.plants.producing.edible.vaccines.


therapeutIc productS

A.range.of.other.therapeutic.agents.have.been.derived.from.transgenic.plants,.includ-ing.potato,.rice,.wheat,.and.corn.77.Recently,.there.has.been.increasing.focus.on.the.use.of.vegetable.crops.to.produce.these.therapeutic.products,.since.they.can.be.con-sumed.raw.(Table.2.1)..The.biopharmaceuticals.produced.to.date.in.vegetables.include.enzymes,.interleukins,.and.antimicrobial.peptides.for.humans.and.animals.as.well.as.blood-clotting.peptides..These.therapeutic.products.were.previously.or.are.currently.produced.in.animal,.yeast,.or.bacterial-cell.cultures..Plant-based.products.have.the.potential.for.significantly.lower.costs.and.reduced.potential.for.contamination.78.Oral.consumption.of.transgenic.tomato.fruit.containing.human.interleukin-12.resulted.in.a.dramatic.reduction.in.the.symptoms.of.tuberculosis.in.mice.79.The.production.of.transgenic.lettuce.expressing.chicken.interferon.has.indicated.the.possibility.of.using.transgenic.forage.crops.for.avian.feed.and.potentially.for.other.livestock.80

For.the.most.part,.the.amount.of.pharmaceutical.proteins.produced.in.the.trans-genic.vegetables.would.be.quite.low.(less.than.1%.of.total.soluble.protein)..This.level.of.expression.is.insufficient.for.compounds.that.are.not.highly.active,.such.as.blood.coagulation.factors.81.These.proteins.would.either.need.to.be.extracted.and.further.purified,.or.an.alternative.expression.system.would.need.to.be.employed.

A. limitation. of. using. edible. biopharmaceuticals. is. the. low. absorption. of. the.active.agent.through.the.mucosal.lining,.without.excessive.degradation..To.increase.the. absorption,. the. use. of. nontoxic. bacterial. adjuvants. has. been. investigated.82.Potentially,.very.promising.results.have.been.seen.in.lettuce.expressing.proinsulin.fusion.protein.with.a.bacterial.adjuvant.in.the.chloroplast.83.The.fusion.allowed.for.easier.uptake.of.the.insulin.and.alleviated.the.symptoms.of.pancreatic. insulitis. in.mice,.a.condition.similar.to.type.I.diabetes.

NUTRITIONAL MODIFICATIONS

enhanced nutrItIonal QualIty

Vegetable.crops.are.a.major.nutritional.source.of.many.essential.human..nutrients,.including.vitamins,.carotenoids,.and.flavonoids,.as.well.as.other.important..nutrients..However,.not.all.vegetables.are.rich.in.all.these.essential.nutrients..Folate.or..vitamin B9.is.an.essential.cofactor.for.the.synthesis.of.many.amino.acids.and.purines..Humans.cannot.synthesize.folates.that.must.be.supplemented.through.the.diet,.which.is.mainly.from.plant.sources..Leafy.greens.contain.high.natural.levels.of.folate;.however,.many.of.the.worldwide.food.staples.(grains.and.tubers).contain.inadequate.levels.of.folate,.which.can.lead.to.widespread.deficiencies.84.Folate.deficiency.can.lead.to.neural.tube.defects. and. other.human.diseases. and. is. a.global. health.problem..Because.plants.are.major.folate.sources.for.humans,. there.is.always.scope.to.enhance.plant.folate.levels.(biofortification)..Plants.synthesize.folate.from.pteridine,.p-aminobenzoic.acid.(PABA),.and.glutamate.moieties..A.synthetic.mammalian.GTP cyclohydrolase1.was.expressed.in.tomato.in.a.fruit-specific.manner.84.As.a.result,.the.fruit.pteridine.and.folate.increased.relative.to.controls.84.PABA.pools.were.depleted.in.engineered.fruit.that.were.higher.in.folate,.and.supplying.such.fruit.with.PABA.further.increased.their.


folate.content.84.When.transgenic.PABA-.and.pteridine-overproduction. traits.were.combined.by.crossing,.vine-ripened.fruit.accumulated.up.to.25-fold.more.folate.than.controls.85.Transferring.the.folate.biosynthetic.branch.developed.in.tomatoes.to.staple.food.plants.such.as.sweet.potato,.could.potentially.increase.the.folate.intake.in.poor.countries.with.limited.access.to.fresh.vegetables.

Modification.of.starch.composition.of.tubers.enables.them.to.be.used.for.diverse.industrial. applications.. Amylose. and. amylopectin. are. the. two. principal. polysac-charide. components. of. most. natural. starches.86. In. sweet. potato. plants,. the. range.of.amylose.content.(10%–20%).is.narrow.in.comparison.with.other.crops,.thereby.making.them.less.amenable.for.industrial.uses..Since.the.ratio.of.amylase.to.amy-lopectin. is. an. important. textural. property. of. starch,. development. of. a. plant. type.that.contains.amylose-free.or.low.amylose.would.envisage.new.industrial.applica-tions.86,87. Amylose-free. transgenic. sweet. potato. plants. were. produced. by. inhibit-ing.sweet potato granule-bound starch synthase I (GBSSI).gene.expression.through.RNA.interference.86,87.GBSSI.is.one.of.the.key.enzymes.catalyzing.the.formation.of.amylose,.a.linear.alpha.(1,4)d-glucan.polymer,.from.ADP-glucose..Due.to.silencing.of.GBSSI.gene,.over.70%.of.the.regenerated.transgenic.plant.roots.contained.amylo-pectin.but.not.amylose..These.reports.suggest.that.RNA.interference.is.an.effective.method.for.manipulating.gene.expression.in.the.starch.metabolic.pathway.86,87

enhanced taSte

Fruit.taste.is.a.complex.genetic.trait.governed.by.multiple.genes.and.gene.interac-tions..Although. taste. improvement. in. food.crops.has.been.classically.achieved.by.careful.selection.and.hybridization.for.centuries,.it.is.one.of.the.traits.that.still.cannot.be.directly.manipulated.by.conventional.plant-breeding.methods.but.may.be.possible.through.genetic.engineering..Taste-modifying.proteins.are.alternative.sweeteners.and.flavor.enhancers,.and.manipulation.of.their.expression.may.enhance.the.nutritional.quality.and.consumer.preference..Thaumatin.is.a.sweet-tasting,.flavor-enhancing.pro-tein.present.in.the.fruits.of.Thaumatococcus daniellii,.an.African.shrub.88.Transgenic.tomato.lines.expressing.recombinant-thaumatin.protein.in.fruits.were.sweeter.than.the.controls,.which.was.confirmed.by.organoleptic.evaluation.88.Miraculin.is.a.taste-modifying.protein.present. in.the.red.berries.of.Richadella dulcifica..Miraculin.by.itself.is.not.sweet;.it.converts.the.sour.taste.of.ascorbic.acid,.acetic.acid,.and.citric.acid.into.a.sweet.taste.after.being.held.in.the.mouth.for.some.time..This.unique.property.has.led.to.increasing.interest.in.this.protein.for.expression.in.other.plants..Miraculin.was.constitutively.expressed.in.lettuce,.and,.as.a.result,.the.transgenic.leaves.accumu-lated.significant.levels.of.miraculin,.which.eventually.attributed.sweetness-inducing.activity.89,90. Similarly,. transgenic. tomato. plants. expressing. recombinant. miraculin.protein.accumulated.high.levels.in.both.leaves.and.fruits..The.purified.recombinant.miraculin.protein.from.transgenic.tomato.plants.showed.strong.sweetness-inducing.activity,.which.was.similar.to.that.of.native.miraculin.90

Similar.to.taste,.flavor.and.aroma.are.traits.that.are.often.rarely.available.within.existing.germplasm.and.hence.have.to.be.imported.from.related.or.alien.gene.pool..Ocimum basilicum. geraniol. synthase. gene. was. expressed. in. tomatoes. in. a. fruit-.specific.manner.using.the.tomato.ripening-specific.polygalacturonase.(PG).promoter..


Various.monoterpenes.that.contribute.to.fruity.and.floral.scents.were.found.to.accu-mulate.in.the.transgenic.tomatoes,.which.eventually.enhanced.the.flavor.and.aroma.of.the.flesh.91

Inulins. are. beneficial. for. human. health. and. are. produced. naturally. in. some.plants.as.storage.carbohydrates..They.serve.as.replacements.for.high.caloric.fats.and.sugars,.since.inulins.have.a.sweet.taste.and.are.not.absorbed.through.the.digestive.tract.. Transgenic. lettuce. lines. expressing. the. asparagine synthetase A. gene. from.Escherichia coli. accumulated. lowered.amounts.of. sucrose,.glucose,. and. fructose,.whereas.inulin.content.increased.up.to.30.times.in.transgenic.leaves.92.These.lettuce.genotypes.with.high.content.of.foliar.inulin.represent.useful.materials.for.breeding.strategies.and.a.potential.source.for.low-molecular-weight.inulin.useful.for.dietary.supplements,.in.addition.to.the.potential.for.enhanced.taste.

enhanced antIoxIdant actIvItIeS

Enhancement.of. antioxidant. content. is. an. important. trait. in. the. light.of.potential.health. benefits. imparted. by. these. chemicals. in. plants. and. animals.. α-Tocopherol.is. the.most.biologically.active.form.of.vitamin.E,.which.is. implicated.in.decreas-ing. the. risk.of. several. types.of.cancers,. coronary.heart.disease,.and.a.number.of..degenerative. human. conditions.. Manipulating. the. tocopherol. biosynthetic. path-way. in. plants. to. convert. tocopherols. into. more. active. α-tocopherol. form. could.have. significant. health. benefits.93. An. Arabidopsis. γ-tocopherol methyltransferase.(γ-TMT).gene.was.overexpressed.in.lettuce.to.improve.the.tocopherol.composition..This.resulted.in.higher.TMT.activity.and.the.conversion.of.the.γ-tocopherol.pool.to.α-tocopherol.in..transgenic.plants.93.In.another.attempt,.in.order.to.increase.tocoph-erol.content.by.increasing.total.flux.to.the.tocopherol.biosynthetic.pathway,.two.dif-ferent.Arabidopsis.genes.were.constitutively.expressed.in.lettuce..Transgenic.plants.expressing.either.of.these.genes.had.total.tocopherol.content.increased.by.more.than.twofold.mainly.due.to.an.increase.in.γ-tocopherol.94

Increased.isoprenoid.levels,.the.precursors.to.many.important.nutrients,.can.be.achieved. in. tomato. through. genetic. manipulation. of. mevalonic. acid. (MVA). and.methylerythritol-4-phosphate. (MEP). pathways,. leading. to. the. formation. of. iso-pentenyl. diphosphate. (IPP),. using. 3-hydroxymethylglutaryl. CoA. (hmgr-1). and.1-deoxy-d-xylulose-5-phosphate synthase.(dxs).genes,.respectively..Transgenic.toma-toes. expressing. the. Arabidopsis. hmgr-1. gene. had. double. the. phytosterol. levels.95.Additionally,. tomatoes. expressing. a. bacterial. dxs. gene. targeted. to. the. plastids.resulted. in. significantly. increased. carotenoid. content,. which. was. attributed. to.enhanced. levels.of.phytoene.and.β-carotene.95. In.order. to. increase. the.carotenoid.and.flavonoid.content.in.tomato.fruits,.an.endogenous.phytomorphogenesis.regula-tory.gene.(DET1).was.suppressed.by.RNAi.in.a.fruit-specific.manner.96.The.resulting.transgenic.fruits.accumulated.higher.levels.of.carotenoids.and.flavonoids.compared.to.the.controls.96.Plastid.expression.of.a.bacterial.lycopene-β-cyclase.gene.in.tomato.resulted.in.the.conversion.of.the.main.storage.carotenoid–lycopene,.to.β-carotene,.leading.to.a.fourfold.increase.in.provitamin.A.content.of.the.fruits.97

Although. β-carotene. (provitamin. A),.α-carotene,. and. lutein. are. widely. preva-lent. in. many. fruits. and. vegetables,. ketocarotenoids,. such. as. canthaxanthin. and.


astaxanthin,.are.rarely.present.in.plants..Ketocarotenoids.are.strong.antioxidants.and.are.chemically.synthesized.and.used.as.dietary.supplements.and.as.pigments.in.the.aquaculture.and.neutraceutical.industries..Ketocarotenoid.biosynthetic.pathway.was.engineered.in.carrot.tissues.by.introducing.a.β-carotene ketolase.gene.isolated.from.the.red.alga.Haematococcus pluvialis.98.In.the.transgenic.carrot.taproots,.up.to.70%.of.total.carotenoids.were.converted.to.novel.ketocarotenoids,.with.accumulation.up.to.2.4.mg/g.root.dry.weight..Astaxanthin,.adonirubin,.and.canthaxanthin.were.most.prevalent,.followed.by.echinenone,.adonixanthin,.and.β-cryptoxanthin,.and.all.these.ketocarotenoids.have.neutraceutical.importance.98

Glucosinolates.are.an.important.group.of.secondary.metabolites.found.in.almost.all.the.members.of.the.family.Brassicaceae..Although.glucosinolates.are.well.known.for.their.toxic.effects.in.both.humans.and.animals.at.high.doses,.at.subtoxic.doses,.their.hydrolytic.and.metabolic.products.act.as.chemoprotective.agents.against.chemi-cally.induced.carcinogens.and.thereby.block.initiation.of.a.variety.of.tumors.in.addition.to.having.a.wide.range.of.bioactivities.99.Sulforaphane,.a.metabolite.of.methionine-derived.4-methylsulfinylbutyl.glucosinolate.(glucoraphanin),.has.attracted.attention.because.of. its. potential.anticarcinogenic.activity.100.Glucoraphanin. is.an.aliphatic.glucosinolate.abundant.in.broccoli.and.Arabidopsis.ecotype.Columbia,.but.nonexis-tent.in.Chinese.cabbage..Three.Arabidopsis.genes,.required.for.aliphatic.glucosino-late.biosynthesis,.were.introduced.into.Chinese.cabbage.101.The.resulting.transgenic.lines.accumulated.significantly.higher.levels.of.the.aliphatic.glucosinolates,.gluco-napin,.and.glucobrassicanapin.101,102

fruIt rIpenInG

Although.ripening.is.a.process,.which.renders.the.fruits.to.become.more.edible,.the.ripening-phenomenon. itself. can. be. either. beneficial. or. detrimental. to. both. fresh-ness. and. processing. quality. of. fruits. and. vegetables.. For. instance,. during. ripen-ing. of. tomato,. cell.wall. disassembly. is. closely. associated. with. loss. of. fresh. fruit.firmness.and.subsequently.with.a.loss.of.viscosity.in.the.processed.products..In.tomato,.PG and.expansin.(Exp).are.among.the.cell.wall.proteins.that.cooperatively.participate.in.ripening-associated.cell.wall.disassembly..Transgenic.suppression.of.either.LePG.or.LeExp1.expression.alone.resulted.in.altered.softening.and.shelf-life.characteristics. of. fruits.103. Suppression. of. LeExp1. or. LePG. alone. did. not. signifi-cantly.increase.fruit.firmness..However,.fruits.suppressed.for.both.LePG.and.LeExp1.expression. were. significantly. firmer. throughout. the. ripening. phase. and. were. less.susceptible.to.deterioration.during.long-term.storage..The.viscosity.of.juice.prepared.from. the. transgenic. tomato. fruit.with. reduced.LePG. and.LeExp1. expression. was.higher.than.that.of.controls.103

DISCUSSION

Although. genetically. engineered. tomato. (Flavr. Savr). with. enhanced. fruit-quality.characteristics.was.the.first.example.of.a.transgenic.vegetable.crop.to.be.marketed.in.the.United.States.and.United.Kingdom.in.the.1990s,.most. transgenic.vegetable.crop.species.have.not.reached.the.market.as.rapidly.as,.for.example,.transgenic.field.


crops. that. include.canola,.corn,. potato,. and.soybean..Transgenic. squash. and.zuc-chini.with.virus.resistance.followed.tomato.as. the.next.vegetable.crop.in.the.U.S..market..This.chapter.has.summarized.many.other.potentially.beneficial.traits. that.have.been.engineered. into.vegetable. crop. species..Some.of. these. traits. can.be.of.direct.benefit.to.the.grower,.e.g.,.insect.or.herbicide.resistance,.while.others.would.have.benefits.to.the.consumer,.e.g.,.enhanced.vitamins,.flavor,.and.quality..While.there.appears.to.be.continuing.interest.in.the.applications.of.biotechnology.to.vegetable.crop.species,.several.requirements.still.must.be.met.to.ensure.success.in.transforma-tion..A.well.defined.and.reproducible.tissue-culture.system.is.needed,.together.with.an.efficient.transformation.method.as.well.as.an.appropriate.selection.system.to.dis-tinguish.transformed.cells.from.non-transformed.cells..While.an.increasing.number.of.vegetable.crop. species. are.becoming.amenable. to. transformation. technologies,.there.are.still.some.limitations..It.appears.that.only.a.few.techniques.are.available.to.introduce.foreign.genes.into.vegetable.crop.species,.with.the.most.popular.being.Agrobacterium-mediated. transformation.. Optimization. of. gene. transfer. methods,.transformation.efficiencies,.and.tissue-culture.procedures.still.remain.a.challenge.for.some.vegetable.species..The.most.amenable.species.appear.to.be.tomato.and.carrot.

There.are.also.other.issues.that.need.to.be.addressed.before.widespread.utility.of.transgenic.vegetable.crop.species.occurs..The.traits.engineered.into.the.respective.crops.need. to.be. evaluated.under. replicated.field. conditions.over. several.years. to.ensure.the.introduced.genes.confer.the.desired.phenotype..Furthermore,.the.horti-cultural.attributes.in.the.transgenic.lines.should.be.similar.to.those.in.the.existing.non-transgenic. cultivar..These. lines. should. demonstrate. a. clear. advantage. (to. the.grower,.the.consumer).compared.to.that.currently.existing.in.the.crop.species.germ-plasm..While. these.criteria.have.been.met.with. the.currently.approved. transgenic.corn,.canola,.soybean,.and.potato.cultivars,.they.still.require.additional.effort.for.the.transgenic.lines.of.vegetable.crops.under.study.

Consumer. acceptance. of. vegetable. crops. that. contain. transgenes. will. require.that.the.same.stringent.criteria.used.to.evaluate.other.transgenic.food.plants.be.met..Perhaps.more.so. than. transgenic.field.crops,. transgenic.vegetable.crops. that.are.consumed.fresh.may.face.additional.consumer.concerns.that.will.require.that.evi-dence. of. their. benefits. be. made. clear.. In. the. case. of. vitamin-enhanced. tomatoes.or.high-antioxidant.carrots,. these.could.provide.a.strong.example.of. the.potential.benefits.of.the.transgenic.produce..The.other.issues.regarding.use.of.marker.genes,.spread.of.the.transgenes.through.pollen,.potential.for.resistance.selection.in.the.case.of.insect.or.fungal.pests,.and.allergenicity.of.the.introduced.protein.are.all.similar.to.those.required.for.other.transgenic.crops.and.need.to.be.satisfied.

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55

3 Transgenic Ornamental Crops

Beverly A. Underwood and David G. Clark

INTRODUCTION

Ornamental.crops.can.be.classified.as.floriculture.and.nursery.plants,.shrubs,.trees,.and.foliage.plants.for.outdoor.and.indoor.use..Ornamental.crops.are.produced.with.the.purpose.of.beautifying,.decorating,.or.enhancing.the.environment,.and.exclude.plants. intended. for. commercial. food. production. such. as. vegetables. and. fruits.1.

CONTENTS

Introduction............................................................................................................... 55Flower.Color............................................................................................................. 56

Flavonoids............................................................................................................56Chalcones.and.Aurones................................................................................... 57Anthocyanins................................................................................................... 58

Carotenoids..........................................................................................................60Plant.Volatiles............................................................................................................ 62

Floral.Fragrance................................................................................................... 62Fragrance.and.Color........................................................................................65

Volatiles.and.Insect.Plant.Defense.......................................................................65Flower.and.Leaf.Longevity.......................................................................................66

Floral.Longevity...................................................................................................66Altering.Ethylene.Responses.Genetically.by.Inhibiting.Ethylene Biosynthesis..................................................................................... 67Inhibiting.Ethylene.Responses.by.Disrupting.Ethylene.Signal.Transduction....... 68

Leaf.Longevity.....................................................................................................69Plant.Architecture...................................................................................................... 70

Plant.Height.......................................................................................................... 71Flower.Morphology............................................................................................. 71Fruit.and.Flower.Size........................................................................................... 72Agrobacterium rhizogenes rol.Genes................................................................... 72

Biotic.Stress.Tolerance.............................................................................................. 73Current.and.Future.Issues.in.Ornamental.Biotechnology......................................... 74Acknowledgments..................................................................................................... 75References................................................................................................................. 75


Production. of. ornamental. crops. is. a. global. industry,. having. a. multibillion. dollar.value. worldwide. driving. the. development. and. introduction. of. novel. ornamental.plants.solely.for.human.use.

Novel.ornamental.plants.have.been.developed.by.traditional.breeding.techniques,.including.hybridization. and.mutation.breeding,. and.more. recently.by. introducing.precise,.direct.genetic.changes.through.genetic.engineering..There.are.major.poten-tial.benefits. to.genetic.engineering. including. the.opportunity. for. the. introduction.of.genes. and. traits.not.present. in. the.natural. gene.pool.of. the. species.of. interest.without.altering.the.genetic.background..A.variety.of.consumer.and.producer.traits.have.been.successfully.modified.in.ornamental.plants.through.genetic.engineering,.including. flower. color,. fragrance,. plant. architecture,. cut. flower. vase. life,. disease.resistance,.abiotic.stress,.and.herbicide.tolerance.2,3.This.chapter.provides.an.over-view. of. the. traits. modified. by. transgenic. technology,. examines. the. benefits,. and.discusses.issues.that.have.limited.the.commercialization.of.transgenic.ornamental.crops.over.the.past.decade.

FLOWER COLOR

Flower.color.and.color.pattern.are.key.elements.in.consumer.selection.of.flowering.ornamentals.. It.has.been.of. considerable. interest. to.develop.commercial. series.of.plants.with.a.full.spectrum.of.flower.colors..Producing.a.series.of.plants.having.flow-ers.with.the.full.color.palette.through.traditional.breeding.is.challenging.for.most.species.since.the.genetics.required.for.producing.all.the.color.compounds.is.usually.limiting..Through. identification.and. introduction.of.color.producing.enzymes.and.pathways.from.other.plants,.new.colors.can.be.introduced.into.varieties.that.would.otherwise.be.difficult.to.obtain.(Table.3.1).

Flavonoids,.carotenoids,.chlorophyll,.and.betalins.are.the.primary.classes.of.com-pounds.that.give.flowers.their.characteristic.color..Flavonoids.are.widely.distributed.in.many.plant.families,.conferring.shades.of.red,.orange,.yellow,.blue,.and.violet..Carotenoids.are. derived. from. isoprenoids. and. give. rise. to. orange. and. yellow. shades,. marigolds.being. the. classic. example.. Betalins. are. water-soluble. nitrogen-containing. com-pounds.derived.from.tyrosine.and.are.taxonomically.restricted.to.the.Caryophyllales.4.Biosynthesis.of.these.compounds.has.been.well.characterized.in.many.plant.species.and.readers.are.referred.to.published.reviews.elsewhere.for.detailed.information.about.their.biosynthesis.4,5

flavonoIdS

Chalcones,.aurones,.anthocyanins,.flavones,.and.flavonols.are.the.major.groups.of.flavonoids.that.impart.the.colorful.floral.display.seen.in.the.angiosperms..Novel.and.complex.coloration.patterns.have.been.achieved.primarily.by.manipulation.of.the.fla-vonoid.biosynthetic.pathway.including.preventing.or.redirecting.flavonoid.synthesis.through.gene. silencing,. introducing.biosynthetic.enzymes.with.different. substrate.preferences.or.novel.activities,.and.modifying.expression.of.regulatory.transcription.factors.controlling.flavonoid.biosynthetic.genes.(Table.3.1).

57Transgenic Ornamental Crops

Chalcones and AuronesChalcone.synthesis.is.the.first.committed.step.in.flavonoid.synthesis..The.chalcones.serve. as. the. substrates. for. anthocyanin. and,. in. some. plant. species,. aurone. synthe-sis..The.first.committed.step.in.flavonoid.synthesis.is.catalyzed.by.chalcone.synthase.(CHS),. which. produces. tetrahydroxychalcone. (THC). from. p-coumaroyl. CoA. and.three. molecules. of. malonyl-CoA. (reviewed. in. Grotewold4).. Silencing. CHS. expres-sion. through. antisense. or. cosuppression. techniques. leads. to. white. or. complex. col-oration.patterns.in.many.plant.species.including.petunia,6,7.lisianthus,8.gerbera,9.and.chrysanthemums.10.THC.has.a.pale.yellow.color,.but.does.not.accumulate.in.plants..

TABLE 3.1Genetically Engineered Ornamental Plants with Modified Flower Color

Plant Initial Flower ColorGenetic

ModificationTransgenic Phenotype References

Petunia.x.hybrida Pale.pink 35S::ZmDFR Red [19]

Purple Antisense.CHS White,.white-purple.patterning

[148]

Purple Sense.CHS White,.white-purple.patterning

[6,7]

White 35S::MtCHR Pale.yellow [12]

Purple Pale.purple

Dianthus caryophyllus

Pink Sense.CHS Pale.pink [149]

Red-orange Antisense.F3H White [58]

White PhF3′5′H.and.DFR Blue [16]

Dendrathema grandiflora

Pink Sense.CHSAntisense.CHS

Pink,.white,.light.pink

[10]

Eustoma grandiflorum

Purple Antisense.CHS White,.white-purple.patterning

[8]

Rosa hybrida Red Sense.CHS Pale.pink [149]

Pink,.mauve 35S::VwF3′5′H Bluish.flowers [150]

Mauve RNAi.DFR,.35S::IhDFR,.35S::VwF3′5′H

Plants.with.all.three.genes.have.bluish.flowers

[150]

Gerbera hybrida Red Antisense.CHS,.DFR Pink [9]

Lotus japonicus Pale.yellow 35S::crtW Bright.yellow,.bright.orange

[29]

Torenia hybrida Purple-blue Sense.CHS White [151]

Purple-blue Sense.DFR White.patterning [151]

Purple-blue Sense.F3′5′H Pink [151]

Purple-blue Sense.F3′5′H Dark.pink [152]

35S::.F3′HPurple-blue Coexpression.of.

Am4′CGT + AmAS1.and.RNAi.DFR

Yellow.flowers [14]


In nonlegumes,.THC.is.quickly.converted.to.naringenin.by.chalcone.isomerase,.which.closes.the.central.ring.of.THC.leading.to.synthesis.of.flavanones..However,.in.legumes,.such.as.Medicago truncatula and Glycine max,. chalcone. reductase. (CHR). reduces.THC.to.6′-deoxychalcone.11.Transgenic.petunias.expressing.Medicago CHR.in.a.white.flowering.background.accumulate.novel.6′-deoxychalcones,.have.lower.levels.of.flavo-nols,.and.exhibit.a.pale.yellow.corolla.color.while.a.pale.purple.color.is.observed.when.the.transgene.is.expressed.in.a.purple.petunia.background.12

Aurones.are.a.class.of.flavonoids.produced.from.chalcones.that.give.flowers.bright.fluorescent.yellow.colors..They.often.provide.nectar.guides.for.visiting.pollinators.and.have.a.more.limited.species.distribution.in.plants.13,14.In.Antirrhinum,.the.chal-cone.THC.undergoes.glucosylation.and.oxidative.cyclization.to.produce.aurones.15.Yellow.Torenia hybrida.flowers.were.created.from.a.line.normally.producing.blue.flowers.by.silencing.native.dihydroflavonol.4-reductase.(DFR).expression.and.intro-ducing.two.Antirrhinum.genes.for.aurone.biosynthesis.14.By.blocking.anthocyanin.accumulation. and. expressing. the. Antirrhinum. aureusidin. synthase. and. chalcone.4′-O-glucosyltransferase.genes,.aurones.accumulated.and.were.not.masked.by. the.blue.malvidin-type.anthocyanins.normally.accumulating.in.the.Summer.Wave.Blue.Torenia.cultivar.

AnthocyaninsAnthocyanins.give.flowers.red,.orange,.blue,.and.violet.hues..They.are.synthesized.in.the.cytosol.from.flavonones.and.accumulate.in.the.vacuole.where.molecular.inter-actions,. vacuolar. pH,. ion. cofactors,. and. cellular. shape. all. contribute. to. the. final.visual.effect..There.are.six.main.groups.of.anthocyanins:.cyanidin,.delphinidin,.mal-vidin,.pelargonidin,.peonidin,.and.petunidin..Their.synthesis.begins.with.the.pro-duction.of.dihydroflavonols.when.hydroxyl.groups.are.positioned.on.the.flavonones.by.flavanone.3-hydroxylase.(F3H),.flavonone.3′-hydroxylase.(F3′H),.and.flavanone.3′5′-hydroxylase.(F3′5′H).(Figure.3.1)..Dihydroflavonols.are.reduced.to.leucoantho-cyanidins.by.DFR..DFR.substrate.preference. is.variable.in.different.plant.species.and.represents.a.critical.entry.point.in.color.production.

Lack. of. F3′5′H. activity. is. associated. with. the. absence. of. blue. coloration. in.many.families.of.flowering.plants,.including.carnation.(reviewed.by.Chandler.and.Tanaka16)..Blue.color.has.been. introduced. into.carnation. through. introduction.of.petunia.F3′5′H.and.DFR.genes.into.a.white.flowered.background.lacking.DFR.and.F3′H.activity.16.Transgenic.carnations.expressing.both.of. these.genes.accumulate.the.delphinidin.type.anthocyanins.17.These.blue-violet.transgenic.carnations,.mar-keted.as.the.Moon.series,.were.developed.by.Florigene.Pty..Ltd..and.Suntory.Ltd..and.are.the.first.and.only.transgenic.ornamental.to.date.that.has.been.successfully.commercialized.. While. F3′H. and. F3′5′H. activities. are. important. for. blue. color.other.factors.including.co-pigments.and.coordinating.metal.ions.are.also.key.fac-tors. in. determining. blue. color.. Cyanidin. type. anthocyanins. are. packaged. into. a.supermolecular.complex.to.give.blue.color.to.flowers.of.blue.cornflower.while.the.same.anthocyanins.give.red.color.to.rose.18.The.brilliant.blue.color. in.cornflower.is.attributed.to.a.tetranuclear.complex.of.cyanidin.anthocyanin,.a.flavone,.and.che-lating. Fe3+,. Mg2+,. and. Ca2+. ions.. An. understanding. of. the. genetics. contributing.


to the formation.of.this.type.of.complex.is.of.interest.for.engineering.blue.color.in.flowers.but.has.yet.to.be.elucidated.

A.novel.orange.color.was.first.introduced.into.Petunia.by.incorporating.a.maize.DFR.gene.into.the.genome.of.a.variety.lacking.F3′H.and.F3′5′H.activity.19.The.back-ground. chosen. for. introducing. the. maize. DFR. was. important. as. petunias. lacking.F3′H.and.F3′5′H.activity.accumulate.the.dihydroflavonol.dihydrokaempferol.since.the.Petunia.DFR.does.not.accept.dihydrokaempferol.as.a.substrate.for.leucoanthocyanidin.synthesis..The.maize.DFR,.which.uses.the.substrate.dihydrokaempferol,.opened.up.a.

O

CoASOH

O

OH OHHO

OH

O

O

OH

HO

OH

O

OHOH

OOH

HOO

OHOH

OOH

HO OH

O

OHOH

OOH

HO

OH

OOH

OH

OOH

HO OHOH

O

O

OH

HO

OH

OH

OOH

OHOH

OH

HO

OH

OH

OOH

OHOH

HO

OH

OOH

OHOH

HO

OH

OH

OOH

OH

OHOH

HO

O

OH

OH

OHOH

HO

OH

OOH

OHOH

HO OOH

OHOH

HO

OH

O

OH

OH

HO

OH

Tetrahydroxychalcone

p-Coumaroyl CoA

Pentahydroxyavanone Naringenin

Dihydromyricetin Dihydroquercetin

Leucodelphinidin

Delphinidin Pelargonidin Cyanidin Luteolinidin

+ 3 Malonyl-CoA

CHS

CHI

F3’H

F3’HF3’5’H

F3’5’H

DFR DFR DFR DFR

LDOX/ANS LDOX/ANS LDOX/ANS

F3H F3H F3H

AB

56

87 5’

4’3’

Chalcones

Flavanones

Dihydro avonols

Leucoanthocyanidins

Anthocyanidins

C

Eriodictyol

LuteoforolLeucocyanidinLeucopelargonidin

Dihydrokaempferol

FIGURE 3.1 Key. steps. of. anthocyanin. pigment. biosynthesis. in. plants.. CHS,. chalcone.synthase;. CHI,. chalcone. isomerase;. F3′5′H,. flavanone. 3′5′-hydroxylase;. F3′H,. flavanone.3′-hydroxylase;. F3H,. flavanone. 3-hydroxylase;. DFR,. dihydroflavonol. 4-reductase;. LDOX/ANS,.leucoanthocyanidin.dioxygenase/anthocyanidin.synthase..(Reprinted.from.Grotewold,.E.,.Annu. Rev. Plant Biol.,.57,.761,.2006..With.permission..©.2006.by.Annual.Reviews.www.annualreviews.org)


pathway.for.orange.pelargonidin.anthocyanin.production..However,.a.commercially.acceptable,.stable.color.was.not.obtained.in.the.initial.report.20.Commercially.accept-able,.stable,.improved.orange.colors.were.achieved.through.introgression.of.the.maize.DFR.into.elite.breeding.lines.accumulating.cyanidin,.peonidin,.delphinidin,.petuni-din,.and.malvidin.anthocyanins.and.three.generations.of.selfing.the.F1.crosses.21

carotenoIdS

Carotenoids.are.plastid.synthesized,.lipid.soluble,.isoprenoid-derived.C40.compounds.essential.to.all.photosynthetic.organisms..Carotenoids.are.structurally.diverse.with.over.700.structures.identified..They.function.in.light.harvesting,.energy.transfer,.and.photoprotection..They.also.provide.pink,.red,.orange,.and.yellow.color.to.flowers.and.fruits,.are.precursors.of.plant.hormones.and.fragrance.compounds,.and.have.signifi-cant.nutritional.value.in.animal.diets.(reviewed.by.Lu.and.Li22).

Carotenoid.synthesis.is.initiated.by.the.condensation.of.two.molecules.of.gera-nylgeranyl. pyrophosphate. (GPP). by. phytoene. synthase. (PSY). to. form. colorless.phytoene..Phytoene.is.converted.to.all.trans.lycopene.through.four.desaturation.reac-tions.catalyzed.by.phytoene.desaturase.(PDS).and.ζ-carotene.desaturase.(ZDS).and.an.isomerization.reaction.catalyzed.by.carotenoid.isomerase.(CRTISO)..The.desatu-ration.reactions.introduce.conjugated.double.bonds,.leading.to.a.shift.in.the.absorp-tion.spectra.that.causes.color.to.appear..Lycopene.represents.a.branch.point.in.the.pathway,.where.either.(1).δ-carotene,.α-carotene,.and.ultimately.lutein.are.produced.through. the.action.of. lycopene.ε-cyclase,. lycopene.β-cyclase,.and.carotenoid.ring.hydroxylases. or. (2). γ-carotene,. β-carotene,. and. zeaxanthin. are. produced. through.the. action. of. lycopene. β-cyclase. and. carotenoid. β-ring. hydroxylases. (Figure.3.2).(reviewed.by.Hirschberg23)..Naturally.occurring.mutants. for.many.of. these.carot-enoid.biosynthetic.genes.exist. in. tomato,.and. fruits. from. these. lines. show.visible.differences.in.color.24.For.example,.the.tomato.R.mutant.has.yellowish-green.fruit.due.to.a.mutation.in.the.phytoene.synthase.gene.25.Mutants.with.ectopic.expression.of.a.lycopene.β-cyclase.have.orange.fruit,.accumulating.high.levels.of.β-carotene.and.less.lycopene.than.control.plants.26

Most.of.the.research.on.engineering.carotenoid.synthesis.in.transgenic.plants.has.focused.on.the.alteration.of.nutritional.characteristics.in.food.plants..For.example,.β-carotene.accumulating.“golden”.rice.varieties.were.developed.by.introducing.daf-fodil.or.maize.phytoene synthase. (Psy).and.a.bacterial.phytoene desaturase (crtl).gene. into. the. rice.genome.27,28.Although. these.plants.may.be.valuable. for.helping.solve. worldwide. vitamin. A. deficiency,. they. also. demonstrate. the. ability. to. drive.heterologous.expression.of.yellow.color.in.plants.

By.transferring.bacterial.genes.for.ketocarotenoid.synthesis,.deep.yellow,.orange,.and. red. colors. have. been. introduced. into. plants. normally. displaying. pale. yellow.colors. (Table.3.1)..Bright.orange.nectaries.were. created. in.Nicotiana. by. transfor-mation. with. the. alga. Haematococcus pluvialis. β-carotene. ketolase. gene,. causing.accumulation.of.astaxanthin.in.nectary.tissue..This.application.has.more.of.a.neu-traceutical.value.since.astaxanthin.has.many.uses.related.to.animal.health..While.nectaries.generally.do.not.contribute.much.to.the.overall.aesthetic.appeal.of.a.plant,.it. demonstrates. that. novel. carotenoids. can.be. expressed. in.plants. to. introduce.


new colors.. Indeed,. bright. yellow. and. orange. Lotus. flowers. were. observed. in.transgenic. plants. transformed. with. the. Agrobacterium aurantiacum. β-carotene.ketolase. (crtW). gene.29. The. CrtW. enzyme. accepts. β-carotene,. β-cryptoxanthin,.and.zeaxanthin.substrates.for.production.of.ketocarotenoids,.which.are.compounds.found.in.bacteria,.fungi,.algae,.and.only.a.few.plant.species..Transgenic.lotus.plants.expressing.crtW.had.altered.carotenoid.profiles.with.orange.lines.exhibiting.a.1.5-fold.increase.in.total.carotenoids.and.ketocarotenoids.making.up.about.one-quarter.of.the.total.carotenoid.content.

Flower. color. range. can. also. be. extended. through. modification. of. carotenoid.breakdown.pathways.(Table.3.1)..Oxidative.breakdown.of.carotenoids.is.catalyzed.by.a.family.of.carotenoid.cleavage.dioxygenases.(CCDs).found.in.plants,.animals,.and.bacteria..The.carotenoid.breakdown.products,.or.apocarotenoids,.are.biologi-cally. important. molecules. functioning. as. hormones,. flavor. and. fragrance. com-pounds,.and.defense.molecules.(reviewed.by.Auldridge.et.al.30)..There.are.multiple.lines.of.evidence.that.support.a.role.for.CCDs.in.carotenoid.turnover..Arabidopsis.ccd1-1.loss.of..function.mutants.have.increased.seed.carotenoid.content.31.Expression.

OH

HO

OH

HOOH

HOO

O

OH

HOOH

O

O P PP P O

GGDP

GGDP

Phytoene

Lycopene

Zeaxanthin

Violaxanthin

Neoxanthin

Lutein

ζ-Carotene

δ-Carotene

α-Carotene

γ-Carotene

β-CaroteneCrtR-eCrtR-b

CrtR-b

aba2

?

CrtL-b

CrtL-b

Psy (crtB)

Pds (crtP)

Zds (crtQ)

CrtL-b

CrtL-e

FIGURE 3.2 Carotenoid.biosynthesis. in.plants..GGDP,.geranylgeranyl.diphosphate;.Psy,.phytoene.synthase;.Pds,.phytoene.desaturase;.Zds,.ζ-carotene.desaturase;.CrtL-e,.lycopene.ε-cyclase;.CrtL-b,.lycopene.β-cyclase;.aba2,.zeaxanthin.epoxidase..(From.Ronen,.G..et.al.,.Plant J.,.17,.341,.1999..With.permission.)


of. the.carotenoid.cleavage.dioxygenase.CCD4.was.demonstrated. to.give.chrysan-themum. flowers. white. color. instead. of. yellow.32. To. date,. this. area. of. research. is.under-explored.and.could.have.considerable.potential.for.development.of.new.color.phenotypes.. Manipulating. CCD. expression. also. opens. up. the. exciting. possibili-ties.of engineering.both.color.and.fragrance.since.many.of.the.apocarotenoids.are..important.floral.fragrance.volatile.compounds.33,34

PLANT VOLATILES

Thousands.of.plant.volatiles.have.been.identified.and.are.generally.classified. into.four.major.groups.including.terpenes,.phenylpropanoids,.fatty.acid.derivatives,.and.amino. acid. derivatives. (reviewed. by. Dudareva. and. Pichersky35).. Only. subsets. of.the.thousands.of.plant.volatiles.identified.are.found.in.any.given.species,.thus.plant.volatiles.are.generally.thought.of.as.secondary.metabolites.with.their.synthesis.being.highly.intertwined.with.primary.metabolism.(reviewed.by.Pichersky.et.al.36)..Plant.volatile. production. occurs. in. many. plant. tissues. including. leaves,. flowers,. fruits,.and.roots.with.functions.relating.to.pollinator.attraction,.seed.dispersal,.inter-.and.intra-plant. communication,. and. defense.. Many. genes. involved. with. plant. volatile.synthesis.have.been.cloned.and.manipulated.in.transgenic.plants.resulting.in.altered.volatile.profiles..These.studies.demonstrate.that.plant.volatile.profiles.can.be.geneti-cally.engineered.and.will.be.useful.in.improving.floral.fragrance.and.plant.defense.

floral fraGrance

Floral.fragrance.has.been.a.source.of.pleasure.and.reverence.in.human.culture.where.fragrant.flowers.have.had.places.in.religious.ceremonies.and.societal.customs.since.ancient.times..Modern.technology.has.allowed.flower.fragrance.profiles.to.be.chemi-cally.copied.for.use.in.cosmetics,.perfumes,.home.fragrances,.aromatherapy.items,.cleaning.products,.and.detergents,. to.name.a. few.popular.uses..Many.of. the.com-pounds.found.in.floral.fragrance.are.documented.to.have.positive.effects.on.human.behavior.and.therefore.could.be.used.as.tools.for.improving.the.health.and.welfare.of.humanity.37–39.There.are.minimal.publicly.available.marketing.research.data.that.investigate.the.value.added.by.making.new.flower.varieties.with.enhanced.fragrance..One.marketing.report.on.Anthurium.suggests.that.about.60%.of.consumers.agreed.fragrance.would. add.value. to. the.product.40.Floraculture. International. reported. in.November. 2008. that. the. demand. for. fragrant. roses. was. increasing. but. the. num-ber. of. “fragrant”. roses. available. is. minimal. (http://www.floracultureinternational.com/index.php?option=com_content&task=view&id=125&Itemid=159&ed=25)..Fragrance.has.historically.not.been.the.focus.of.ornamental.plant.breeding.programs.and. flowers. have. lost. their. characteristic. fragrance. since. breeders. are. primarily.concentrating.on.flower.color.and.longevity,.plant.growth.habit,.disease.resistance,.among.other.genetic.factors..Genetic.engineering.could.help.overcome.the.hurdles.of.reintroducing.this.trait.into.crops.where.there.is.demand.for.fragrant.flowers.and.would.also.provide.testing.material.for.determining.the.real.market.value.of.this.trait.

Over.the.past.decade,.our.understanding.of.the.biochemistry.and.physiology.of.floral.scent.has.progressed.rapidly..Floral.scent.is.a.highly.variable,.complex.mixture.


of. low.molecular.weight,.volatile.organic.compounds. that.attracts. insect.and.ani-mal.pollinators..Hundreds,.perhaps.thousands,.of.different.volatile.compounds.are.emitted.from.flowers.in.varying.levels.and.mixtures.41.In.many.species,. including.Antirrhinum,.Petunia,. and.Rosa,. petals. are. the.primary. site.of.floral. scent. emis-sion42–44.with.fragrance.compounds.emitted.from.both.the.adaxial.and.abaxial.petal.epidermal.layers.of.Rosa.x.hybrida.‘Anna’.44.Fragrance.is.a.dynamic.trait,.with.quan-titative.and.qualitative.changes.occurring.during.flower.development,.in.response.to.pollination,.through.day–night.cycles,.and.in.response.to.the.environment.(reviewed.by.Clark.et.al.45).

Petunia. x.hybrida. has.emerged.as.a.popular.model. system.for.characterizing.floral.volatile.benzenoid/phenylpropanoid.synthesis.(Table.3.2).(reviewed.by.Clark.et. al.45).. The. floral. perfume. of. Petunia. cultivar. ‘Mitchell’. is. dominated. by. ben-zyl. alcohol,. benzaldehyde,. methylbenzoate,. benzylbenzoate,. phenylacetaldehyde,.2-phenylethanol,.phenylethylacetate,.phenethylbenzoate,. isoeugenol,.and.eugenol,.

TABLE 3.2Published Reports of Genetically Engineered Plants with Modified Floral Fragrance

Plant Genetic Modification Effect on Floral Volatiles References

Petunia.x.hybrida 35S::CbLIS Accumulation.of.nonvolatile.linalool.conjugate

[153]

Petunia.CCD1.RNAi Reduced.β-ionone [33]

35S::SAAT No.effect [55]

Petunia.BSMT.RNAi Reduced.methylbenzoate [53]

Petunia.ODO1.RNAi Reduced.volatile.benzenoids [47]

Petunia.PAAS.RNAi Reduced.phenylacetaldehyde [50]

Reduced.2-phenylethanol

Petunia.BPBT.RNAi Reduced.benzylbenzoate [51,52]

Reduced.phenethylbenzoate

35S::RhAAT Higher.benzyl.acetate [154]

Higher.phenethyl.acetate

Petunia.CFAT.RNAi Reduced.isoeugenol [49]

35S::LePAR Reduced.phenylacetaldehyde [54]

Higher.2-phenylethanol

35S::AtPAP Higher.benzaldehyde [60]

Higher.phenylacetaldehyde

Higher.benzaldehyde.4-hydroxy

Higher.benzaldehyde.3,4-dimethoxy

Higher.vanillin

Dianthus caryophyllus 35S::CbLIS Linalool.production [57]

35S::antisenseF3H Higher.methyl.benzoate [58]

Eustoma grandiflorum 35S::CbBEAT No.effect [155]

Nicotiana tabacum 35S::ClTER,.35S::ClLIM,.35S::ClPIN

Plants.with.all.three.genes.emit.β-pinene,.limonene,.γ-terpinene,.and.side.products

[153]


which.are.compounds.derived.from.the.amino.acid.phenylalanine..Small.amounts.of. the. apocarotenoids. β-ionone. and. 6-methyl-5-hepten-2-one. are. also. detectable.in. the. fragrance.profile.33,46. Many.of. these. compounds. have. been. removed. from.the.petunia.volatile.profile.through.silencing.of.regulatory.and.biosynthetic.genes.(Table.3.2)..A.reduction.in.multiple.benzenoid.compounds.was.achieved.by.silenc-ing.odorant1. (ODO1),. a. transcription. factor. that. regulates. transcription.of.genes.important.for.supplying.substrates.for.floral.volatile.production.47.β-Ionone.emis-sion.was. reduced.by.approximately.50%. in. transgenic.petunias.with.RNA.inter-ference. (RNAi)-suppressed. CCD1.33. While. emission. of. β-ionone. is. low,. only.detectable. in. picogram. quantities,. the. published. human. odor. threshold. is. low.(0.007.nL.L−1).compared.to.other.more.abundant.volatiles.in.petunia.33,48.Therefore,.β-ionone.could.potentially.be. important. in. the.overall. fragrance.of.petunia..The.clove-like.component.of.petunia.scent.was.removed.from.the.fragrance.profile.by.suppression.of.coniferyl alcohol acyltransferase.(CFAT).expression.49.CFAT.cata-lyzes.the.acetylation.of.coniferyl.alcohol.to.form.coniferyl.acetate,.the.precursor.of.isoeugenol.and.eugenol..The.two.volatile.compounds.imparting.rosy,.floral.notes.to.petunia.fragrance.were.reduced.by.silencing.phenylacetaldehyde synthase (PAAS)..Transgenic. RNAi. suppressed. PAAS. plants. had. reduced. phenylacetaldehyde. and.2-phenylethanol.levels.50.Dexter.et.al.51.and.Orlova.et.al.52.removed.benzyl.benzoate.and.phenethylbenzoate.from.the.petunia.fragrance.by.RNAi-induced.silencing.of.benzyl CoA:benzyl alcohol/phenylethanol benzoyl transferase. (BPBT)..Silencing.of. benzoic acid/salicylic acid carboxyl methyltransferase1. and. 2. (BSMT1. and.BSMT2). led. to. a. reduction. in. methylbenzoate,. the. most. predominant. volatile. of.petunia. fragrance.. Flowers. lacking. normal. methylbenzoate. levels. had. a. distinct,.detectable.change.in.fragrance,.and.80%.of.human.olfactory.panelists.were.able.to.distinguish.them.from.control.flowers.53

Increasing. flux. through. existing. biosynthetic. pathways. and. introducing. new.fragrance.pathways.are.a.means.to.make.novel. fragrance.profiles. (Table.3.2)..For.example,.petunia.flowers.that.smell.more.like.a.rose.could.be.developed.by.increas-ing.2-phenylethanol.synthesis.and.decreasing.levels.of.endogenous.volatiles,.which.may.mask.the.perception.of.rose.aroma..Petunias.that.produced.more.intense.rose-like. 2-phenylethanol. were. developed. by. introducing. tomato. phenylacetaldehyde reductase.(LePAR).into.the.petunia.genome.54.Introduction.of.a.strawberry.alcohol.acyltransferase.(SAAT).gene.into.petunia.did.not.affect.the.volatile.profile.55.Feeding.isoamyl.alcohol,.a.substrate.of.SAAT,.to.the.transgenic.tissue.resulted.in.emission.of.acetyl.ester.products..Lücker.et.al.56. introduced. the.Clarkia breweri S-linalool synthase.gene.into.petunia..However,.plants.did.not.produce.linalool.and.instead.accumulated. a. nonvolatile. conjugated. form.. Linalool. was. detected. in. transgenic.CbLIS Dianthus caryophyllus.cultivar.‘Eilat’.plants.57.While.these.plants.produced.linalool. and. derivatives. thereof,. the. transgenic. flowers. did. not. smell. differently.from.control.flowers.in.human.olfaction.tests..It.will.be.of.interest.to.introduce.the.LIS.gene.into.a.different.background.of.carnation.that.has.less.fragrance.since.it.is.possible.that.there.was.masking.by.other.volatile.compounds.

The.reports.discussed.in.this.section.unequivocally.demonstrate.that.floral.fra-grance.can.be.engineered..A.number.of.important.issues.can.be.highlighted,.includ-ing. (1).altering. fragrance.profiles.with.heterologous.genes. is.possible.and.will.be.


useful,. (2). substrate. availability. needs. to.be. considered. when.altering. volatile.pathways,.and.(3).endogenous.activities.can.convert.products.to.nonvolatile.forms..The.opportunities.for.significant.increases.in.floral.volatile.production.most.likely.lie.with.controlled.expression.of.genes.early.in.the.biosynthetic.pathways.and.regulatory.factors.controlling.gene.expression.of.several.biosynthetic.pathways.

Fragrance and ColorFragrance.benzenoids.and.anthocyanin.color.metabolites.are.produced.from.phen-ylpropanoid.metabolism..Therefore,.altering.fragrance.metabolism.could.influence.the. production. of. color. and. vice. versa.. While. there. are. no. consistent. trends. for.direct. relationships. between. color. and. fragrance,. there. is. evidence. that. this. does.occur. in. carnation.. White. flowers. from. antisense. F3H. carnation. plants. produced.higher.amounts.of.benzenoids.compared.to.the.red-orange.control.flowers.58.These.transgenic.flowers.produced.five-.to.sevenfold.higher.levels.of.methyl.benzoate.and.2-hydroxy.methyl.benzoate..Thus,. in.carnation,. loss.of.F3H.function.may. reduce.flux.through.anthocyanin.synthesis.causing.phenylpropanoid.metabolism.to.be.redi-rected.toward.benzenoid.production.

Interplay.between.color.and.fragrance.production.is.less.clear.in.petunia..Vivid.pink.color.was.introduced.into.a.white.flowered,.nocturnally.fragrant.Petunia axil-laris. background. by. transformation. with. the. AN2. flavonoid. biosynthesis. regula-tor.59.The.transgenic.colored.flowers.did.not.exhibit.significant.changes.in.benzenoid.volatile.production..Likewise,.colored.petunias.with.reduced.expression.of.ODO1,.a.transcription.factor.thought.to.control.substrate.flux.to.benzenoid.fragrance.produc-tion. in.petunia,. did.not. exhibit. any.visible.differences. in. pigmentation. compared.to.controls.47.Recently,.Ben.Zvi.et.al.60.showed.that.heterologous.expression.of.the.Arabidopsis thaliana PAP1. Myb. transcription. factor. in. Petunia hybrida. cultivar.‘Blue.Spark’. led. to. increased.flower.pigmentation. and. increased. levels.of.certain.fragrance.volatiles..There.were. three. to.five. times.more.benzaldehyde.and.higher.internal. pools. of. phenylacetaldehyde,. benzyaldehyde. 4-hydroxy,. benzaldehyde.3,4-dimethoxy,.and.vanillin.60.The.mechanism.of.how.PAP1.is.regulating.these.two.processes.in.the.‘Blue.Spark’.petunias.remains.to.be.elucidated.

volatIleS and InSect plant defenSe

Plants.produce.a.myriad.of.volatiles.in.response.to.herbivores.that.serve.to.intoxicate,.attract.predators.and.parasitoids,.and/or.elicit.an.avoidance.response. in. the. insect.inflicting.damage.to.the.plant..There.are.only.a.handful.of.documented.examples.of.engineering.of.plant.defense-related.volatiles..However,.as.this.will.likely.be.a.useful.method.in.the.future.for.improving.plant.defense.in.both.agronomic.and.ornamental.crops,.a.few.examples.in.model.systems.will.be.highlighted.here..Increased.para-sitoid.attraction.and.increased.pest.repellant.properties.have.been.achieved.through.introducing.single.genes.into.Arabidopsis..For.example,.increased.aphid.parasitoid.attraction.and.aphid.repellant.behavior.was.induced.in.Arabidopsis.plants.expressing.(E)-β-farnesene.(Eβf).synthase.cloned.from.Mentha.x.piperita.61.The.Eβf.express-ing.plants.emitted.high.levels.of.Eβf,.an.aphid.alarm.pheromone..Arabidopsis.plants.overexpressing. a. strawberry. linalool/nerolidol. synthase. (FaNES1). gene. produced.


high.levels.of.linalool,.which.was.a.repellant.to.aphids.62.By.targeting.FaNES1.to.the.mitochondria. instead.of. the. cytosol,. synthesis.of.4,8-dimethyl-1,3(E),7-nonatriene.((E)-DMNT).and.nerolidol.was. induced.making. transgenic.plants.more.attractive.to.predatory.mites.63.Both.of. these.studies.with.FaNES1.report.a.growth.retarda-tion.phenotype.that.could.be.a.phytotoxic.effect.of.the.new.volatiles.being.produced.or.the.new.pathway.causing.a.metabolic.drain.on.metabolites.essential.for.normal.growth.and.development..In.the.case.of.the.latter,.fine.tuning.expression.through.use.of.promoters.conferring.lower.or.more.specific.transgene.expression.may.be.useful.for. balancing.metabolite. flow. between. the.newly. introduced.pathway. and.endog-enous.pathways.

FLOWER AND LEAF LONGEVITY

Retention.of.high.quality.flowers.and.leaves.for.visual.appearance.is.of.utmost.impor-tance.to.the.ornamental.plant.industry..Flowers.that.are.short-lived.or.highly.sensi-tive.to.postharvest.handling.conditions.are.limited.in.their.marketability..Similarly,.leaf.yellowing.or.senescence.negatively.affects.the.consumer.appeal.of.potted.plants.and. bedding. plants.. Plant. and. cut. flower. quality. has. been. improved. through. the.use.of.proper.postharvest.handling.procedures.and.chemical.preservatives..Applying.transgenic.technology.to.ornamentals.to.improve.their.postharvest.attributes.presents.the.opportunity.for.saving.on.postharvest.costs.associated.with.labor,.chemical,.and.environmental.control,.contributing.to.environmentally.sustainable.practices..With.such.great.importance,.there.are.now.many.examples.where.genetic.engineering.has.been.successfully.employed.to.obtain.flowers.with.longer.vase.life.and.plants.with.leaves.that.stay.green.

floral lonGevIty

The. plant. hormone. ethylene. negatively. affects. flower. longevity. in. many. orna-mentally. important. plant. species.64,65. This. gaseous. phytohormone. is. produced. in.response.to.pollination,.stress,.and.common.postharvest.handling.procedures.caus-ing.petal. in-rolling,.abscission,.and.senescence. in.many.species.of.monocots.and.dicots,.ultimately.eliminating.their.marketability..Van.Doorn.et.al.66.surveyed.over.300.species.of.flowering.plants.from.50.different.families.and.found.that.over.64%.of.these.were.highly.ethylene.sensitive..To.address.this.issue,.the.molecular.genetics.and.biochemistry.of.ethylene.synthesis.and.perception.has.been.the.focus.of.much.research. and. chemical. and. genetic. methods. for. altering. ethylene. responses. have.been.developed.(Table.3.3).

Flower.longevity.can.be.extended.through.the.use.of.chemicals.that.inhibit.ethyl-ene.biosynthesis.and.perception..Chemicals.such.as.aminoethoxyacetic.acid.(AOA).and.aminoethoxyvinylglycine. (AVG). significantly. reduce.ethylene.production. and.delay.petal. senescence.and.abscission.of.many.species.67–70.However,. the.concern.of. potential. toxic. effects. and. lack. of. control.of. effects. due. to. ethylene. generated.from.outside.sources.halted.their.commercial.use.67.Therefore,. inhibiting.ethylene.perception.is.more.desirable.for.preventing.effects.of.exogenous.ethylene.that.may.occur. during. storage. and. transportation.. Silver. thiosulfate,. Ag2S2O3. or. STS,. has.


become. widely. used. for. blocking. ethylene. effects. in. the. ornamental. industry. for.several.decades.67,71–73.Unfortunately,.STS.is.a.heavy.metal.pollutant,.and.in.recent.years.commercial.use.has.become.more.restricted.because.of.environmental.con-cerns..1-Methylcyclopropene.(1-MCP).is.a.more.recently.developed.chemical.found.to.block.ethylene.perception74.and.has.been.commercialized.and.is.effective.in.delay-ing.flower.senescence.in.many.ornamentals.(reviewed.by.Sisler.and.Serek75).

Altering Ethylene Responses Genetically by Inhibiting Ethylene BiosynthesisThe.biosynthetic.pathway.of.ethylene.has.been.elucidated76.and.ethylene.biosynthetic.genes.have.been.altered.in.transgenic.ornamental.plants.to.reduce.the.effects.of.eth-ylene..Ethylene.biosynthesis.is.a.two-step.process.beginning.with.the.conversion.of.S-adenosylmethionine.(SAM).to.1-aminocyclopropane-1-carboxylic.acid.(ACC).by.the.enzyme.ACC.synthase.(ACS)..ACC.is.then.converted.to.ethylene.by.ACC.oxi-dase.(ACO)..Reducing.expression.of.either.of.these.enzymes.through.RNAi.or.cosup-pression.technology.decreases.ethylene.production.and.extends.vase.life..Carnation.plants.with.silenced.ACS.or.ACO.expression.produce.less.ethylene.from.flowers.and.

TABLE 3.3Publications Documenting Extended Flower Life through Genetic Engineering Ethylene Biosynthesis or Signaling

PlantGenetic

Modification Effect References

Petunia.x.hybrida 35S::Atetr1-1 Flowers.lasted.∼4×.longer.than.controls

[87]

35S::sense.PhEIN2 Flowers.lasted.∼4×.longer.than.controls

[88]

RNAi.PhEIN2 Flowers.lasted.∼6×.longer.than.controls

[88]

Dianthus caryophyllus Antisense.ACO ∼1.7×.longer.vase.life [77]

Sense.ACO ∼2×.longer.vase.life [78]

Antisense.and.sense.suppression.of.ACS

∼2×.longer.vase.life [79]

PhFbp1::Atetr1-1 ∼2.7×.longer.vase.life [89]

35S::Atetr1-1 ∼2–3×.longer.vase.life [89]

Etr1::etr1-1 ∼2–2.4×.longer.vase.life [89]

DcCmb2::Atetr1-1 ∼2×.longer.vase.life [80]

Torenia fournieri Antisense.and.sense.suppression.of.ACO

Sense.flowers.lasted.0–3.5× longer.than.controls

[81]

Nemesia strumosa 35S::Cmetr1 Flowers.lasted.1–3.days.longer.than.controls

[90]

Campanula carpatica Phfbp1::Atetr1-1 Variable..Lines.ranging.between.0–6×.longer.than.control.flowers

[92]

Kalanchoe blossfeldiana Phfpb1::Atetr1-1 Variable..10×.longer.flower.retention.for.40%–50%.of.transgenic.flowers.in.two.lines

[93]


have.extended.vase.life.77–80.ACO.cosuppressed.carnation.plants.had.approximately.a. twofold. longer. vase. life. than. control. flowers.78. Suppression. of. ACO. in. Torenia fournieri.extended.flower.life.up.to.3.5-fold.longer.than.controls.81.While.these.flow-ers.produce.less.ethylene.and.have.longer.vase.life,.the.flowers.are.still.sensitive.to.exogenous.sources.of.ethylene..Thus,.much.of. the.focus.on.engineering-improved.flower.life.has.centered.on.manipulations.of.ethylene.receptor.and.signaling.proteins.

Inhibiting Ethylene Responses by Disrupting Ethylene Signal TransductionEthylene.is.perceived.by.plants.through.transmembrane.receptor.proteins,.which.are.encoded.by.the.ethylene resistant.(ETR).gene.family,.typically.consisting.of.five.or.more.genes.in.plants..The.receptors.have.been.identified.in.expressed.sequence.tag.(EST).collections.from.more.than.20.plant.species,.including.monocots,.dicots,.and.lower.vascular.plants.82.Binding.of.the.hormone.has.been.demonstrated.in.cyanobac-teria,.fungi,.green.alga,.lower.vascular.plants,.and.angiosperms.83,84.While.studies.of.receptor.function.at.the.molecular.level.have.not.been.carried.out.in.many.plant.spe-cies.outside.of.Arabidopsis.and.tomato,.the.proteins.are.highly.conserved.in.regions.that.are.functionally.important.82,84.It.is.therefore.likely.that.the.receptors.act.in.a.similar.fashion.in.plant.species.other.than.tomato.and.Arabidopsis,.that.is,.as.nega-tive.regulators.of.ethylene.responses..In.the.absence.of.ethylene,.ethylene.responses.are.repressed,.and,.in.the.presence.of.ethylene,.repression.of.ethylene.responses.is.lifted..Arabidopsis.plants.carrying.the.etr1-1.mutation.are.almost.completely.insen-sitive.to.ethylene.85.Another.central.component.of.the.pathway,.ethylene insensitive 2.(EIN2),.is.critical.for.ethylene.signaling..Arabidopsis.EIN2.loss-of-function.mutants.are.unable.to.respond.to.ethylene.86.Both.ETR.and.EIN2.are.critical.to.plant.ethylene.responses,.making.them.good.candidates.for.manipulating.ethylene.responses.at.the.molecular.level.

Floral.longevity.has.been.extended.in.transgenic.plants.with.altered.ethylene.sig-naling..Petunias.with.longer-lasting.flowers.have.been.attained.by.overexpression.of.the.mutant.ethylene.receptor.allele,.etr1-1,.from.Arabidopsis thaliana87.and.by.sup-pressing.expression.of.EIN2.by.RNAi.or.cosuppression.88.Petunia.corollas.expressing.the.Arabidopsis.etr1-1.gene.do.not.wilt.after.pollination.or.exposure.to.ethylene.and.often.stay.attached.to.the.receptacle.until.the.developing.fruit.detaches.the.corolla,.up.to.10.days.after.pollination.compared.to.2–3.days.for.control.flowers..EIN2.RNAi.and.EIN2.cosuppression.petunia.plants.also.display.delayed.corolla.senescence.after.pollination.or.exposure.to.exogenous.ethylene..These.flowers.do.not.exhibit.visual.signs.of. senescence.until.more. than.9–12.days. after.pollination.88.Similarly,.con-stitutive.expression.of. the.Arabidopsis.etr1-1. in.carnation.doubled.the.vase.life.of.carnation.flowers.80,89.Constitutive.expression.of.a.melon.etr1-1-like.mutant.receptor.in.Nemesia strumosa.resulted.in.flowers.that.lasted.1–3.days.longer.than.controls.90

Transgenic.plants.expressing.constitutive.ethylene.insensitivity.not.only.prove.the.concept.that.genetically.disrupting.ethylene.signaling.will.extend.flower.life.but.have.also.been.useful.as.a.tool.for.learning.about.the.myriad.of.processes.that.ethylene.is.involved.with.in.a.plant.life.cycle..For.example,.35S::etr1-1.petunias.are.unable.to.be.vegetatively. propagated. due. to. an. inability. to. form. adventitious. roots91. and. exhibit.higher.mortality.than.control.plants.88.Both.of.these.issues.are.huge.horticultural.limi-tations..The.problems.likely.can.be.overcome.through.the.use.of.promoters.that.restrict.


expression.to.floral.tissues..Use.of.flower-specific.promoters.to.drive.ethylene.insensi-tivity.has.been.demonstrated,.but.exhaustive.physiological.characterization.and.horti-cultural.examination.have.not.been.documented.for.these.transgenic.lines..For.example,.flower-specific.expression.of.the.Arabidopsis.etr1-1.in.carnation.with.the.petunia.fbp1.promoter.extended.flower.longevity.to.twice.that.of.control.flowers.89.Flower-specific.expression.of. the.Arabidopsis.etr1-1.gene.with. the.petunia. fbp1.promoter.extended.floral.display.in.Campanula carpatica.92.The.petunia.fbp1.promoter.was.also.used.to.drive.Arabidopsis.etr1-1. expression. in.Kalanchoe blossfeldiana.93.While. transgenic.Kalanchoe. plants. had. a. longer. flower. display,. expression. of. the. transgene. did. not.appear.to.be.entirely.flower.specific.with.some.expression.in.root.tissue..There.was.no.consistent,.obvious.difference.in.all. transgenic.ethylene-insensitive.Kalanchoe. lines.for.shoot.dry.weight,.leaf.area,.or.root.length;.however,.exhaustive.physiological.and.horticultural.characterization.was.not.performed..These.results.demonstrate.that.trans-genic.ethylene.insensitivity.can.be.engineered.in.specific.plant.tissues.

leaf lonGevIty

Leaf.senescence.is.a.highly.ordered,.deteriorative.process.where.cellular.components.are.broken.down.and.nutrients.are.remobilized.from.dying.tissue.to.other.parts.of.the.plant..Although.this.process.involves.macromolecular.breakdown,.synthesis.of.new.proteins,. from.genes.known.as.senescence.associated.genes.(SAGs),. is. required.94.The.initiation.of.the.senescence.process.is.known.to.involve.alteration.in.the.levels.of.the.phytohormone.cytokinin.(reviewed.by.Sakakibara95)..In.addition.to.their.role.in.senescence,.cytokinins.regulate.a.number.of.developmental.processes.in.plants..These. include. promoting. cell. division,. transducing. nutritional. signals,. promoting.shoot.initiation,.and.increasing.crop.productivity96.(reviewed.by.Sakakibara95)..The.first. cytokinin.biosynthetic.gene. identified. and.characterized.was. the. isopentenyl transferase.(ipt).gene.from.Agrobacterium tumefaciens.97.The.IPT.protein.catalyzes.synthesis.of.the.cytokinin.Δ2-isopentenyladenosine.monophospate.through.a.trans-ferase. reaction. between. dimethylallylpyrophosphate. (DMAPP). and. 5′AMP.. The.gene.encoding.IPT.is.located.on.the.Ti-plasmid.of.A. tumefaciens.and.is.integrated.into.the.host.genome.after.infection..The.enzyme.is.active.in.plants;.increased.cyto-kinin.production.is.observed.in.plants.expressing.ipt.98.Since.the.Agrobacterium ipt.was.cloned.and.characterized,.cytokinin.biosynthetic.genes.have.been.isolated.from.plants.99,100.Work.to.date.has.centered.on.heterologous.use.of.the.Agrobacterium ipt.gene.in.transgenic.plants.

Increasing.cytokinin. levels. in.plants. is.a.strategy.for.delaying.leaf.senescence,.but.most.early.attempts.resulted.in.plants.with.irregular.growth.patterns.101–103.These.reports.made.use.of.modified.constitutive.promoters,.Cu2+.inducible,.or.heat.shock.promoters. for. driving. expression. of. ipt.. Gan. and. Amasino104. designed. constructs.for.auto-regulated,.senescence-specific.expression.of.ipt.using.the.SAG12.promoter.from.Arabidopsis..With.this.construct,.expression.of.ipt.is.restricted.until.the.plant.initiates.senescence..Expression.of.ipt.is.self-attenuating.as.leaf.senescence.ceases.thereby.preventing.overproduction.of.cytokinins.and.eliminating.abnormal.growth..Tobacco.plants.expressing.SAG12::ipt.had.longer.lasting.leaves.and.plants.were.mor-phologically.normal.104


Petunia.has.been.used.as.a.model.for.engineering.delayed.leaf.senescence.using.different.SAG.promoters.and.the.ipt.gene..The.SAG12::ipt.construct.was.introduced.into. ‘V26’.petunia.and.over.20. lines.were. screened.by.drought. stress. for.delayed.leaf.senescence..Drought.stress. is.a.common.problem.encountered.in.commercial.and.home.garden.environments.and.induces.the.leaf.senescence.program,.but.does.not. induce. SAG12. expression.105. Two. lines. out. of. 20. SAG12::ipt. transgenic. lines.displayed.a.normal.growth.phenotype.and.had.delayed.leaf.senescence.2,106–108.The.numbers.of.chlorotic.basal.leaves.on.transgenic.SAG12::ipt.plants.were.about.14.times.less.that.of.control.plants.2,106

To.engineer.plants.for.extended.cold.storage.tolerance,.Khodakovskaya.et.al.109.utilized.cor15a,.a.cold-inducible.promoter.from.Arabidopsis,.to.drive.ipt.expression.in. petunia. and. chrysanthemum.. During. horticultural. propagation,. plant. parts. are.commonly. stored. in. cool,. dark. environments.. These. conditions. cause. leaf. senes-cence.and.ultimately.increase.mortality.when.plants.are.in.prolonged.storage..Plants.transformed.with.cor15a::ipt.had.morphologically.normal.growth.at.a.normal.tem-perature.(25°C),.similar.to.that.of.controls..When.a.propagation.environment.was.simulated. with. cold. storage,. the. transgenic. plants. resisted. senescence. exhibiting.greener. leaves. with. elevated. cytokinin. levels. and. leaves. with. normal. chlorophyll.levels.while.control.tissue.was.visibly.senescing.

Other.approaches.for.delaying.leaf.senescence.have.been.successful.and.have.resulted. in. interesting.phenotypes.useful. for.manipulation. in.ornamental.plants..The.SAG12.promoter.was.used.to.drive.expression.of. the.maize.Knotted1.gene..KN1.is.a.homeobox.protein.involved.in.meristem.formation,.cytokinin.synthesis,.and.repression.of.senescence..Another.SAG.promoter,.SAG13,.was.used.to.drive.ipt.expression.in.petunia..Plants.expressing.SAG12::KN1.or.SAG13::ipt.exhibited.delayed.leaf.senescence.after.drought.stress.107.Compared.to.controls,.SAG13::ipt.transgenic.plants.were.more.branched,.leaves.were.thicker.and.larger,.root.growth.was. reduced,. and. flowering. was. delayed.. These. phenotypes. were. ascribed. to.“leakier”. expression. of. the. ipt. transgene.107. Nutrient. stress. experiments. showed.that.source–sink.relationships.were.altered.in.SAG13::ipt.and.SAG12::KN1. lines.as. new. growth. after. nutrient. stress. was. chlorotic. and. necrotic.107. Interestingly,.SAG13::ipt.and.SAG12::KN1.plants.also.showed.enhanced.tolerance.to.pathogens..In.controlled.greenhouse.experiments.in.which.Cercospora.was.spray-inoculated.onto.plants,.control.plants.had.sixfold.more.senescing.leaves. than.SAG13::ipt.or.SAG12::Knotted1. plants,. with. transgenic. lines. exhibiting. local. hypersensitive.responses.to.infection.107.These.experiments.highlight.the.value.of.exhaustive.hor-ticultural.evaluations.of.multiple.lines.of.transgenic.plants..Selection.of.elite.culti-vars.will.be.required,.similar.to.conventional.breeding.

PLANT ARCHITECTURE

Plants. have. evolved. to. grow. a. fascinating. array. of. floral. and. vegetative. forms.that.are. influenced.by.genetic.and.environmental. factors..Plant.height,.branching,.flower.number,.and.floral.morphology.are.just.some.of.the.readily.apparent.forms.giving. individual. plants. their. unique. architectural. characteristics.. Understanding.how.a.plant.develops.its.architectural.attributes.is.of.economic.importance.to.both.


agronomic. and. ornamental. industries. since. creating. plants. with. more. desirable.architecture.can.increase.yield,.decrease.the.amount.of.input.in.growing.a.crop,.and.improve.the.overall.aesthetic.value.of.a.plant.

plant heIGht

Plant.height.is.routinely.regulated.during.greenhouse.crop.production.through.the.use.of.chemical.growth.regulators.that.control.the.synthesis.of.gibberellic.acid.(GA),.a.phytohormone.that.promotes.stem.elongation..Ideally,.plants.would.produce.inter-nal.growth.regulators.to.decrease.labor.and.chemical.costs.and.create.a.product.that.would.be.easier. to. ship. to. the.consumer. and.easier. to.maintain. in. the. landscape..Transgenic.control.of.plant.height.through.manipulation.of.gibberellin.synthesis.has.been.proven.as.a.successful.means.for.controlling.plant.height.

Plant.height.can.be.transgenically.controlled.by.altering.signaling.of.the.phyto-hormone.GA.or.lowering.the.levels.of.endogenous.GA.in.the.plant.through.inhibit-ing.GA.synthesis.or. increasing.GA.breakdown..GAs.are. chemically.diverse.with.more. than.120.different.GAs. identified;.many.are.precursors.or.breakdown.prod-ucts.and.only.a.handful.are.biologically.active.in.plants.(reviewed.by.Yamaguchi110)..GA-induced.cell.elongation.has.been.attenuated.in.Chrysanthemum morifolium.by.using. the. Arabidopsis. gibberellic insensitive. (gai). gene.. Transgenic. gai. chrysan-themum.plants.continually.repress.GA.responses,.exhibit.dwarfism.with.transgenic.plants. growing. to. about. 30%. as. tall. as. the. control. plants.111. Several. GA. oxidase.enzymes.that.hydroxylate.GA.precursors.to.form.inactive.GA.products.have.been.identified.in.Arabidopsis.112.Two.GA2-oxidases.(AtGAox7.and.AtGAox8).hydroxyl-ate.C20-GA.precursors,.but.not.active.C19-Gas.112.Based.on.work.by.Schomburg.et.al.,112.petunias.with. reduced.plant.height.were.produced.by.overexpressing.GA.oxidase.(35S::AtGAox7.and.AtGAox8),.thereby.reducing.endogenous.levels.of.GA..Transgenic.GA.oxidase.petunias.have.a.range.of.dwarf.phenotypes.2.The.dwarf.phe-notype. can. be. rescued. by. application. of. exogenous. C19-Gas,. a. feature. useful. to.commercial.growers.as.a.means.of.elongating.cuttings.for.vegetative.propagation.2

flower morpholoGy

Over. the. past. two. decades. genes. responsible. for. floral. organ. identity. have. been.identified.and.models. for.flower.development.are.rapidly.progressing..In.general,.floral. organ. identity. is. controlled.by. the. sole. and.combinatorial. activity. of. mul-tiple.classes.of.organ. identity.genes.encoding.A,.B,.C,.D,.and.E.class. transcrip-tion.factors..The.A class.transcription.factors.are.responsible.for.sepal.formation..Interaction.of.A and.B.class.factors.specifies.petals..B.and.C.class.together.specify.stamens,.while.the.C class.gives.rise.to.carpels.113.Ovule.development.requires.D.class. genes,. possibly. in. combination. with. C. class. genes. (reviewed. by. Angenent.and. Immink114).. The. E. class. functions. in. combination. with. A,. B,. and. C. factors.to.determine. identity.of.petals,.stamens,.and.carpels. (reviewed.by.Angenent.and.Immink114).. This. mechanism. of. flower. development,. known. as. the. ABC. model,.is.generally.conserved.among.flowering.plants..Mutants.with.ectopic.or.repressed.expression.of.these.genes.have.altered.floral.form..Loss.of.function.of.PMADS3,.


an.ortholog.of. the.Arabidopsis AGAMOUS.class.C.gene,. in.petunia.gives.rise.to.flowers.with.stamens.converted.into.petal-like.structures.115

Transgenic.manipulation.of.floral.organ.identity.genes.has.led.to.a.greater.under-standing.of.the.molecular.mechanisms.underlying.flower.development.in.diverse.spe-cies,.giving.us.the.required.knowledge.for.creating.morphologically.novel.flowers.with.the.tools.of.biotechnology..Mutation.screens.have.been.the.primary.means.for.identi-fying.lines.with.altered.floral.morphology.in.many.ornamental.crops..For.example,.a.transposon.insertion.into.Duplicated.(DP),.a.C.class.gene,.caused.a.double.flower.phe-notype.in.Japanese.morning.glory.116.Although.it.is.possible.to.obtain.altered.flower.morphology.mutants,.biotechnology.will.be.useful.in.developing.lines.with.novel.flo-ral.appearance.where.mutant.populations.are.difficult.to.generate.and.for.understand-ing. the. basic. molecular. mechanisms. underlying. flower. development.. Sepals. were.converted. into. purple. colored. petal-like. organs. when. green petal,. a. class B. func-tion.gene,.was.overexpressed.in.petunia.117.Antisense.repression.of. the.chrysanthe-mum.AGAMOUS-like.gene.caused.conversion.of.pistils.to.petal.tissue.and.pistil-like..tissue.118.Interesting.changes.to.petal.floral.morphology,.such.as.changes.in.ray.floret.shape,.have.also.been.observed.when.expression.of.floral.homeotic.genes.is.altered.in.Gerbera.(e.g.,.Yu.et.al.119)..Altering.petal.position.and.number.through.manipulation.of.floral.organ.identity.genes.can.result.in.a.more.colorful,.showier.flower.and.flowers.with.new.morphology,.both.of.which.are.traits.of.interest.for.consumers.

fruIt and flower SIze

While. larger. flowers. are. often. selected. in. ornamental. breeding. programs,. only.recently.has.this.trait.been.modified.through.biotechnology..As.mentioned.in.Leaf.Longevity.section,.cytokinins.are.a.class.of.phytohormones.that.promote.cell.divi-sion;.therefore,.increasing.cytokinin.biosynthesis.is.a.strategy.toward.increasing.cell.numbers.in.target.tissues..This.was.achieved.by.using.the.Arabidopsis thaliana AP3.promoter.to.drive.flower-specific.expression.of.the.Agrobacterium tumefaciens ipt..cytokinin.biosynthetic.gene.in. transgenic.petunia.120.AP3. is.a.class.B.floral.organ.identity. gene. specifying. petal. and. stamen. identity.. The. AP3. promoter. restricts.expression.to.the.petals.and.stamens.in.Arabidopsis121.and.to.floral.organs.in.petu-nia.120.Five.independent.transgenic.AP3::ipt.petunia.lines.had.increased.flower.diam-eter.compared.to.control.flowers..Flowers.from.these.lines.had.a.20%–30%.increase.in.the.diameters.of.individual.floral.organs.due.to.radial.expansion..Transgenic.lines.had.flowers.with.over.90%.increase.in.fresh.weight.compared.to.controls..Some.lines.had.>60%.increase.in.overall.fruit.weight.while.others.had.fruit.weights.similar.to.those.of.controls.

AgrobActerium rhizogenes rol GeneS

The. rol. genes. from. Agrobacterium rhizogenes. have. been. useful. for. changing..multiple.aspects.of.plant.architecture.(reviewed.by.Casanova.et.al.122)..There.are.four.rol.genes.(rolA,.B,.C,.and.D),.with.rolC.(cytokinin-β-glucosidase).having.the.most.potential. for. transgenic. enhancement. of. ornamental. plants.. In. vivo. the. rol. genes.interfere.with.phytohormone.metabolism.and.modify.secondary.metabolism,.which.


is. phenotypically. manifested. as. a. dwarf. habit,. increased. rooting,. darker. leaves,.increased.lateral.shoots,.and.increased.flowering.(reviewed.by.Casanova.et.al.122)..The.rol.genes.have.been.transformed.into.Antirrhinum,123.Begonia,124.Rosa,125.Petunia,126.Dianthus,127.Osteospermum,128.Angelonia,129.Lilium,130.and.Pelargonium.131.In.some.instances,.transgenic.plants.had.smaller.flowers.and.reduced.fertility,.which.could.be.a.limitation.from.a.production.standpoint,.but.may.be.useful.in.addressing.environ-mental.concerns.since.transgene.flow.would.likely.be.more.restricted.

BIOTIC STRESS TOLERANCE

Engineering.plants.to.tolerate.biotic.stresses.has.the.potential.for.large.cost.savings.in.pesticide.chemicals. leading. to.more.environmentally. friendly.production.prac-tices.(reviewed.by.Redenbaugh.and.McHughen132)..Pesticide.use.data.collected.from.farmers.growing.insect.resistant.transgenic.corn.and.cotton.indicate.biotech.crops.have.decreased.pesticide.usage,.increased.yield,.higher.quality.crops,.and.decreased.labor.133.These.are. clear.benefits. that. are. currently.only. realized.by.producers.of.biotech.food.crops,.but.are.applicable.to.ornamental.crops..While.there.are.no.com-mercially.available.biotech.ornamentals.engineered.for.biotic.stress.tolerance,.this.trait. is. being.engineered. in. some.ornamentals. and. a. few.examples.will. be. high-lighted.here.

There.are.a.number.of.strategies.for.genetic.engineering.plants.to.be.more.resis-tant.to.insect.attack..Engineering.plants.to.produce.insect.toxins.has.been.the.most.successful.means.to.date.and.is.widely.used.today.in.corn.and.cotton.production..Genes.encoding. insect.control.proteins. from.Bacillus thuringiensis,.know.as.Cry.genes,.have.been.cloned.and.modified.for.efficient.production.in.plants134.(reviewed.by.Schuler.et.al.135)..These.proteins.bind.to.gut.receptors.in.lepidopteran,.dipteran,.and.coleopteran.insects.causing.disfunction.of.the.insect.gut.and.lysis.of.gut.epithe-lial.cells.leading.to.insect.death.(reviewed.by.Gill.et.al.136)..The.Bacillus cryIA(b).gene.has.been. introduced. into.chrysanthemum.causing.feeding. insects. to.die.and.significantly.less.consumption.of. leaf.area.137. Insects. feeding.on. transgenic.plants.consumed. less. than. 5%. of. the. leaf. area. while. 95%. or. more. of. control. leaf. area.was.consumed..Similarly,.transgenic.petunias.expressing.the.cryIA(c).gene.caused.significant.reduction.in.insect.fecundity.138.While.the.BT.toxins.have.proven.to.be.very. successful. for. insect. management,. other. biotech. insecticidal. strategies. have.been.developed.and.will.be.important.considerations.for.crop.management.to.avoid.insect.resistance.issues..As.discussed.in.“Plant.Volatiles”.section,.plants.can.be.engi-neered.to.produce.volatiles.that.modify.insect.behavior.or.attract.predatory.insects..Protease.inhibitors,.α-amylase.inhibitors,.lectins,.and.nonvolatile.secondary.metab-olites,.such.as.tryptamine,.are.also.means.for.engineering.insect.defense.in.plants.(reviewed.by.Llewellyn. and.Higgins139)..However,. examples.of. these.methods. for.engineering.insect.defense.in.ornamental.plants.are.few.

While.disease.resistance.in.ornamental.crops.has.largely.been.achieved.through.traditional. breeding,. this. trait. has. been. successfully. genetically. engineered. using.a. number. of. approaches.. Transgenic. expression. of. genes. commonly. induced. by.disease.infection.(pathogenesis-related.genes,.PR).confers.enhanced.disease.resis-tance..For.example,.chitinases.degrade.fungal.cell.walls. into.breakdown.products.


that. elicit. plant. defense. responses. and. are. often. induced. in. response. to. pathogen.attack.(reviewed.by.Stintzi.et.al.140)..Blackspot.is.a.common,.problematic.disease.in.Rosa hybrida. and. is.caused.by. the. fungus.Diplocarpon rosae..Transformation.of.rose.via.particle.bombardment.with.a. rice.basic.chitinase.gene.reduced.blackspot.severity. by. 13%–43%. with. lesion. size. reduced. by. up. to. half. of. those. of. control.blackspot. lesions.141.A.PR.gene.encoding.for.a.cysteine-rich.antimicrobial.protein.(AMP).confers.disease.resistance.in.plants..Enhanced.tolerance.to.powdery.mildew.was.observed.in.transgenic.rose.plants.constitutively.expressing.an.AMP.gene.from.onion.142.The.onion.AMP.was.also.introduced.into.Pelargonium.for.enhanced.resis-tance.to.Botrytis.143.In.leaf.disc.assays,.Botrytis.sporulation.was.slightly.reduced.in.transgenic.lines..Greater.disease.protection.is.observed.in.transgenic.plants.expressing.more.than.one.PR.gene.and.this.should.be.considered.in.development.of.transgenic.plants.with.enhanced.disease.resistance.144

CURRENT AND FUTURE ISSUES IN ORNAMENTAL BIOTECHNOLOGY

Despite. the. documented. technical. successes,. utility,. and. the. enormous. benefits.genetic. engineering. rewards,. there. are. only. a. limited. number. of. commercialized.transgenic. ornamental. plants. on. the. market.. Presently,. the. only. commercialized.ornamental.plants.on. the.market.are.carnations.engineered. for.blue-violet.color.145.This.is.in.stark.contrast.to.the.rapid.adoption.of.genetically.engineered.agronomic.crops.since.their.introduction.in.1996.with.transgenic.plants.accounting.for.well.over.50%–90%.of.planted.acreage.of.corn,.soybean,.and.cotton.in.2008.(http://www.ers.usda.gov/Data/BiotechCrops/)..The.lack.of.commercialized.transgenic.ornamentals.is.not.due.to.lack.of.potential.products,.but.is.impeded.by.regulatory.issues.concern-ing.safety,.consumer.acceptance,.intellectual.property,.postcommercialization.moni-toring.of.transgenic.crops,.and.the.diversity.of.germplasm.requiring.development.of.a.biotech.trait.2.While.ornamental.crops.are.grown.for.aesthetic.purposes.and.do.not.require.human.health.safety.testing,.they.are.subject.to.strict.handling.procedures,.field-testing,.tracking,.and.postcommercialization.stewardship..All.of.these.increase.the.costs.of.developing.a.biotech.crop.to.at.least.$1.million.for.one.transgenic.event.per.country,132.making.it.difficult.to.justify.use.of.biotechnology.in.the.ornamental.market.where.crop.values.are.significantly.lower.than.agronomic.crops..With.turn-over.of.new.varieties.in.ornamental.crops,.sales.are.not.high.enough.to.generate.a.profit.that.would.offset.the.costs.to.bring.a.transgenic.plant.to.market.

Regulatory.issues.pose.a.significant.obstacle.to.the.development.and.commercial-ization.of.genetically.engineered.crops..Any.organism.that.has.been.modified.through.genetic.manipulation.technology.is.subject.to.regulation.by.overseeing.agencies,.such.as.the.United.States.Department.of.Agriculture.(USDA),.Animal.and.Plant.Health.Inspection.Service.(APHIS),.Environmental.Protection.Agency.(EPA),.and.Food.and.Drug.Administration.(FDA).in.the.United.States,.which.oversees.introduction,.trans-port,.risks.to.human.health,.and.environmental.risk.assessment.of.the.genetically.or.living.modified.organism.(GMO.or.LMO,.respectively)..The.regulations.and.proce-dures.vary.by.country,.creating.additional.complications.in.releasing.a.GMO.interna-tionally.(reviewed.by.Chandler.and.Tanaka16),.but.the.goals.of.regulating.GMOs.are.


unified.in.that.negative.environmental.and.health.effects.want.to.be.avoided..A.cen-tralized,.international.resource,.known.as.the.Cartagena.Protocol.on.Biosafety.(CPB),.has.been.developed.for.promoting.the.safe.handling.and.use.of.GMOs.internationally.(http://www.cbd.int/biosafety/).. However,. the. protocol. is. still. being. developed. and.ratified.by.many.countries.and.countries.such.as.the.United.States.and.Australia.are.not.yet.contributing.to.the.development.of.this.centralized.protocol.

Continued.consumer.education,.outreach,.and.perception.assessment.are.impor-tant.avenues.to.continue.pursuit.of.making.commercialization.of.ornamental.GMOs.worthwhile..Reports.indicate.that.consumer.perception.of.GMOs.is.mixed,.but.most.surveys.are.conducted.with.regard.to.the.presence.of.GMOs.in.food.and.not.ornamen-tal.plants..A.survey.of.master.gardeners.revealed.that.∼73%.of.survey.respondents.expressed. interest. in.purchasing.a. GM. plant.product. for. the.garden.146. This. said,.there.were.also.clear.opinions.regarding.how.different.sources.of.transgenes.affected.acceptability.of.the.GMO,.with.plant-derived.transgenes.being.more.acceptable.than.those.derived.from.bacteria,.fungus,.virus.or.animal.sources..Respondents.were.also.neutral. about. the. effect. of. landscape. GMOs.. This. latter. point. should. be. empha-sized. in.education.programs.since.many.applications.of.biotechnology.will. result.in.decreased.water.use,.decreased.fossil.fuel.use,.and.decreased.chemical.use,.all.of.which.would.have.positive.effects.on.our.environment..For.example,.Redenbaugh.and.McHughen132.cite.data.showing.declining.pesticide.sales.with.increased.sales.of.biotech-based.varieties.of.plants.

Ornamental.biotechnology.will.clearly.benefit.from.the.evolution.of.newly.devel-oping.DNA.sequencing.technologies..New.sequencing. technologies.including.454.sequencing. and. real. time. single. molecule. long. read. sequencing. soon. to. be. com-mercialized,147.will.make.it.possible.to.quickly.have.genome.data.for.most.species.of. interest. at. very. low.costs..The. limitation.will.no. longer.be.on. sequence.avail-ability.but.rather.on.data.management.and.data.mining..The.availability.of.genome.data.will.facilitate.discovery.of.regulatory.elements.that.will.be.useful.for.delivering.more.precise.transgene.expression.patterns.in.plants,.rapidly.progress.the.discovery.of.enzymes.involved.in.important.biotechnological.applications,.and.make.develop-ment.of.transgenic.ornamental.plants.more.economically.feasible.

ACKNOWLEDGMENTS

The.authors.thank.Dr..Thomas.A..Colquhoun.for.critical.reading.of.the.manuscript.and.Dr..Bart.Schutzman.for.assistance.with.figure.preparation.

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83

4 Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants

Qiang Chen

CONTENTS

A.Brief.History.........................................................................................................84Plants.Provide.a.New.Manufacturing.Technology.for.Pharmaceutical.Proteins......... 85Protein.Pharmaceutical.Production.Using.Plant.Systems.........................................88

Monoclonal.Antibodies........................................................................................ 89Vaccines...............................................................................................................92

Glycosylation.of.Plant-Made.Pharmaceuticals......................................................... 95Downstream.Processing.of.Plant-Derived.Protein.Pharmaceuticals........................96

Column.Chromatography.....................................................................................97Nonchromatographic.Separations...................................................................... 101

Plant.Species.Used.for.PMP.Production................................................................. 103Leafy.Crops........................................................................................................ 104Seed.Crops......................................................................................................... 105Fruit.and.Tuber.Crops........................................................................................ 106Aquatic.Plants.................................................................................................... 107Plant.Suspension.Cell.Cultures.and.Single-Cell.Cultures.of.Algae................... 107

Regulatory.Concerns.and.Public.Acceptance......................................................... 108Better.Stewardship.of.the.PMP.Community...................................................... 109Alternative.PMP.Production.Technology.and.Risk.Control.............................. 109

Nonbiological.Containment.......................................................................... 109Biological.Containment................................................................................. 110

Regulatory.Policies.regarding.PMP................................................................... 112Conclusion.............................................................................................................. 112References............................................................................................................... 113


A BRIEF HISTORY

Humans.have.been.using.plants.as.a.natural.factory.of.pharmaceutical.molecules.for.many.centuries..Many.secondary.metabolites.of.plants.exhibit. a.diversity.of.therapeutic.effects.including.anti-inflammatory,.wound-healing,.and.antimicrobial.properties..These.natural.plant.medicines.were.widely.used.around.the.world.by.early.civilizations.to.protect.and.maintain.human.and.animal.health..Even.today,.with.our.highly.sophisticated.pharmaceutical.technologies,.more.than.one-quarter.of. .prescription.pharmaceutics. are. still. derived. from.plants.1,2.These.plant-based.natural.pharmaceuticals.include.single-molecule.drugs.such.as.the.painkiller.mor-phine.from.the.opium.poppy.and.botanicals.that.are.a.mixture.of.active.plant.mol-ecules.acting.synergistically.in.the.treatment.of.specific.conditions..Traditionally,.these.plant.molecules.are.extracted.from.either.wild.or.cultivated.species..Large-scale.production,.however,.is.usually.relying.on.modern.advancement.of.plant.tissue.culture.since.the.medicinal.plants.are.sometimes.rare.in.nature.or.are.difficult.to.cultivate..Therefore,.plants.and.plant.tissue.cultures.have.been.serving.as.impor-tant. bioproduction. systems. for. traditional. pharmaceutical. bulk. substances. for. a.long. time..With. the. advancement.of.modern. molecular. biology. and.biotechnol-ogy,.it.became.possible.to.alter.the.plant.genome.by.inserting.expression.cassettes.including. the. target. transgene. and. associated. regulatory. genetic. elements.. This.technological.breakthrough.allowed.the.creation.of.transgenic.plants.that.carry.the.transgene.and.produce.recombinant.proteins.3.The.first.recombinant.pharmaceuti-cal.protein.of.plant.origin.was.a.human.growth.hormone.produced.in.transgenic.tobacco.in.1986.4.Three.years.later,.the.success.of.expressing.monoclonal.antibod-ies.(mAbs).in.transgenic.tobacco.plants.was.published,.demonstrating.the.ability.of.plant.cells. in.expressing.and.assembling.of.complex. functional.multi-subunit.proteins.5.Our. research. group.directed.by.Dr..Charles.Arntzen. led. the. effort. in.plant-made.vaccines.and.published.our.first.finding.in.1992..We.demonstrated.that.transgenic. tobacco.could. successfully.produce.hepatitis.B.virus. surface. antigen.(HBsAg).with.similar.biochemical.properties.and.antigenicity.to.that.produced.in.human.serum.or.recombinant.yeast.6.Soon.after,.research.from.our.group.showed.that.E. coli.heat-labile.enteroxin.(LT-B).derived.from.transgenic.tobacco.or.potato.had. equivalent. functionality. to. E. coli–derived. LT-B. and. was. orally. immuno-genic.in.mice,.which.provided.the.first.proof.of.principle.for.plant-derived.edible.vaccines.7.The.successes.of.these.researches.opened.up.a.completely.new.applica-tion.area.for.plants.as.factories.for.human.and.animal.pharmaceutical.production..In.the.last.two.decades,.a.broad.range.of.functionally.active.vaccines.and.thera-peutic. proteins. have. been. produced. in. an. increasingly. diverse. species. of. crops.with.a.variety.of.different.plant.expression.systems..Recent.improvements.in.plant.expression.vectors.have.allowed.for.significant.strides.in.a.variety.of.new.plant-based.expression.systems.and.their.broad.application..Meanwhile,.the.importance.of. the. downstream. processing. for. extraction. and. purification. of. pharmaceutical.proteins.from.plant.materials.has.been.realized,.and.increasing.efforts.have.been.applied.to.address.this.critical.issue..The.public.opinion.and.regulatory.landscape.have.been.evolving.to.a.more.welcoming.climate.where.scientists.and.regulatory.agencies.can.work.together.to.develop.regulations.and.guidelines.for.plant-made.

85Expression and Manufacture of Pharmaceutical Proteins

recombinant.pharmaceutical. proteins..At.present. stage,. plants.provide. a. serious.viable..alternative.to.the.traditional.mammalian.or.microbial.cell.culture.technolo-gies,.allowing.for.cost-effective,.highly.scalable,.and.safe.production.of.pharma-ceutical.proteins.

PLANTS PROVIDE A NEW MANUFACTURING TECHNOLOGY FOR PHARMACEUTICAL PROTEINS

The.majority.of.pharmaceutical.proteins.are.currently.produced.in.mammalian.cell.cultures,.with.a.few.exceptions.produced.in.Escherichia coli,.yeast,.or.insect.cells.8.As.one.of.the.fundamental.technological.revolutions.in.the.pharmaceutical.indus-try,.the.development.and.optimization.of.mammalian.cell.culture.allow.for.uniform.production.of.pharmaceutical.proteins.with.high.efficacy..However,.mammalian.and.other. cell. culture-based.production. systems. rely.on. the. fermentation.of.cells. sus-pended.in.bioreactors,.which.requires.a.large.initial.capital.investment.and,.there-fore,. severely. constrains. their. economic. effectiveness. and. scalability.. Application.of.such.technologies.in.the.developing.world.will.not.only.face.intellectual.property.challenges,.but.undoubtedly.will.also.encounter.further.financial.and.logistical.bar-riers.that.are.difficult.to.overcome..Meanwhile,.the.worldwide.demand.for.protein-based.pharmaceuticals.has.greatly.increased.for.the.last.two.decades..If.the.demand.for.such.products.cannot.be.met.or.if.they.are.too.costly.to.produce.for.health.care.systems,. all. of. the. advances. in. recombinant. pharmaceutical. protein. creation. and.optimization,.as.well.as.the.vast.therapeutic.potential.of.these.agents,.are.made.irrel-evant..This.urgent.situation.calls.for.the.development.of.new.production.platforms.that.are.cost-effective,.scalable,.and.safe.

Plant-based.protein.production.technology.combines.the.innovations.in.medicine.and.plant.biology.and.has.a.great.potential. to.produce. affordable.pharmaceutical.proteins. at. a. large. scale..Plants. can.produce. large.volumes.of.proteins. efficiently.and.sustainably,.and,.under.certain.conditions,.with.significantly.lower.manufactur-ing.costs.than.mammalian.cell.cultures.9–13.Plants.are.also.far.less.likely.to.intro-duce.human.or.animal.pathogens.to.humans.compared.to.mammalian.cells.or.whole.transgenic.animal.systems..Unlike.bacterial.and.other.prokaryotic.systems,.plants.share.a. similar.endomembrane. system.and.secretory.pathway.with.human.cells.14.Therefore,.plant.cells.are.able.to.efficiently.assemble.multiple.subunit.proteins.and.perform. necessary. posttranslational. modifications. on. transgenic. pharmaceutical.proteins..The.low-cost,.high-scalability,.and.safety.characteristics.of.plant.produc-tion.system.offer.an.attractive.alternative.technology.platform.for.both.commercial.pharmaceutical.production.and.for.manufacturing.products.for.the.developing.world..In.fact,.an.increasing.number.of.academic.and.industrial.laboratories.are.investing.in.plant-made.protein.pharmaceuticals.(PMPs)..While.much.research.is.still.required.to.optimize.plant.production.of.pharmaceuticals.and.to.validate.them.in.large-scale.clinical. trials,. the. results. to.date.show.a.promising. technology. that. justifies.com-mercial.development.

Currently,.three.strategies.are.primarily.employed.for.the.expression.of.pharma-ceutical.proteins.in.plants.including.the.stable.transformation.of.the.nuclear.genome,.


the. stable. transformation. of. the. chloroplast. genome,. and. transient. transformation.(Figure.4.1)..In.the.first.two.strategies,.an.expression.cassette.harboring.the.gene.of.interest.is.used.to.transform.plant.cells.to.enable.the.insertion.of.transgene.into.the.nuclear.or.chloroplast.genomes..The.stable.integration.of.the.cassette.into.either.the.nuclear.or.plastid.genome.allows.the.Mendelian. inheritance.of. the. transgene.over.generations.and,.in.turn,.the.stable.expression.of.transgenic.proteins..The.resulting.transgenic.lines.can.be.propagated.to.establish.a.master.seed.bank.for.future.large-scale. production. of. pharmaceutical. proteins.. For. most. plant. species,. exogenous.transgene.cassette.can.be.delivered.into.their.genome.by.using.Agrobacterium tume-faciens,.which.inserts.DNA.into.the.plant.genome.in.a.somewhat.random.fashion..Alternatively,.ballistic.methods.with.DNA-coated.microprojectiles.can.be.used. to.bombard.plant.cells..The.ballistic.“gene.gun”.is.especially.useful.for.plant.species.that.are.difficult.to.transform.efficiently.by.Agrobacterium.15.Up.to.today,.the.bal-listic.method.with.tungsten.or.gold.particles.remains.to.be.the.only.efficient.way.for.the.transformation.of.the.chloroplast.genome.

In. the. third.plant.expression.strategy,. transgenes.are.not. integrated.into.one.of.the.plant.genomes,.but.instead.they.are.present.in.the.plant.nucleus.transiently.while.being.transcribed,.and.later.the.transcripts.are.transported.into.the.cytoplasm.and.the.transgenic.proteins.are.translated..While.transient.expression.can.be.carried.out.with.both.nonviral.and.viral.vectors,. the. latter.are.more.widely.used.due. to. their.robustness. in. replication,. transcription,. and. translation.16. Plant. viral. vectors. have.been.used.in.several.major.formats.to.transiently.express.PMPs..For.example,.many.vaccine.epitopes.have.been.fused.to.the.viral.coat.protein.so.that.they.will.be.dis-played.of.on. the.surface.of. the.virus.17,18.PMP.genes.can.also.be. inserted. into.an.

Pharmaceutical protein gene

Clone into planttransformation

vector

Transient expression:infect plant to initiate

viral replication

Stable expression:transgene

integrates intochloroplast genome

Stable expression:transgene

integrates intonuclear genome

Stable expression:regulated release ofviral replicon from a

viral genome integratedinto plant genome

Stable expression:integrate into nuclear

genome, high-levelprotein expression

Transient expression:high-level protein

expression withoutforming infectious viral

particles

Incorporate intodeconstructed viralsequences as partof Agrobacterium

transformationvector

Integrate into aviral coding sequence

for expression as a“by product” ofviral replication

FIGURE 4.1 Strategies.for. the.expression.of.pharmaceutical.proteins.in.plants..(Adapted.from.Chen,.Q.,.Biol. Eng.,.1,.291,.2008..With.permission.)


expression.cassette,.and.the.target.protein-coding.sequences.will.be.expressed.as.a.by-product.of.the.viral.genome.replication.cycle.under.the.control.of.subviral.pro-moters.19,20.In.a.variation,.recombinant.viral.genomes.can.be.integrated.into.plant.chromosomes.via. transformation..The.subsequent. transcription.of.viral.RNA.will.initiate.vial.replication.and,.in.turn,.drive.the.expression.of.the.target.pharmaceutical.protein.21.Overall,.the.robust.transcription.and.amplified.translation.of.the.plant.viral.components.allow.the.transient.expression.systems.to.yield.high-level.accumulation.of.recombinant.proteins.

Viral. “deconstruction”. is. a. recent. application.of. using.viral. genomes. to. cre-ate.transient.transgene.expression.22,23.This.newly.deconstructed.viral.vector.sys-tem.is.based.on.in planta.assembly.of.replication-competent.tobacco.mosaic.virus.(TMV).and.potato.virus.X.(PVX).genomes.from.separate.provector.modules.23,24.Agrobacterium tumefaciens. is.used. to.deliver.DNA.modules.along.with.a. sepa-rate.construct. that.produces.a. recombination. integrase..Upon. its.expression,. the.integrase.assembles.separate.DNA.modules.into.a.replication-competent.TMV.or.PVX.genome.under.the.control.of.a.plant.promoter..This.assembled.DNA.construct.is. then. transcribed.and. spliced. to. generate. a. functional. infective. replicon.. This.“Maginfection”.system.combines.the.advantages.of. three.biological.systems.22,24.First,.the.laborious.in.vitro.process.of.generating.RNA-based.vectors.is.eliminated.due.to.the.use.of.Agrobacterium.as.viral.vector.delivery.tool..Second,.the.deletion.of.viral.coat.protein.genes.in.this.system.allows.the.speed.and.high.protein.yield.of.a.viral.system.without.the.threat.of.creating.functional.infectious.particles..In.addition,. “Maginfection”. integrates. the. posttranslational. processing. capacity. of.eukaryotic.plant.cells.for.producing.complex.proteins..Peaks.of.PMP.accumula-tion.are.usually.detected.between.7.and.14.days.after.vector.delivery.24.This.system.thus.gains.the.flexibility.of.nuclear.gene.expression.with.the.speed.and.expression.amplification.of.viral.vectors.

All.three.plant.expression.strategies.are.likely.to.have.their.place.in.the.com-mercial. realm. of. producing. pharmaceutical. proteins.. Expression. systems. using.stably. transformed.plants. represent.an.inexpensive.and.permanent.genetic.prop-agation. source. for. pharmaceutical. protein. production.25. However,. this. develop-ment.of.technology.has.been.hindered.by.several.scientific.and.regulatory.issues..First,.it.requires.a.relatively.long.time.frame.to.create.and.select.the.initial.stable.transgenic.plants..In.addition,.the.expression.level.of.the.transgene.product.can.be.fluctuated.wildly.or.unstable.not.only.between.individual.plants.but.also.between.different.generations.of.the.same.plant.line..These.uncertainties.are.often.caused.by.the.randomness.of.transgene.insertion.into.the.plant.nuclear.genome.(the.“posi-tion.effect”).and.by.the.phenomenon.of.posttranscriptional.or.siRNA-dependent.gene.silencing..The.latter.may.be.triggered.by.the.presence.of.a.particular.mRNA.in.high.concentration,.leading.to.both.immediate.and.potential.permanent.produc-tion.instability.26–31.The.potential.risk.of.unwanted.transgene.outflow.from.fields.with.genetically.modified.(GM).plants. to.neighboring.fields.with.non-GM.crops.or.their.wild.relatives.has.also.raised.regulatory.and.public.acceptance.issues.for.this.technology..These.scientific.and.regulatory.hurtles.have.been.addressed.by.a.growing.number.of.academic.and.industrial.laboratories..For.example,.in.response.to.regular.concerns.for.potential.transgene.escape.through.outcrossing,.transgenic.


plants.are.increasingly.being.grown.in.containment.or.in.areas.with.natural.geographic.barrier.to.isolate.transgenic.plants.from.their.agricultural.or.wild.relatives..Once.the.transgenic.plant.lines.are.selected.and.the.“master.seed.banks”.are.established,.however,.stable.transgenic.plants.will.provide.the.most.scalable.technology.when.large.amounts.of.pharmaceutical.protein.are.required.for.commercial.production..Since.there.are.thousands.of.chloroplasts.in.each.cell,.the.copies.of.transgene.per.cell.in.transplastomic.plants.are.significantly.higher.than.that.of.nuclear.transfor-mation,. which,. in. turn,. drive. higher. level. of. transgenic. protein. production.32–34.The.prokaryotic.origin.of.chloroplasts.also.supports.facile.homologous.recombina-tion.and.polycistronic.transcript.production..These.features.allow.the.production.of. multiple. transgenic. proteins. from. one. polycistronic. mRNA. as. well. as. better.transgene.targeting.in.chloroplast.genome.to.avoid.the.“position.effect”.and.trans-gene. silencing,. which. cause. inconsistent. level. of. transgenic. protein. production..Chloroplasts.are.maternally.transmitted.in.most.crops,.which.exclude.chloroplast.transgene.escape.from.pollen.transmission..Therefore,.transplastomic.expression.of.transgene.is.considered.a.superb.alternative.for.regulatory.compliance.in.ensur-ing.transgene.containment.and.improving.the.biosafety.of.transgenic.plants.35.Due.to.lack.of.posttranslational.machineries.in.chloroplasts,.however,.plant.lines.with.stably.transformed.chloroplast.genomes.will.mostly.be.applicable.to.produce.pro-teins,.which.do.not.require.posttranslational.modifications.for.their.function.or.pro-teins.for.diagnostic.purposes.36.The.transient,.viral-based.expression.systems21,23.are.focused.on.production.speed,.and. therefore,. the.most.convenient. technology.for.obtaining. the. initial. research.material. (mg–g. level).used. in.preclinical. stud-ies..These. transient. expression. systems. rely. on.Agrobacteria’s. ability. to. deliver.the.“deconstructed”.viral.genome.to.majority.of.plant.cells,.therefore,.eliminating.the.need.for.viral.systemic.spreading.function..This.characteristic.not.only.elimi-nates.the.concern.of.transgene.loss.during.systemic.spreading.but.also.allows.the.application.of. this. technology. to.a.diversity.of.plant. species.beyond. the.natural.virus.hosts..Since.nontransgenic.plant.materials.are.used.for.viral.vector.delivery,.they.can.be.readily.stocked.up.for.semi-large-scale.productions..Overall,.a.rapid.evaluation.of.pharmaceutical.candidates.and.transition.to.a.large-scale.commercial.production.platform.can.be.accomplished.by.employing.the.combination.of.both.transient.and.stable.transgenic.plant.technologies.

PROTEIN PHARMACEUTICAL PRODUCTION USING PLANT SYSTEMS

Unlike. mammalian. cell. cultures. or. bacterial. fermentation. systems,. which. require.capital-demanding. bioreactors. and. expensive. tissue. culture. media. and. operations,.pharmaceutical. protein. production. by. transient.or. stable. transgenic.plants. is.more.economical.in.both.initial.setup.and.subsequent.scale-up..This.is.due.to.decrease.in.large.hardware. investment.and.expensive.culture.media. regardless.of.whether. it. is.produced.in.the.field.or.greenhouses..As.a.result,.a.variety.of.pharmaceutical.proteins.have.been.expressed.and.characterized.in.plant.systems..Here,.we.will.focus.on.the.production.of.the.two.most.important.classes.of.pharmaceuticals:.mAbs.and.vaccines.


monoclonal antIbodIeS

Monoclonal. antibodies. (mAbs). have. a. wide. range. of. important. pharmaceutical.applications.. MAb.market. was.projected. to. reach.$16.7.billion.by. 2010,. account-ing.for.over.a.quarter.of.the.total.protein.therapeutics.market.37,38.As.a.result,.new.expression. systems.have. to.demonstrate. their.effectiveness. in.producing.mAbs. in.order.to.be.considered.seriously.as.contenders.for.commercial.production.of.pharma-ceutical.proteins..MAbs.are.extremely.expensive.to.produce.in.mammalian.culture.system.and,.therefore,.are.costly.to.patients.and.health.care.systems..Development.of. new. production. platforms. is. indeed. necessary. to. reduce. the. cost. and. increase.the. scalability. of. mAb. manufacture.. MAbs. have. been. successfully. expressed. in.stable.transgenic.plants.since.the.early.stage.of.PMP.development,.despite.they.are.complex.heterotetrameric.glycoproteins.5.Since. then,.a.variety.of.mAbs.and. their.derivatives,.such.as.secretory.IgAs,.single-domain.fragments,.single-chain.variable.fragment.(scFv),.and.diabodies.and,.more.recently,.recombinant.immune.complexes.(RICs),.have.been.successfully.produced.in.many.host.plant.species,.with.increas-ing.number.and.type.of.mAb.being.produced.each.year.(Table.4.1).39–62.Our.group.recently.published.the.first.report.that.demonstrated.the.efficacy.of.a.plant-produced.mAb.against.a.potentially.lethal.infection.several.days.after.exposure.in.an.animal.challenge.model.58.Several.mAbs.have.been.selected.for.clinical.evaluation.with.two.having.reached.Phase.II.clinical.studies.(Table.4.2).63,64.For.example,.a.TMV-based.vector.was.demonstrated.to.be.an.efficient.vector.to.express.a.human.scFv.for.the.treatment.of.non-Hodgkin’s. lymphoma.(NHL).in.tobacco.plants.65,66.The.tobacco-produced. scFv. is. an. effective. anti-idiotype. vaccine. candidate. in. a. murine. NHL.tumor. challenge.model. and. has.been. further. evaluated. in.human.clinical. trials.67.This.patient-specific.anti-idiotype.vaccine.requires.a.speedy.system.for.rapid.pro-duction. and.patient. treatment. evaluation.. In. contrast. to.mammalian. cell. cultures,.the.TMV-based.plant.expression.system.has.successfully.provided.such.speedy.and.versatile.production.platform.

Similar.to.stable.transgenic.plants,.transient.expression.systems.have.been.very.successful.in.producing.single.subunit.therapeutics.such.as.scFvs.68,69.However,.until.recently,.viral.vector-based.transient.expression.systems.are.incapable.of.producing.full-length.mAbs.or.any.other.heterooligomeric.proteins.efficiently..This.is.due.to.the.fact.that.viral.vectors.built.on.the.same.virus.backbone.are.typically.“competing”.with.each.other,.resulting.in.early.segregation.and.subsequent.preferential.amplifica-tion.of.one.of.the.vectors.in.one.cell.23,70–72.The.MagnICON™.system.composed.of.two.sets.of.noncompeting.vectors.(TMV.and.PVX).has.successfully.resolved.this.problem.22,73.TMV.and.PVX.do.not.compete.with,.nor.dominate.over,.each.other,.because.they.interact.with.different.host.factors.for.their.movement.and.replication..As.a.result,.the.efficient.co-expression.of.light.chain.and.heavy.chain.of.mAb.in.the.same.cells.occurs.when.two.vectors.harboring.the.two.mAb.genes.are.co-delivered.into. plants.23. Extensive. studies. have. shown. that. this. transient. system. can. rapidly.(within.10.days.of.vector.delivery).produce. fully.assembled.mAbs. in.high. levels,.reaching.up.to.0.8.g.mAb/kg.of.fresh.tissue.weight.(FW).23,58.Therefore,.MagnICON.system.provides.a.potential.large-scale.production.platform.for.rapid.manufacturing.of.mAbs.and.other.oligomeric.proteins.


TABLE 4.1Representative Antibodies, Antibody-Fragments, and Antibody Fusions Produced in Plants

AntigenAntibody

TypePharmaceutical

Target Crop References

Streptococcal.surface.antigen.SA I/II

Secretory.IgA/G

Tooth.decay N. tobacum [43]

Herpes.simplex.virus 2

IgG Microbicide.for.genital.herpes

G. max [59]

glycoprotein.B O. sativa

Tobacco.mosaic.virus.coat.proteins

Bispecific-scFv

Model.bispecific-scFv.molecule

N. tobacum [242]

Human.creatine.kinase-MM

Fab Neurone.and.rheumatic.diseases

A. thaliana [44]

Human.carcinoembryonic.antigen.(CEA)

Diabody Tumor.imaging.for.colorectal,.lung,.breast,.and.pancreatic.carcinomas

N. tobacum [60]

Human.rhesus.D IgG1 Alloimmunization,.haemolytic.disease.of.new.born

A. thaliana [61]

Human.chorionic.gonadotropin.(HCG)

scFv,.diabody.and.IgG1

Diagnostic.and.therapeutic.for.HCG-expressing.cancers.or.as.contraceptive

N. tobacum [69]

Tumor.surface.antigen

scFv Therapeutic.vaccine.for.NHL

N. benthamiana [66,67]

Rabies.virus IgG Rabies.virus N. tobacum [45]

Herpes.simplex.virus.glycoprotein.D

LSC.(IgA.type)

Genital.herpes.diagnostics

C. reinhardtii [248]

Human.epidermal.growth.factor.receptor

IgG Therapeutics.for.refractory.colorectal.cancer

Z. mays.(corn.seeds)

[46]

Hepatitis.B.surface.antigen

IgG Hepatitis.B.virus.infections

N. tobacum.BY2 cells

[62]

Protective.antigen.(PA).of.Bacillus anthracis

IgG Therapeutics.for.anthrax.exposure,.antiterrorism.agent

N. benthamiana [72]

Lipopolysaccharide.(LPS).of.S. enterica.Paratyphi.B

scFv Salmonella enterica.diagnostic.and.therapeutic

N. tobacum [48]

Tumor-associate.antigen.GA733

IgG Colorectal.carinomas N. tobacum [40]


TABLE 4.1 (continued)Representative Antibodies, Antibody-Fragments, and Antibody Fusions Produced in Plants

AntigenAntibody

TypePharmaceutical

Target Crop References

Human.epidermal.growth.factor.receptor.HER2

scFv Diagnostic.for.breast,.ovary,.prostate.and.lung.cancer

N. tobacum [49]

Tetanus.toxin.C.fragment

RIC Vaccine.for.tetanus.toxin

N. tobacum [186]

Botulinum.toxin.A scFv Antidote.for.botulinum.toxin.A,.anti-bioterrorism.agent

N. tobacum [50]

Tumor-associated.antigen.oligosaccharide.Lewis.Y

IgG2a Breast.and.colorectal.cancer

N. tobacum [55]

HIV.p24 p24-IgA.Hc.fusion

HIV.vaccine N. tobacum [185]

CD30 IgG Immunotherapy.for.Hodgkin.lymphoma.(HL).and.anaplastic.large.cell.lymphoma

Lemna.minor [136]

TGEV Minibody,.Full.IgA

Therapeutic.for.TGEV

N. clevelandii [51]

Hepatitis.A.virus scFv-Fc Hepatitis.A.virus.infection

A. thaliana [54]

Human.epidermal.growth.factor.receptor.HER1

scFv Targeting.agent.for.breast,.ovary,.prostate.and.lung.cancer

N. tobacum [52]

P. aeruginosa.serotype.O6ad.PS.O.side.chain

IgG P. aeruginosa infection

N. tobacum [39]

HIV.gp.41 IgG HIV.infection N. tobacum.and.BY2.cell.culture,.Z. mays

[42,143]

HIV.gp.120 IgG HIV.infection N. benthamiana,.Z. mays,.A. thaliana

[53,64,134,135]

Ebola.GP1 IgG,.RIC Therapeutic.for.Ebola N. benthamiana [21,263]

West.Nile.Virus.E.DIII

IgG Therapeutic.for.WNV.infection

N. benthamiana [58]

Source:. Adapted.from.Chen,.Q.,.Biol. Eng.,.1,.291,.2008..With.permission.Note:. scFv,.single-chain.variable.fragment;.RIC,.recombinant.immune.complex;.LSC,.large.single.chain.

antibody;.NHL,.non-Hodgkin’s.lymphoma.


Some.heterooligomeric.pharmaceutical.targets.such.as.secretory.IgA,.IgM,.and.certain.viral-like.particle.(VLPs).vaccine.candidates.contain.more.than.two.hetero-subunits.74,75. High-level. co-expression. and. assembly. of. these. types. of. proteins. in.plant.systems.have.not.been.reported.as.of.today..A.Gemini.virus-based.expression.system. developed. recently. by. our. group. and. a. system. based. on. a. disabled. cow-pea.mosaic.virus.RNA-2,.however,.may.provide.the.potential.for.expressing.these.heterooligomeric.proteins.21,76.Successful.production.of.mAbs.by.both.transient.and.stable.transgenic.plants.clearly.indicates.that.a.plant.production.system.is.a.viable.alternative.technology.in.producing.pharmaceutical.proteins.

vaccIneS

The. World. Health. Organization. estimates. that. approximately. 30. million. children.are.born.each.year.without.adequate.immunization,.contributing.to.the.majority.of.deaths..One.cost-effective.strategy.for.this.incredible.health.concern.is.to.expand.the.

TABLE 4.2Plant-Derived Human Pharmaceuticals That Have Reached Clinical Trial Stage

Organization ProductPharmaceutical

Target CropClinical

Trial Stage

Arizona.State.University

E. coli.heat-liable.toxin Traveler’s.diseases Potato Phase.I

Hepatitis.B.virus.surface.antigen

Hepatitis.B Tobacco Phase.I

Norwalk.virus.capsid.protein

Norwalk.virus Potato.and.tobacco

Phase.I

Biolex.Therapeutics Alpha.Interferon.(Locteron)

Hepatitis.C Duckweeds Phase.II

Large.Scale.Biology.Corporation

scFvs NHL Tobacco Phase.I

Meristem.Therapeutics Gastric.lipase Cystic.fibrosis Corn Phase.II

Lactoferrin Gastrointestinal.diseases

Corn Phase.I

Planet.Biotechnology sIgA.(CaroRx) Tooth.decay Tobacco Phase.II

Protalix.Biotherapeutics Glucocerebrosidase.GCD

Gaucher’s.disease Carrot.cell.culture

Phase.III

SemBiosys Acetylcholinestrase Nerve.agents Carrot.cell.culture

Phase.I

Insulin Diabetes Safflower Phases.I/II

Thomas.Jefferson.University

Hepatitis.B.virus.surface.antigen

Hepatitis.B Lettuce Phase.I

Rabies.glycoprotein Rabies.virus Spinach Phase.I

Source:. Adapted. from.Chen,.Q..et. al.,.Subunit.vaccines.produced.using.plant.biotechnology,. in.New Generation Vaccines,.Levine,.M.M..Ed.,.4th.edn.,. Informa.Healthcare.USA,. Inc.,.New.York,.2009,.p..77..Copyright.2009..With.permission.from.Informa.Healthcare.USA,.Inc.


utilization.of.available.and.emerging.vaccine.manufacturing.technologies..Therefore,.there.is.a.tremendous.need.for.affordable.vaccines.in.the.world,.especially.in.devel-oping.countries..An.ideal.vaccination.program.should.thus.not.only.contain.the.vac-cine. components. that. can. generate. a. neutralizing. antibody. response,. a. long-term.memory.B.cells.stimulation,.and.a.T.cell-mediated.immunity,.but.should.also.include.a.cost-effective.production.technology.that.can.produce.large.quantities.of.vaccines.affordable.to.people.in.all.parts.of.the.world..Plants.offer.unique.advantages.for.the.production.of.subunit.vaccines.in.terms.of.scale,.speed,.costs,.yield,.and.safety..Our.first. work. in. 1992,. describing. the. expression. of. the. hepatitis. B. surface. antigen. in.transgenic. tobacco,.marked. the.beginning.of.developing. low-cost.strategies. for. the.production. and.delivery. of. vaccines. in. plants.6.Since. then,.more. than.80.vaccine.candidates.have.been.expressed.in.a.variety.of.plant.hosts.and.vector.systems.13,77.For.example,.plant-produced.vaccines.for.an.Influenza.A.M2E.epitope,78.a.human.immu-nodeficiency.virus.1.(HIV-1).epitope,79,80.HBsAg,81,82.and.the.S.protein.of.transmis-sible.gastroenteritis.coronavirus. (TGEV)83,84.have. successfully.demonstrated. their.immunogenicity.. Immunization.with.heat. labile.enterotoxin.B. (LTB),85,86. the.FP1.epitope.of.foot.and.mouth.disease.virus.(FMDV),87,88.and.P. aeruginosa.epitopes89,90.yielded.successful.challenge.trials..In.addition,.vaccine.candidates.for.the.respira-tory.syncytial.virus.(RSV).G.and.F.proteins,91.the.VP6.protein.of.rotavirus,92,93.the.measles.virus.(MV),.hemagglutinin.(H).protein,94,95.an.epitope.from.the.major.sur-face.antigen.of.Plasmodium falciparum. (PfMSP1),96,97. the.VP7.protein.of. rotavi-rus,98. F1. and. V. antigen. of. Yersinia peptis,99,100. E. Domain. III. of. dengue. virus,101.ESAT6—Ag85B.antigens.of.Mycobacterium tuberculosis,102.E7.protein.of.human.papilloma.virus,103.pB5.antigenic.domain.of.smallpox,103.and.PA.and.LF.domains.of.Bacillus anthracis,105,106.have.also.been.successfully.expressed.in.plants..These.data.collectively.demonstrated.that.(1).plants.are.capable.to.express.a.broad.portfolio.of.diverse.vaccines.and.(2).plant-derived.antigens.were.able.to.induce.active.protective.humoral.and.cell-mediated.immune.responses.

For.current.licensed.vaccines,.they.are.the.products.of.complicated.and.expen-sive.downstream.purification.and.their.transport. to.final.point.of.use.and.storage.require.continuous.refrigeration.“the.cold-chain.”.Both.of.these.requirements.add.significant. cost. to. the. immunization. program. and. are. especially. detrimental. to.developing.countries..Fresh.or.dried.plant.parts.containing.subunit.vaccines.may.present.an.ambient.temperature-stable.product.similar.to.the.storage.and.transport.of.fresh.fruits.or.dehydrated.food.products,.thereby.providing.a.possible.solution..This.motivation.and.advances.in.our.understanding.of.mucosal.immunity.and.toler-ance.have.facilitated.further.exploration.of.edible.vaccines.107–112.Studies.have.found.that.when.oral.or.other.mucosal.surfaces.such.as.nasal,.intestinal,.rectal,.and.vagi-nal.mucosal.surface.are.exposed.to.vaccines,.a.strong.mucosal.(sIgA).as.well.as.sys-temic.(IgG).immune.response.could.be.elicited.113–115.Therefore,.at.least.in.theory,.oral.immunization.can.be.achieved.by.simply.eating.edible.parts.of.plants.contain-ing.the.expressed.subunit.vaccines.(Figure.4.2)..The.edible.vaccine.strategy.is.very.appealing.because.it.allows.plants.to.not.only.serve.as.a.low-cost.expression.system.but.also.as.a.novel.delivery.vehicle.for.vaccines..It.will.eliminate.or.reduce.the.need.for.downstream.processing.and,.in.turn,.further.reduce.the.overall.vaccine.produc-tion.cost..In.addition,.the.needle-free.delivery.method.and.natural.preservation.of.


vaccines. in. plant. tissue. will. circumvent. major. infrastructural. challenges. in. vac-cine. storage,. distribution,. and.delivery. and.allow. the. realistic. implementation.of.an.immunization.program.in.countries.where.the.“cold-chain”.and.other.medical.supplies. are. limited.116.The.optimal.candidates. for.oral. subunit. vaccine.develop-ment. in. plants. are. aggregated. pathogen. protein. complexes. as. they. are. naturally.recognized.at.mucosal.sites..For.example,.viral.surface.proteins.that.assemble.into.VLPs. spontaneously. and.bacterial. toxins. that.naturally.aggregate. to. form.multi-meric.complexes.have.been.explored.as.plant-derived.oral.vaccine.candidates.117–119.These.natural.mucosally.targeted.protein.complexes.have.also.been.studied.as.car-riers.for.developing.other.plant-derived.oral.vaccines.as.they.can.be.linked.to.other.antigens.by.protein.fusion.technologies.99,120,121.So.far,.moderate.success.has.been.achieved. in. the. development. of. plant. oral. vaccines. as. many. vaccine. candidates.have.been.expressed.in.edible.plant.parts,.and.five.human.clinical.trials.have.been.performed.with.orally.delivered.plant-produced.vaccines.(Table.4.2).122–125.Results.from.clinical. trials. indicated. that. serum.antibodies. and,. in. some.cases,.mucosal.antibodies.can.be.successfully.stimulated.by.plant-derived.oral.vaccines..Although.

FIGURE 4.2 (See color insert.).Human.volunteers.ate.transgenic.potatoes.in.a.clinical.trial. study. by. Tacket. et. al.238. Transgenic. potatoes. expressing. LTB. antigen. were. peeled,.diced,.and.consumed.by.human.volunteers..Non-transgenic.potatoes.were.used.as.negative.controls.


protective.efficacy.in.humans.has.yet.to.be.determined.for.a.plant-derived.vaccine,.challenge.studies.have.yielded.promising.results.in.animal.models.

Commercial.success.of.this.strategy,.however,.has.not.been.achieved.due.to.chal-lenges.including.the.possible.degradation.of.vaccines.by.the.digestion.system,.poor.rec-ognition.of.certain.antigen.at.mucosal.immune.effector.sites,.and.persistent.concerns.about.whether.edible.vaccines.would.cause.inappropriate.antigenic.tolerance..Technical.difficulties.also.contributed.to.the.lack.of.commercial.success..For.example,.challenges.in.controlling.the.fluctuation.of.antigen.content.among.individual.plants.or.plant.tissue.often.cause.inconsistent.vaccine.protein.content.per.unit.of.plant.tissue..This,.in.turn,.complicates.the.dosage.control.process.and.ultimately.hinders.the.commercial.applica-tion.of.this.strategy.115.This.particular.problem,.however,.is.being.resolved.as.progress.in.genetic.element.manipulations.has.allowed.more.precise.control.of.vaccine.expres-sion.in.plants.126.While.oral.vaccines.still.offer.an.attractive.option.for.vaccine.delivery.in.developing.countries,.the.application.of.this.strategy.in.the.United.States.will.face.regulatory.obstacles,.since.a.vaccine.without.a.strictly.controlled.dose.would.have.tre-mendous.difficulty.in.getting.approval.by.the.Food.and.Drug.Administration.(FDA)..As.a.result,.increasing.efforts.have.been.shifted.to.downstream.processing.to.identify.innovative.and.low-cost.ways.to.purify.or.partially.purify.vaccines.from.plant.tissue.(see.below)..Purified.vaccines.can.be.formulated.with.adjuvants.and.delivered.through.different.immunization.routes.including.oral.and.other.mucosal.surfaces..Thus,. this.new.strategy.is.driven.not.only.by.the.necessity.to.develop.PMP.vaccines.with.defined.dosage.content.but.also.to.address.the.needs.of.a.creative.formulation.with.adjuvants.to.enhance.potency.as.well.as.creating.multivalent.and.multicomponent.vaccines.77,127–129.On.going.research.by.our.group.has.allowed.us.to.establish.downstream.processing.standard.operating.procedures.(SOPs).for.extracting.and.purifying.Norwalk.virus.cap-sid.protein.(NVCP).from.N. benthamiana.plants.under.stringent.federal.current.Good.Manufacturing.Practices.(cGMP).guidelines.13.We.anticipate.that.our.NVCP.material.produced.under.the.cGMP.regulation.will.be.used.in.a.human.clinical.trial.as.a.vaccine.candidate.for.sexually.transmitted.infections.later.in.2011.130

Due.to.its.advantages.in.speed,.scale,.and.cost.for.subunit.vaccine.production,.the.application.of.plant.expression.systems.has.recently.been.expanded.to.develop.and.produce.vaccines.for.emerging.and.re-emerging.diseases,.cancers.as.well.as.agents.of. biological. warfare.. For. example,. vaccines. for. smallpox,104. anthrax,106. dengue.virus,101.avian.influenza.A.virus,131.and.a.personalized.cancer.vaccine.for.follicular.B-cell.lymphoma67.are.some.of.these.new.vaccine.candidates.produced.in.plants.

GLYCOSYLATION OF PLANT-MADE PHARMACEUTICALS

As. with. recombinant. proteins. produced. in. other. systems,. glycosylation. of. plant-derived.pharmaceutical. proteins. is. a. critical. issue..Glycosylation. is.depending.on.a.series.of.posttranslational.modification.steps.by.host.cells,.and.the.outcome.can.seriously.impact.pharmacokinetics,.antigen/receptor.binding,.stability,.effector.func-tions, and. efficacy. of. the. mAbs. and. other. PMPs.. As. such,. recombinant. proteins.produced.by.heterologous.systems.may.have.appreciable.structural.and.functional.dif-ferences.from.the.native.molecules.and.could.be.immunogenic.in.humans..In.general,.the.glycosylation.of.proteins.in.plants.is.similar.to.that.of.mammalian.cells..However,.


plants.do.have.unique.plant-specific.β-1,2-xylose.and.core.α-1,3-fucose.residues.on.complex.N-linked.glycans.and.lack.terminal.β1,4-Gal.and.N-acetylneuraminic.acid.(Neu5Ac).residues.57.Extensive.studies.have.shown.that.variations.in.glycosylation.patterns.do.not.always.lead.to.loss.of.in.vitro.or.in.vivo.function.or.cause.side.effects.in.humans.132.Despite.this.fact,.a.variety.of.strategies.have.been.developed.to.circum-vent.the.potential.problems.associated.with.the.difference.between.plant.and.human.glycosylation. patterns.. For. example,. glycoengineering,. involving. sophisticated.genetic.and.metabolic.engineering.of.plant.with.mammalian.glycosylation.enzymes,.has.recently.emerged.as.the.most.promising.approach.to.“humanized”.glycosylation.in. plants.133. In. one. of. these. studies,. a. “humanized. knockout”. Arabidopsis thali-ana.plant.line.has.been.generated.to.express.a.mAb.134.Similar.transgenic.tobacco.(N. benthamiana). lines. with. humanized. glycans. were. generated. later.135. In. these.plants,. enzymes. for. the.biosynthesis.of.plant-specific.glycans.are. inactivated,.and.results.have.shown.that.the.mAb.was.glycosylated.with.a.mammalian-like.pattern.and.was.structurally.equivalent.to.the.same.mAb.derived.from.mammalian.cells.

In.the.last.few.years,. the.feasibility.of.glycoengineering.at.a.biomanufacturing.scale. was. demonstrated.136. For. example,. an. RNA. interference. (RNAi). strat-egy. was. employed. to. suppress. the. expression. of. endogenous. enzymes. (α-1,.3-fucosyltransferase.and.β-1,2-xylosyltransferase).for.plant-specific.glycans..Results.showed.that.mAbs.produced.in.plant. lines.with.mutated.α-1,3-fucosyltransferase.and.β-1,2-xylosyltransferase.had.no.detectable.plant-specific.N-glycans..More.sig-nificantly,.the.plant-produced.mAb.contained.only.a.single.major.N-glycan.species.and.showed.improved.antibody-dependent.cell-mediated.cytotoxicity.(ADCC).and.effector.cell-binding.activities. in.comparison.with. the.equivalent.mAb.produced.in.mammalian. cells.135.For. certain. class.of. therapeutic.proteins,. the. presence.of.the. terminal. Neu5Ac. residues. is. required. for. their. biological. activities. and. sta-bility..Adding. the. terminal.Neu5Ac. residues. to.plant-derived.proteins. is. the. last.remaining.challenge. for. the. full.humanization.of.plant.N-glycosylation.pathway..Recently,.attempts.were.made.to.express.the.three.key.enzymes.of.the.mammalian.Neu5Ac. biosynthesis. pathway,. namely,. UDP-N-acetylglucosamine. 2-epimerase/.N-acetylmannosamine. kinase,. N-acetylneuraminic. acid. phosphate. synthase,. and.CMP-N-acetylneuraminic.acid. synthetase,. in.plants..Results.showed. that. signifi-cant.amounts.of.Neu5Ac.were.generated.when.the.three.enzymes.are.simultane-ously.expressed.in.plants.137,138.Plant.glycoengineering.has.demonstrated.its.ability.in.producing.mAbs.with.better.glycoform.uniformity.and.enhanced.functionality.than.mammalian.cell.cultures,.therefore.demonstrating.its.potential.in.producing.pharmaceutical.proteins.with.fully.humanized.glycans.

DOWNSTREAM PROCESSING OF PLANT-DERIVED PROTEIN PHARMACEUTICALS

In.the.last.decade,.significant.progress.has.been.realized.in.boosting.PMP.expression.levels.through.the.enhancement.of.genetic.elements.for.stable.transgenic.expression.and.the.development.of.viral-based.transient.expression.systems..As.a.result,.PMP.field. has. increasingly. shifted. its. attention. to. developing. technologies. for. efficient.PMP.recovery.and.delivery..While.“immunization-by-eating”.still.presents.a.viable.


opportunity.to.deliver.plant-derived.vaccines.in.the.developing.world,.considerations.of.licensure.and.regulatory.issues.have.pointed.to.necessity.of.developing.process-ing.technologies.to.produce.vaccines.and.therapeutics.with.defined.unit.dosage.139.Developing.new.technologies.to.minimize.cost.associated.with.downstream.pro-cessing. is. crucial. for. the. full. realization.of.economic.effectiveness.of.PMP. tech-nology,.since.it.can.account.for.up.to.80%.of.the.total.cost.in.a.therapeutic.protein.production.140.A.well-developed. downstream.process.will. increase.manufacturing.productivity,.reduce.cost.of.goods,.enhance.scalability,.and.ensure.the.compliance.of.the.manufacturing.procedures.with.FDA’s.cGMP.regulations.

Similar.to.other.production.systems,.the.goal.of.PMP.downstream.processing.is.to.recover.the.maximal.amount.of.highly.purified.target.protein.with.minimal.number.of.steps.including.tissue.harvesting,.protein.extraction,.purification,.and.product.formula-tion.(Figure.4.3)..The.unique.properties.of.plant.tissues,.however,.present.both.specific.challenges.and.opportunities.for.each.of.the.processing.steps..Column.chromatogra-phy.has.been.extensively.employed.in.PMP.purification,.while.nonchromatographic.methods.are.being.explored.aiming.to.provide.alternatives.for.large-scale.manufacture.

column chromatoGraphy

Chromatography.has.been. the.method.of.choice.for.PMP.purification.due. to. its.resolving. power.. As. a. result,. many.PMPs. have. been. successfully. purified. from.a. diversity. of. host. plants. with. a. variety. of. chromatographic. techniques. tai-lored. for. each. individual. PMP. protein. based. on. its. solubility,. size,. pI,. charge,.

Workingcell bank

Mastercell bank

–80

1 2 3

Agrobacterium productionMedia

Inoculumpreparation Fermentation

(A)(B)

(C)

Seedbank

FIGURE 4.3 (See color insert.).General.large-scale.production.steps.for.plant-made.pharma-ceuticals.from.green.leafy.materials.using.deconstructed.viral.vector.transient.expression.strat-egy..(A).Plant.cultivation..(B).Infiltration.and.growth..(C).Downstream.processing..(Adapted.from.Chen,.Q..et.al.,.Subunit.vaccines.produced.using.plant.biotechnology,.in.New Generation Vaccines,.Levine,.M.M..Ed.,.4th.edn.,.Informa.Healthcare.USA,.Inc.,.New.York,.2009,.p..77..Copyright.2009..With.permission.from.Informa.Healthcare.USA,.Inc.)


hydrophobicity,.and.affinity.to.specific.ligands.and.the.parallel.characteristics.of.plant.host.proteins.13,64,141–143

For.mAb-based.PMPs,.Protein.A.or.G.affinity.chromatography.provides.a.superb.purification. step144. and.has. allowed. successful.purification. of. many. plant-derived.mAbs. and. their. derivatives.21,23,64,145. Usually,. a. two-column. purification. scheme.including.a.protein.A.capture.step.and.an.anion.exchange.polish.step.is.sufficient.to.achieve.high.purity.58,146.However,.direct.loading.of.plant.extracts.onto.Protein.A.(or.G).columns.often.causes.resin.fouling.and.poor.binding.of.target.protein.due.to.the.fact. that.plants.produce.much.more.solid.debris.than.other.organisms.and.are.rich. in.phenolics.and.alkaloids.142,147,148.For.example,.clarified. tobacco.extract.was.found.to.foul.Protein.A.Sepharose.and.reduce.the.resin.capacity.147.Ion.exchange,.gel. filtration. chromatography,. and. ultrafiltration. methods. have. been. employed. to.remove. the. interfering. molecules,. but. with. very. little. success.147. The. solution. for.this.problem.came.from.a.nonchromatographic.method.called.aqueous. two-phase.partitioning.system.(ATPS)..It.was.found.that.ATPS.could.effectively.remove.phe-nolics,.toxic.alkaloids.and.other.plant.compounds.from.tobacco.extract..This.extract.could.then.be.directly.loaded.to.a.Protein.A.column.without.causing.resin.fouling,.which.allowed.efficient.separation.of. target.mAbs.(anti-HIV.mAb.2F5.and.2G12).from.tobacco.host.proteins.149.ATPS.has.also.been.shown.to.be.effective.in.removing.interfering.plant.molecules.from.extracts.of.other.plant.species.150–152

Protein.A.is.an.expensive.resin.and.requires.extreme.low.pH.(2.0–2.5).in.the.elu-tion.step..In.large-scale.productions,.the.resins.are.routinely.recycled.up.to.50.times.to.maintain.a.reasonable.cost.153.This.practice,.however,.often.leads.to.additional.cost.in.cleaning.and.revalidation.of.the.resins,.which.is.necessary.to.prevent.leaching.of.degraded. ligands. into.products.145,149.Extensive.researches.have.been.conducted. to.identify.alternative.resins.with.equivalent.or.better.affinity.to.antibodies.or.cheaper.ways.to.produce.protein.A.resin.

One.alternative.is.to.create.a.single-use.protein.A-related.resin.with.low.produc-tion.cost..For.example,.an.attempt.has.been.made.to.produce.transgenic.protein.A.or.its.fragments.in.plants.to.take.advantage.of.the.inexpensive.nature.of.plant.expression.systems.145.Low-cost.Protein.A.alternatives.have.also.been.developed.in.the.form.of.fusion.proteins..For.instance,.Protein.A.or.its.affinity.components.were.genetically.fused.to.molecules. that.are.capable.of.polymerizing.or.binding.to.high.molecular.weight. structures.. Antibodies. that. bound. to. these. Protein. A-decorated. large. par-ticles. can. then. be. separated. from. plant. host. proteins. by. simple. centrifugation.or.ultrafiltration.(see.discussion.of.“affinity.precipitation”.on.page.102)..Examples.of. this. strategy. include. fusion. proteins. of. Protein. A. with. self-polymerizing. bac-teriophage. capsid. proteins,154,155. high. molecular. weight. bacterial. S-layer. proteins,.cellulose-binding. proteins,. and. starch-binding. domains.156–160. Oleosins,. a. class. of.plant.seed.oil-body-associated.small.proteins,.have.also.been.explored.as.Protein.A.fusion.partners.161–163.This.technology.has.been.developed.and.matured.to.the.com-mercially.ready.stage.by.a.Canadian.Biotechnology.company.Sembiosys,.in.which.the.oleosin–Protein.A.fusion-mAbs.complex.can.be.purified.by.extracting.with.oil.bodies.(www.sembiosys.com)..The.yield.of.oleosin–Protein.A.fusion.molecules.in.safflower.plants.is.relatively.low.and.needs.further.improvement.145.An.immunoab-sorbent.nanoparticle. technology.based.on.a. tobamovirus,. displaying.a.133-amino.


acid.protein.A.fragment,.has.also.been.developed.recently..These.nanoparticles.can.be.produced.at.high.levels.in.plants,.and.the.density.of.the.Protein.A.fragment.on.the.nanoparticle.is.high,.satisfying.the.matrices.of.high.mAb-binding.capacity.and.low-cost.economics.required.for.a.successful.processing.technology..A.simple.mAb.puri-fication.process.can.be.performed.with.these.plant-derived.nanoparticles.to.achieve.>90%.product.purity.145

In.addition.to.protein.A,.many.conventional.chromatographic. resins.have.been.investigated.for.mAb.purification..These.resins.are.less.expensive,.easier.to.scale.up,.and.more.resistant.to.chemical.and.biological.degradations.and,.therefore,.are.more.suitable.for.industrial.manufacturing..It.was.showed.that.a.simple.S-Sepharose.FF.cation.exchange.chromatography.followed.by.immobilized.metal.affinity.chro-matography.(IMAC).on.Zn-iminodiacetic.acid.(IDA)-agarose.can.efficiently.purify.an.anti-HIV.mAB.(2G12).from.corn.seeds.with.90%.purity.64.Histamine.Sepharose.was.also.used.as.an.affinity.resin.in.purifying.mAbs.from.plant.extracts.143.A.>95%.purity.of.a.mAb.can.be.achieved.by.this.resin.in.a.single.purification.step.when.used.with.a.maize.seed.extract..When.the.same.resin.was.used.to.purity.the.mAb.from.tobacco.leaf.extracts,.however,.the.binding.capacity.and.the.purification.efficiency.were. slightly. reduced. due. to. the. interference. of. tobacco-specific. phenolics,. alka-loids,.and.host.proteins.143

“Pseudobiospecific. ligands”. represent. another. class. of. non-Protein. A. resins.for. mAb. purification.. They. include. biologically. engineered. peptides. and. protein.domains.as.well.as.synthetic.mimetics.exhibiting.different.degree.affinity.to.anti-bodies..Comparative.studies.showed.that.the.mAb.affinity.of.these.ligands.(includ-ing.hydrophobic,.thiophilic,.hydroxyapatite,.chelating.metal.ions,.and.mixed.mode.affinity.ligands).is.generally.lower.than.that.of.Protein.A,.but.high.enough.for.mAb.selectivity.164.Overall,.under.optimized.conditions,.the.mAb-binding.specificity.and.capacity.of.these.resins.are.comparable.to.those.of.the.Protein.A,.but.vary.depend-ing.on.the.source.and.the.isotype.of.mAbs.165–167.Advantages.of.pseudobiospecific.ligands.include.reduced.cost.of.production,.mild.elution.condition,.facile.steriliza-tion. and.validation,. resistance. to. chemical. and.biological.degradation,. and. lower.toxicity,.all.of.which.makes.them.favorable.as.large-scale.mAb.purification.alterna-tives..Furthermore,.chemical.modifications.allow.these.affinity.ligands.to.acquire.new.affinities.to.mAb-derivatives.that.traditional.Protein.A.lacks,.therefore.broad-ening.the.range.of.application..For.example,.artificial.PpL.is.able.to.purify.a.Fab.to. >90%. purity. with. 77%. recovery.168. MEP. Hypercel,. IDA-Ni,. epitope. peptides,.Mabsorbent,.affibodies,.and.artificial.Protein.A.are.some.examples.of.pseudobio-specifc.ligands.146.Not.all.artificial.affinity.ligands.have.been.tested.for.plant-derived.mAb.purification..However,.there.is.no.doubt.that.they.will.contribute.substantially.to.the.development.of.economical.Protein.A.alternatives.for.large-scale.manufactur-ing.of.plant-derived.mAbs.

For. purification. of. non-mAb-based. vaccines. and. therapeutics,. there. are. no.platforms. based. on. a. universal. affinity. resin. equivalent. to. Protein. A.. Usually,.the.purification.is.carried.out.by.multiple.steps.of.conventional.chromatographic.methods.and.has.to.be.developed.individually.based.on.the.properties.such.as.the.pI,. size,. hydrophobicity,. and. stability. of. the. target. protein. and. the. contaminat-ing.host.molecules.142.This.time-consuming.and.challenging.process.calls.for.the.


need.to.develop.more.“universal”.or.versatile.purification.methods..Technologies.employing. on. affinity. tags. provide. possibilities. for. such. more. universal. solu-tion.169–173. In. this. strategy,. instead. of. attempting. to. identify. a. specific. ligand. to.the.target.protein,.a.highly.selective.affinity.tag.with.known.ligand.is.fused.to.the.target.protein.by.genetic.engineering..Successful.examples.of.this.strategy.include.pharmaceutical. proteins. tagged. with. biotin,. histidine,. glutathione. S-transferase.(GST),. and. c-Myc.174–179. In. addition,. tags. developed. for. mAb. purification. (see.antibody. purification. discussed. above). such. as. bacteriophage. capsid. proteins,.S-layer. proteins,. cellulose-binding. domains,. starch-binding. domains,. oleosins,.and.tobamovirus.nanoparticle.have.also.been.successfully.used.to.purify.vaccines.and.other.transgenic.proteins.156,162,180–183.The.high.affinity.of.the.Fc.fragment.of.immunoglobulin.to.Protein.A.provides.an.excellent.tag.for.PMP.purification..As.a.result,.many.antigen.proteins.have.been.fused.to.the.heavy.chain.of.antibodies.and.purified.by.Protein.A.affinity.chromatography.184,185. In. fact,.genetic. fusions.of.antigens.to.mAbs.against.them.have.created.a.new.class.of.highly.potent.vac-cines.called.RICs.186.The.mAb.component.in.RICs.allows.not.only.facile.purifi-cation. but. also. enable. them. to. induce. significantly. stronger. immune. responses.in.host.animals.without. the.help.of.adjuvants..In.addition.to.providing.a.conve-nient.purification.tool,.affinity.tag.often.adds.several.other.benefits.to.the.target.protein. such. as. enhancing. its. yield,. solubility,. stability,. and. promoting. its. cor-rect.folding.184,187.However,.no.individual.affinity.tag.alone.is.ideal.in.delivering.all.and.every.possible.benefit..Consequently,.the.tandem.affinity.tag.purification.(TAP).or.combinatorial.tagging.strategy.was.developed.to.deliver.the.maximum.possible.benefit.175,188–193.For.example,.a.dual.affinity.His6-MBP.affinity.tag.vec-tor.was.developed,. in.which. the. MBP. moiety. improves. the. yield. and.enhances.the. solubility.of. the. recombinant.protein.while. the.His. tag. facilitates. its.purifi-cation.194,195.Since. all. tags.have. the.potential. to. interfere.with. the. structure. and.biological.activity.of.the.target.recombinant.protein,.for.certain.applications,.they.have.to.be.removed.from.the.purified.protein.by.site-specific.protease..Challenges.still. remain. in. identifying.proteases.with.both.high.efficiency.and.precision. for.tag.removal.196,197.Some.endoproteases.such.as. thrombin,.TEV.protease,.and.3C.protease.often.leave.one.or.two.extra.amino.acid.residues.at.their.cleavage.sites.and.produce.unnatural.N-terminus.on.the.target.protein.198.In.contrast,.proteases.like.enterokinase.and.factor.Xa.have.better.precision.and.leave.target.proteins.with.their.native.N-termini.197.However,. these.enzymes.have.relatively.low.efficiency.and. routinely. require. high. enzyme. concentrations. and. long. incubation. time,197.which.often. lead. to. nonspecific. cleavage. at. cryptic. sites..Regardless,. the. inclu-sion.of.proteases.in.the.manufacturing.process.should.be.avoided.whenever.it.is.possible.as.it.would.require.extra.step(s).to.eliminate.the.protease.from.the.final.product..This.additional.step(s).would,.in.turn,.increase.the.overall.processing.cost.and.raise.additional.regulatory.concerns.196,197.Consequently,.nonenzymatic.affin-ity.tag.removal.techniques.have.been.explored.to.avoid.such.problems.200–202.For.example,.intein,.a.self-splicing.protein.element,.has.been.used.as.a.fusion.partner.of.affinity.tags.to.replace.protease.cleavage.sites.202.The.preliminary.results.of.this.strategy. are. promising.. However,. further. testing. in. high-throughput. settings. is.necessary.before.it.can.be.applied.for.large-scale.PMP.manufacturing.


One.of.the.most.critical.strategies.for.PMP.purification.is.to.take.early.measures.to.prevent.undesirable.host.proteins,.microorganisms,.and.other.contaminants.from.entering.purification.feed.streams..For.example,.bioburden,.environmental.contami-nants,.and.certain.plant.host.proteins.should.be.reduced.as.early.as.possible.prior.to.the.chromatographic.purification.steps..These.early.measures.are.not.only.important.in.simplifying.the.subsequent.purification.process,.but.are.also.essential.in.ensuring.the.regulatory.compliance.of.the.final.product..For.example,.whole.leaf.homogeniza-tion.should.be.avoided.for.isolating.apoplast-targeted.proteins.with.molecular.weight.<50.kDa..Instead,.they.should.be.extracted.directly.into.the.extraction.buffer.by.a.specialized.centrifugation.technique.203.Since.this.technique.allows.the.extraction.of.target.protein.without.breaking.plant.cells,.the.purification.process.is.greatly.simpli-fied.as.a.result.of.a.reduction.in.host.protein.contaminants..The.carbon.assimilation.enzyme. ribulose-1,5-bisphosphate. carboxylase-oxygenase. (RuBisCo). is. the. major.contaminating.protein. in.plant. leaves,. and. it. should.be. removed. from. the. extract.by.centrifugation.under.low-pH.buffer.conditions.(∼pH.5.3).prior.to.the.chromato-graphic.steps.117.Phenolics,.alkaloids,.and.other.plant.specific.molecules.should.also.be.eliminated.by.nonchromatographic.separation.methods.(see.below).prior.to.col-umn.chromatography.to.prevent.resin.fouling.149.Ongoing.research.by.our.group.has.established.a.purification.process.for.vaccine.candidate.NVCP.based.on.low-pH.precipitation.and.conventional.chromatographic.steps.130.SOPs.based.on.this.purifi-cation.scheme.have.been.established.and.used.in.a.cGMP.production.of.NVCP.for.human.clinical.trials.

nonchromatoGraphIc SeparatIonS

The.promise.of.PMP.production.on.an. agricultural. scale.demands. a.downstream.processing.platform.with.extraordinarily.large-scale.capabilities..Despite.the.wide-spread.use.of.chromatographic.separations.in.PMP.purification,.there.are.growing.concerns.that. this.approach.cannot.be.scaled.up.sufficiently.for.very.large-scale.production..As.a.result,.alternative.technologies.have.been.explored.for.very.large-scale. downstream. processing. of. PMPs,. aiming. to. not. only. enhance. manufacture.scalability,.but.also.to.provide.separation.power.similar.or.better.than.that.of.column.chromatography,.while.reducing.separation.steps.and.the.manufacture.cost..These.technologies.include.ATPS,.precipitation,.and.membrane.chromatography.204

ATPS.has. tremendous.scalability.potential.and.is.able. to.handle.large.biomass.load.compared.to.other.separation.methods.205,206.It.relies.on.the.incompatibility.of.certain.polymer.mixtures. in. forming.homogeneous.solutions,.but. instead.forming.two.immiscible.phases.under.specific.concentrations.207.As.discussed.earlier,.ATPS.is.an.effective.method.in.eliminating.phenolics,.toxic.alkaloids,.pigments,.and.other.compounds.from.plant.extracts.149,150.ATPS.has.been.successfully.used.in.purifying.tobacco-derived.anti-HIV.mAbs.and.protein.subunit.vaccine.candidates.as.well.as.nanoparticles.including.VLPs.149,208–210.ATPS.offers.high.separation.rates.and,.there-fore,.minimizes.the.contact.time.between.PMP.protein.and.deleterious.plant.extract..Inclusion.of.this.system.in.downstream.processing.will.facilitate.plant.extract.clari-fication,.removal.of.phenolics,.alkaloids.and.other.harmful.compounds,.and.partial.purification.of.target.proteins..Overall,.the.advantages.in.low-cost,.scalability,.high.


biocompatibility,.and.ability.for.continuous.processing.qualify.this.system.as.an.ini-tial.fractionation.strategy.for.large-scale.processing.

Precipitation.is.a.simple.and.robust.method.to.separate.a.precipitable.protein.from.other.soluble.protein.contaminants..One.of. the.strategies.to.make.a.target.protein.precipitable.is.to.fuse.it.to.a.stimulus-responsive.polymer..Since.this.type.of.polymer.can.transit.reversibly.between.water-soluble.and.water-insoluble.state.depending.on.temperature,.pH,.or.other.environmental.conditions,.the.target.protein.can.be.easily.separated.from.host.proteins.by.a.simple.centrifugation.or.filtration.step.while.the.polymer.is.in.a.water-insoluble.state.211.Both.centrifugation.and.filtration.work.well.in. separating.precipitated.proteins. from.soluble. ones;. the. latter,. however,. has. the.advantage.of. the.possibility.of.being.operated. in. a.continuous.process.212.Several.PMPs.including.human.interleukin-4,.10,213.scFv214.and.anti-HIV.mAbs215.have.been.purified.by.precipitation.using.an.elastinlike.peptide.(ELP)..A.temperature-induced.“inverse.transition.cycling”.allowed.ELP-fusion.proteins.to.transition.to.an.insoluble.state..As.a.result,.the.fusion.proteins.were.easily.purified.by.centrifugation.216

Affinity. precipitation. is. a. special. case. of. precipitation. methods. and. has. been.suggested.as.one.of.the.most.promising.nonchromatographic.techniques.for.large-scale.processing.146.The.power.of.affinity.precipitation.is.derived.from.the.unique.combination. of. the. specificity. and. selectivity. of. biorecognition. with. the. simplic-ity,. robustness,. and. high. concentration. factors. associated. with. precipitation.. This.method. does. not. require. the. fusion. between. target. protein. and. the. precipitation.carrier..Instead,.an.affinity. ligand. is.covalently. linked. to.a.stimulus-responsive.polymer. or. other. molecules. capable.of. forming.high.molecular.weight.particles..Since.there.is.no.fusion.between.the.PMP.and.the.precipitation.carrier,.structural.and.functional. integrity.of. the. target.protein.is.better.preserved,.and.the.need.for.proteases.to.remove.fused.precipitation.carriers.is.eliminated..Affinity.precipitation.has.been.extensively.tested.with.plant-derived.mAbs..As.discussed.earlier,.protein.A.or.its.affinity.components.have.been.fused.to.bacteriophage.capsid.proteins,.bacte-rial.S-layer.proteins,.oleosins,.cellulose,.and.starch-binding.domains,.and.recently.to.tobamovirus.nanoparticles..Binding.of.mAbs.to.these.Protein.A-precipitation.carrier.fusions.has.allowed.efficient.purification.of.a.variety.of.mAbs.from.plants.with.com-parable.purity.and.recovery.as.mAbs.purified.by.Protein.A.chromatography,.but.with.much.superior.cost.effectiveness.and.scalability.145.Similarly,.other.affinity.ligands.have.also.been.successfully.used. to.create.agents. for.affinity.precipitation.of.affin-ity-tagged. vaccines. and. non-mAb. therapeutics.162,183. Another. variation. of. affinity.precipitation.is.magnetic.separation,.in.which.magnetic.nanoparticles.are.decorated.with.affinity.ligands,.and.nanoparticle-bound.target-protein.can.be.then.separated.from.other.soluble.proteins.by.using.magnets.217,218.Plant-derived.mAbs.have.been.successfully.purified.by.such.method.24.In.addition.to.using.magnetic.crystals,.mag-netic.particles.can.also.be.produced.by.certain.bacteria,.which.can.be.engineered.to.display.Protein.A.or.other.affinity.ligands.on.their.surface.219,220

Another. attractive. alternative. to. column. chromatography. is. membrane. separa-tion or. membrane. chromatography.. The. effectiveness. of. these. membrane-based.separation. techniques.has. been. successfully. demonstrated. in.purifying. mAbs.and.other.therapeutic.proteins.221,222.For.example,.an.anion-exchange.membrane.efficiently.removed.impurities.from.the.feed.stream.when.used.in.a.flow-through.mode.223,224.


The.binding.capacity.of. this.membrane.is.much.higher. than.that.of. the.equivalent.anion-exchange.resins..One.of.the.attractive.features.of.membranes.is.that.they.espe-cially.have.higher.binding.capacity.for.large-size.impurities,.which.are.abundant.in.feed.streams.and.a.major.cause.for.column.clotting.225.Since.plant.feed.streams.have.unusually. abundant. high-molecular-weight. impurities,. membrane. separation. may.provide.a.promising.noncolumn.chromatographic.solution.for.problems.associated.with.large.chromatographic.columns,.such.as.low.flow.rate.and.expensive.operations.in.column.packing,.cleaning,.and.validation..In.fact,.preliminary.tests.have.shown.that. this. technique. can. be. successfully. applied. in. purifying. PMPs.. For. example,.separations.with.a.combination.of.cation-exchange.and.protein.A.membranes.have.successfully.purified.a.tobacco-derived.antipseudomonas.human.mAb.(06ad).226.The.high.binding.capacity.and.scalability.of. this. technique.make. it.highly. suitable. for.very.large-scale.manufacturing.operations..Many.speculated.that.membrane.separa-tion. is. likely. to. be. the. first. nonchromatographic. method. used. in. production-scale.processing.of.biopharmaceuticals.164

Regardless.of.the.purification.method.used.or.the.nature.of.a.specific.target.pro-tein,.the.overall.extraction.and.purification.design.has.to.be.robust,.scalable,.cost-effective,.and.compliant.to.cGMP.regulations..An.ideal.purification.scheme.should.have.no.more.than.three.processing.steps.in.order.to.achieve.high.product.purify.while.maintaining.a.practical.recovery.rate..In.addition,.the.PMP.purification.and.manu-facturing.process.must.be.governed.by.an.independent.quality.management.system.(QMS).similar. to.regulations.used.by.other.expression.systems.227,228.Accordingly,.materials.and.processes.widely.accepted.by.the.pharmaceutical.industry.should.be.considered.first.during.process-development.to.minimize.future.regulatory.expendi-ture..Furthermore,.in.spite.of.the.lack.of.animal.viruses.and.other.infectious.agents.in.plants,.it.must.be.validated.that.the.purification.process.is.adequate.in.removing.endotoxin,. phenolics,. and. alkaloids. as. well. as. potential. herbicide. and. insecticide.contaminations.from.the.final.product.

PLANT SPECIES USED FOR PMP PRODUCTION

A.diversity.of.plant.species.and.expression.systems.has.been.used.to.express.phar-maceutical.proteins..The.choice.of.a.system.for.a.particular.transgenic.protein.pro-duction.depends.not.only.on.specific.biological.properties.of.the.target.protein/host.plant. species. but. also. on. economical,. regulatory,. and. social. factors.. The. optimal.plant.expression.system.for.a.target.pharmaceutical.protein.must.have.(1).one.or.more.expression.cassettes.with.genetic.elements.that.can.drive.the.high-level.expression.of.the.transgene,.(2).the.ability.to.target.the.transgenic.protein.to.a.cellular.compart-ment.where.it.is.stable,.and.(3).technologies.to.isolate.and.purify.the.target.protein.from.host.plants..The.optimal.plant.species.coupled.to.the.optimal.expression.system.must.also.(1).produce.high-yield.biomass,.(2).have.facile.scalability,.(3).have.suffi-cient.biomass.stability.during.the.required.storage.period,.and.(4).have.a.reasonable.land.and. labor. requirement. for.plant.growth.and.harvesting..Other.economic. fac-tors.may.include,.for.example,.the.value.of.the.protein.product,.which.will.dictate.the.affordability.of. investment. in.new.equipment.and. labor.as.well.as. in.convert-ing. existing. farming. and. food. processing. infrastructure. for. molecular. pharming..


Other factors such as the. distance. between. sites. of. biomass. production. and. pro-cessing.will.determine.the.required.period.for.biomass.storage,.which,.in.turn,.will.determine.the.choice.between.leafy.and.seed.production.system..In.addition,.consid-erations.have.to.be.in.place.in.choosing.an.expression.system/species.to.address.regu-latory.compliance.and.public.concerns.on.issues.of.transgene.containment,.product.biosafety,.and.others.related.to.GM.plants.in.general.

leafy cropS

Tobacco.and.its.related.Nicotiana.species.have.been.one.of.the.earliest.crop.systems.for.successful.transgenic.plant.pharmaceutical.expression,.in.which.target.proteins.can.be.extracted.directly.from.harvested.leaves..Tobacco.is.one.of.the.easiest.plants.to.transform,.and,.therefore,.the.molecular.tools.for.transgene.expression.in.this.spe-cies. are. well. established.. Biomass. yield. for. tobacco. is. higher. than. most. of. other.leafy.crops..Tobacco.is.also.a.prolific.seed.producer.allowing.rapid.scalability..The.existence. of. large-scale. processing. infrastructures. in. traditional. tobacco. industry.provides.the.advantage.of.quick.adaptation.for.processing.pharmaceutical-containing.biomass.. Since. tobacco. is. a. nonfood,. nonfeed. crop,. the. risk. of. contamination. of.human.food.chain.or.animal.feed.chain.by.transgenic.tobacco.is.relatively.low..All.these.advantages.of.tobacco.make.it.one.of.the.most.advanced.candidates.for.molec-ular.farming.in.commercial.production.of.pharmaceutical.proteins.

Other.green. leaf.expression.systems.also. include.alfalfa,.soybean,.Arabidopsis thaliana,.lettuce,.and.spinach..They.share.many.common.advantages.with.tobacco.including.easy.to.engineer.and.high.biomass.yield..One.of.the.most.advantageous.properties.of. the. leaf.expression.systems. is. that. it.allows. the.flexibility. to.choose.from.all.three.plant.expression.technology.platforms.including.stable.nuclear,.chlo-roplast.transformation,.and.viral.vector-based.transient.expressions..This.flexibility.increases.the.chance.of.success.for.many.target.proteins,.since.multiple.options.are.available. to. identify.an.optimal. expression.platform.based.on. the.property.of. the.target.protein.or.the.nature.of.the.particular.project.

Alfalfa. and. soybean. are. legumes. and,. therefore,. can. obtain. organic. nitrogen.through.nitrogen.fixation..This.may.provide.some.advantages. in. reducing. the.con-sumption.of.nitrogen-containing.chemicals..Alfalfa.also.can.be.harvested.up.to.nine.times.a.year,.adding.to.a.higher.biomass.yield..Arabidopsis thaliana.has.been.a.model.plant.for.plant.molecular.genetics.and.molecular.biology.research..It.has.been.used.to.produce.a.variety.of.pharmaceutical.proteins..However,.the.small.size.of.this.plant.will.limit.its.scalability..Lettuce.and.spinach.are.palatable.without.cooking.and,.there-fore,.have.been.particularly. targeted.to.produce.edible.vaccines. for.oral.delivery.124.Because.they.are.food.crops,.the.biosafety.and.containment.of.the.transgene.are.major.concerns.for.their.commercial.application..One.of.the.drawbacks.for.leaf.expression.systems.is.the.potential.interference.of.expressed.transgenic.proteins.on.plant.growth.and.development..For.this.type.of.proteins,.inducible.promoters.may.be.advantageous.as. transgenic. proteins. are. not. expressed. during. plant. growth. under. the. control. of.such.promoters.but.will.only.be.produced.upon.application.of.specific.inducer.agent.post-biomass.harvest.229.Leaves. also. tend. to. contain.more.pigments,. alkaloids,.and.polyphenols,. which. sometimes. may. be. difficult. to. remove. from. the. target. protein..


The.amount.of.these.compounds.varies.between.plant.species..Tobacco.and.related.Nicotiana.plant.leaves.generally.contain.high.levels.of.phenolics.and.toxic.alkaloids..However,.tobacco.cultivars.with.low-alkaloid.and.other.toxic.metabolites.have.been.developed. for. pharmaceutical. protein. production.230. Protein. stability. is. one. of. the.major.issues.for.leafy.crops..Transgenic.proteins.expressed.in.leaves.accumulate.in.a.more.aqueous.environment.and.thus.tend.to.be.unstable..This.translates.into.a.short.target.protein.shelf.life.in.harvested.green.biomass.due.to.proteolysis..They,.therefore,.must.be.quickly.transported.to.a.processing.facility.at.a.controlled.temperature.to.be.extracted.for.target.protein.or.to.be.processed.by.freezing,.drying,.or.other.means.for.short-term.storage..In.contrast,.pharmaceutical.proteins.produced.in.cereal.seeds.can.remain.stable.for.a.long.period.at.ambient.temperature.(see.below)..Regulatory.con-cerns.for.nonfood,.nonfeed.pharmaceutical.producing.leafy.crops.include.the.potential.leaching.of.recombinant.products.into.the.environment.and.the.exposure.of.herbivores.to.transgenic.proteins..Again,.this.type.of.risks.can.be.reduced.by.using.inducible.pro-moters.to.drive.the.expression.of.the.transgene.so.that.transgenic.proteins.can.only.be.synthesized.in.a.controlled.environment.post.biomass.harvest.229,231

Seed cropS

Expression.of.pharmaceutical.protein.has.been.explored. in.seeds.of.several.crops.including.maize,.rice,.wheat,.barley,.tobacco,.oilseed.rape,.and.a.variety.of.beans.232–234.For. certain. transgenic.proteins. that. required. long-term. storage. in. biomass.before.processing,.expression.in.seeds.driven.by.seed-specific.promoters.is.preferred.over.leafy.expression.systems..In.contrast.to.the.“watery”.leaves,.seeds.are.natural.desic-cators,.which.preserve.functional.proteins.by.a.drying.process.during.their.matura-tion. and,. therefore,. allow. long-term.protein. stability.during. storage..Studies.have.shown.that.mAbs.produced.in.corn.seeds.remain.stable.after.3.years.of.storage.at.room.temperature.and.retain.their.biological.activities.235.Phenolic.and.alkaloid.com-pounds.are.not.present.in.cereal.crop.seeds,.thus.simplifying.downstream.purifica-tion.process.and.reducing.the.overall.production.cost.accordingly..Cereal.seeds.do.contain.higher.levels.of.lectins.that.could.be.difficult.to.remove.from.the.final.prod-uct.especially.if. the.target.proteins.are.glycosylated..Legumes.and.other.oil.crops.such.as.oilseed.rape.are.useful.alternative.systems.for.pharmaceutical.production,.because.their.oil.bodies.can.be.exploited.to.facilitate.target.protein.purification..For.example,.SemBioSys.Genetics.has.developed.an.oleosin-fusion.technology.to.target.pharmaceutical.proteins.to.oil.bodies..As.a.result,.these.proteins.can.be.purified.by.a. simple.extraction.process.236.The. level.of.expression.of. recombinant.proteins. in.seeds.is.generally.lower.than.that.in.tobacco.leaves..This.is.mostly.due.to.the.fact.that.many.of.the.seed.expression.systems.are.still.at.early.developing.stage.and.have.ample.rooms.for.further.optimization.234.An.inherent.disadvantage.of.producing.pro-tein.in.seeds.is.that.it.can.only.be.evaluated.until.seeds.are.produced.after.a.long.growth.cycle..Within.the.three.expression.platforms,.only.nuclear.stable.transforma-tion.is.feasible.for.seed.expression..Transient.expression.techniques.for.seed.expres-sion.have.not.been. well. established,. therefore,. hindering. the.quick. assessment.of.expression.feasibility.for.a.given.product..Seed.crops.offer.both.biosafety.advantages.and.drawbacks.for.expression.of.recombinant.proteins..On.one.hand,.it.reduces.the.


risk.of.product.exposure.to.herbivores.and.other.nontarget.organisms..On.the.other.hand,.however,.the.necessity.of.pollination.for.seed.formation.presents.a.new.risk.for.potential.transgene.escape.into.the.environment.

Maize.was.one.of.the.first.seed.crops.used.for.recombinant.protein.production.237.Compared.with.other.cereal.plants,.it.has.several.advantages.including.high.biomass.yield,.ease.of.transformation,.and.the.availability.of.large-scale.processing.technol-ogy..Since.maize.has.been.a.model.plant.for.genetic.studies,.there.is.a.vast.genetic.and. molecular. knowledge. base. available. for. its. genetic. manipulation.. Maize. has.been.a.successful.seed.crop.for.producing.a.variety.of.recombinant.proteins.includ-ing.mAbs,.biotech.reagents,.even.commercial.grade.b-glucuronidase.and.avidin.10.For.example,.mAb.(2G12),.a.promising.anti-HIV.microbicide.candidate,.was.produced.in.transgenic.maize.64.This.research.showed.that.maize-produced.mAb.neutralized.HIV.as.effectively.as.the.same.antibody.produced.in.Chinese.hamster.ovary.(CHO).cells.. Despite. these. advantages,. disastrous. transgene. outflow. incidents. happened.in.this.crop.have.raised.serious.regulatory.and.public.concerns.on.using.maize.or.other.food.crops.for.recombinant.protein.production.(see.“Regulatory.Concerns.and.Public.Acceptance”.section.of.this.chapter)..A.comparative.study.was.conducted.to.examine.the.yield.of.a.scFv.in.different.seed.and.leafy.crops.including.wheat,.rice,.pea,.and.tobacco..Results.showed.that.the.target.protein.yield.per.unit.of.biomass.was.in.the.order.of.rice.seeds.>.tobacco.leaf.>.wheat.seeds.=.pea.seeds.234.The.highest.yield.per.unit.of.land,.however,.belongs.to.tobacco.plants.because.of.its.high.biomass.production..Since.different.promoters.were.used.for.seed.and. leaf.expression,. the.results.have.to.be.evaluated.with.caution.and.need.further.validation..This.study.did.demonstrate.that.choosing.a.optimal.production.crop.was.relying.on.multiple.factors.such.as.target.protein.yield.per.unit.biomass.and.biomass.yield.per.acre.and.can.be.only.determined.on.a.protein-by-protein.basis.

fruIt and tuber cropS

Pharmaceutical.protein.production.in.fruit.and.tuber.crops.provides.the.opportunity.for.a.novel.approach.of.pharmaceutical.delivery.through.oral.route.by.ingesting.palatable.crop.organs..This.unique.advantage.makes.them.ideal.hosts.for.the.production.of.oral.vaccines.or.topical.therapeutics..For.example,.potato.plants.have.been.explored.as.one.of.the.earliest.plant.systems.for.expression.of.a.diversity.of.vaccines.81,238,239.Our.group.and.others.also.conducted.at.least.three.clinical.trials.by.administering.uncooked.trans-genic.potato.tubers.expressing.exterotoxic.E. coli.LT-B.or.VLPs.of.NVCP.to.human.volunteers..Fruits.such.as.tomatoes.and.strawberries.are.more.edible.than.raw.potatoes.and.thus.are.more.feasible.for.edible.vaccine.development..Tomatoes.have.been.used.for.both.vaccine.and.therapeutic.mAb.expression.due.to.its.high.biomass.yield.and.ease.of.production.in.greenhouses.for.transgene.containment.240.Bananas.have.been.envi-sioned.for.a.long.time.as.hosts.for.edible.vaccine.production.and.delivery.especially.in.Africa.where.vaccination.for.infectious.diseases.is.urgently.needed..However,.difficul-ties.in.generating.transgenic.banana.plants.and.the.long.time.frame.needed.to.grow.fruit-bearing.crops.still.present.hurdles.for.its.practical.application..Since.fruits.and.edible.tubers.are.food.crops,.one.of.the.biggest.obstacles.for.their.commercialization.is.the.regulatory.and.public.concern.for.biosafety.and.transgene.containment.


aQuatIc plantS

One.of.the.most.innovative.and.the.simplest.whole-plant. transgenic.expression.systems.may.be.that.of.employing.the.common.duckweed,.Lemna..Largely.devel-oped.by.Biolex.Therapeutics.(Pittsboro,.NC),.Lemna-based.LEX.Systems.expres-sion. technology. allows. rapid. pharmaceutical. product. development. and. potential.seamless. transition. to. large-scale.manufacture.under.GMP.conditions..This. tech-nology. is. enabled. by. several. advantages. of. the. duckweed. plants. including. small.genome.for.facile.molecular.manipulation,.ease.of.transformation.and.regeneration,.rapid.clonal.propagation,.and.biomass.growth.requiring.only.water.and. inorganic.nutrients..Furthermore,.smaller.pharmaceutical.proteins.produced.in.duckweed.can.be. directly. secreted. into. the. culture. medium.. This. would. eliminate. the. need. for.homogenization.of.plant.tissue.and.significantly.simplify.target.protein.purification.and,.in.turn,.reduce.the.overall.processing.cost..Since.Lemna.plants.produce.neither.pollen.nor.seeds,.but.are.propagated.vegetatively.in.simple.containers,. the.system.also.offers.high.degree.of.containment..The.clonal.nature.of.Lemna.plants.and.the.feasibility.of.growing.them.in.a.controlled.environment.also.allow.better.batch-to-batch.product.reproducibility..Like.any.other.bioreactor.or.container-based.pro-duction.systems,.the.scalability.of.the.duckweed.system.could.be.limited.compared.with. those. of. other. crops.. Biolex. Therapeutics. has. produced. 35. proteins. with.their. LEX Systems. and. their. leading.drug.candidate,.Locteron®,.an.α-interferon.for.the.treatment.of.hepatitis.C,.is.being.examined.in.a.Phase.2b.human.clinical.trial.in.Europe.(http://www.biolex.com,.2011)..Duckweeds.have.also.been.shown.as.an.excellent.host.system.for.therapeutic.mAb.production..Because.of.its.small.genome,.its.glycosylation.pathway.can.be.manipulated.with.relative.ease..This.added.advan-tage.allows.Lemna-derived.mAbs.to.have.uniform.and.“humanized”.glycans.136.It.was.shown.that.Lemna-produced.CD30.mAb.contained.only.a.single.major.N-glycan.species.and.showed.more.superior.ADCC.and.effector.cell-binding.activities. than.those.of.equivalent.mAb.produced.in.mammalian.cells..In.addition.to.Lemna minor.used.by.Biolex,.other.species.in.the.Lemnaceae.family.such.as.Pirodela oligorhiza.and. several. Wolffia. are. also. being. exploited. as. a. source. for. recombinant. protein.production.241

plant SuSpenSIon cell cultureS and SInGle-cell cultureS of alGae

Plant. suspension. cells. are. liquid. culture. of. cells. or. cell. aggregates. derived. from.callus.tissue..Transgenic.plant.cell.cultures.can.be.established.by.transforming.sus-pension. cells. with. Agrobacterium. or. by. using. stable. transgenic. plant. to. produce.the.callus. tissue..Since. these.suspension-cultured.plant.cell. lines.can.be.grown.in.a.controlled.environment,.they.allow.better.product.reproducibility,.a.high.level.of.containment,.and.the.potential.to.manufacture.protein.under.cGMP.similar.to.mam-malian.cell.culture.practices..Even.though.these.culture.systems.share.similar.limita-tions.in.scalability.with.current.bacterial,.yeast,.and.mammalian.cell.cultures,.they.still.provide.useful.alternatives.in.producing.pharmaceutical.proteins.owing.to.the.fact. that. plant. cell. lines. require. less. expensive. culture. media. to. grow.. Moreover,.pharmaceutical.proteins.smaller.than.20.kDa.can.be.directly.secreted.into.the.culture.


medium.readily.for.purification..As.a.result,.many.pharmaceutical.proteins.includ-ing.mAbs,.scFv,.and.their.fusion.proteins.have.been.produced. in.tobacco.or. rice-cell-suspension.cultures.242.In.fact,.the.first.plant-made.pharmaceutical.approved.by.the.FDA.was.a.poultry.vaccine.against.Newcastle.disease.produced.in.tobacco.suspension.cells.243.In.addition.to.tobacco.cells,.rice.and.carrot.cells.are.also.being.developed.for.pharmaceutical.production..For.example,.carrot.cells.are.being.used.by. Protalix. Biotherapeutics,. a. biotechnology. company. in. Israel,. to. produce. the.enzyme.glucocerebrosidase.(GCD).as.an.enzyme.replacement.therapy.agent.to.treat.the.Gaucher.disease..They.have.shown.that.GCD.produced.in.cultured.carrot.cells.has.several.advantages.over.GCD.derived.from.CHO.cells,.including.increased.gly-can.efficacy.and.consistency.and.longer.product.half-life.in.blood.serum.244

Single-cell. cultures.of.algae. such.as.chlorella. and.chlamydomonas. offer. short.generation. time,. fast. vegetative. biomass. growth,. and. require. inexpensive. cultiva-tion.in.water.and.inorganic.nutrients.with.sunlight..The.biased.alga-specific.genetic.codon.usage.has.been.problematic.for.expressing.human.or.animal.genes.in.these.algae,.but.has.been. resolved.by.using. alga-codon.optimized.synthetic.genes.245. It.was.initially.proposed.that.the.algae-based.protein.expression.systems.could.be.eas-ily.maintained.in.ponds.or.larger.water.bodies.for.large-scale.biomass.production..However,.biosafety.and.environmental.concerns.have.limited.the.use.of.these.cul-tures.in.controlled.simple.containers.or.more.sophisticated.flow-through.transparent.“reactors.”.The.green.alga.Chlamydomonas reinhardtii.has.been.a.successful.model.alga.in.expressing.several.pharmaceutical.proteins.including.foot-and-mouth.disease.virus.VP1-CTB.fusion.protein.and.a.scFv.for.herpes.simplex.virus.glycoprotein.D.with.transgenes. transformed. into.either. its.nuclear.or.chloroplast.genomes.246–247.Other.algae. species. such.as.Chlorella ellipsoidea. and.Dunaliella salina.have.also.been.explored.for.PMP.production.246,249

REGULATORY CONCERNS AND PUBLIC ACCEPTANCE

PMP.is.a.potentially.revolutionary.technology.for.providing.vast.affordable.pharmaceu-tical.proteins.to.the.world..However,.as.other.transformative.technological.develop-ments,.PMP.is.also.plagued.with.controversy.and.has.stimulated.significant.public.debate..One.of.the.major.issues.confronting.commercial.PMP.production.is.the.pub-lic.perception.that.the.bioproduction.of.pharmaceuticals.with.food.or.feed.crops.could.potentially.contaminate.the.human.or.animal.food.chains..Some.in.the.agriculture.and.food.industry.also.share.such.concerns.for.reasons.that.any.potential.contamina-tion.could.negatively.impact.their.businesses.250.Many.of.these.concerns.are.caused.by.several.contamination.incidents.that.have.special.resonance.for.the.PMP.field.251.

The.most.publicized.episode.was.the.one.involving.a.Texas-based.biotech.company,.ProdiGene.Corp..In.2002,.corn.and.soybean.harvested.in.Nebraska.and.Iowa.were.found. contaminated. with. ProdiGene’s. transgenic. corn. seeds. intend. to. express. a.transgene. of. TGEV. for. a. candidate. pig. diarrhea. vaccine.. As. a. result. of. the. con-tamination,.more.than.12,000.tons.of.soybeans.have.to.be.destroyed,.and.ProdiGene.was.penalized.by.the.U.S..government.with.a.fine.of.$250,000.251.In.addition.to.the.potential.contamination.of.human.food.supplies,.other.concerns.among.the.general.public.and.scientists.include.the.potential.escape.of.transgene.into.the.environment.


through.outcrossing.with.conventional.crops.or.their.related.wild.species,.exposure.of microorganisms.and.insects.or.other.herbivores.to.transgene.and.transgenic.prod-ucts,.and.possible.immunological.tolerance.caused.by.edible.vaccines.

To.ultimately.address.these.public.concerns,.it.requires.the.diligent.stewardship.by. the.PMP.community,.scientific.and.technical.advancement. in.PMP.production.platforms,.and.a.close.collaboration.between.different.regulatory.agencies.to.estab-lish.clear.and.effective.policies.and.guidelines.for.transgenic.crops.and.PMPs.

better StewardShIp of the pmp communIty

One.of.the.embarrassing.lessons.learned.from.the.ProdiGene.incident.by.the.PMP.community.was.the.need.to.improve.their.stewardship.on.PMPs..Practices.without.consideration.and.common.sense.caused.several.incidents.and.controversies,.which.are.unnecessary.and.avoidable..For.example,.when.Ventria.Bioscience.planned.to.test. their.PMP.carrying. transgenic. rice. in.field.plots,. instead.of.growing. them. in.containment.or.in.a.location.far.away.from.areas.of.major.food.rice.production,.it.proposed.to.grow.them.in.California.Central.Valley.where.rice.production.for.human.food.is.a.major.industry.250.This.proposal.angered.some.local.farmers,.and.the.con-troversy.forced.Ventria.to.abandon.its.original.plan.and.moved.its.testing.plots. to.Kansas..The.ProdiGene.and.Ventria.controversies.have.alarmed.PMP.community.to. develop. stricter. self-disciplinary. guidelines. on. PMP. containment.. As. a. result,.the. Biotechnology. Industry. Organization. established. a. “Reference. Document. for.Confinement.and.Development.of.Plant-Made.Pharmaceuticals.in.the.United.States”.to.express.their.commitments. in.ensuring.the.safety.of.PMPs.during.all.stages.of.development. and. production,. including. full. compliance. with. all. applicable. laws,.regulations.and.guidance,.and.the.regulatory.framework.from.regulatory.agencies.that.oversee.the.PMPs.250.This.document.also.provided.guidelines.for.practical.mea-sures. in.preventing.inadvertent.human.exposure.to. the.transgenic.plants.and. their.expression.product(s). through. food.and. feed.and. in.minimizing.occupational. and.environmental.exposure..This.has.led.the.PMP.community.to.reconsider.the.risks.associated.with.using.food.crops.as.PMP.hosts.and.to.develop.new.expression.plat-forms.to.reduce.such.risks.

alternatIve pmp productIon technoloGy and rISk control

Nonbiological ContainmentTo.prevent.human.and.environmental.exposure.of.transgene.or.PMP.products,.the.simplest. form. of. containment. would. be. having. a. physical. barrier. between. PMP.host.plants.and.their.surroundings..The.barrier.can.be.green.houses.or.geographic.distances..For.example,.SemBioSys,. a.Canadian.PMP.company,.has. adopted. this.approach.and.decided.to.grow.its.insulin-producing.transgenic.safflower.in.an.iso-lated.geographical.area.of.Chile.far.away.from.any.food.or.feed.safflower.crops.250.

Crops.such.as.tomato,.which.is.routinely.grown.in.large-scale.greenhouses.for.food.production,. are. being. explored. as. expression. platforms. for. PMPs. as. they. can. be.easily.adapted.for.PMP.production.in.containment.240.Other.physical.measures.can.


also.be.used.to.prevent.the.exposure.of.environment.to.the.transgene.and.transgenic.products..For.example,.PMP.tobacco.plants.are.routinely.harvested.through.mowing.before.reproductive.organs.are.formed,.and.transgenic.corn.is.detasseled.mechani-cally.to.prevent.transgene.flow.to.the.environment.through.pollen.outcrossing.252.In.addition.to.meeting.the.traditional.requirements.such.as.high.PMP.yield.and.easy.scalability,. many. new. expression. systems. were. developed. with. the. added. advan-tage. for. containment..For. example,. since.duckweeds.produce.no.pollen.or. seeds,.but.propagate.vegetatively.and.can.grow.in.closed.containers,.the.duckweeds-based.LEX.Systems.offers.a.higher.degree.of.containment.than.other.systems..Similarly,.plant.suspension.cultures.are.also.grown.in.controlled.containers,. allowing.better.biosafety.control..Furthermore,.the.feasibility.of.growing.aquatic.plant.and.plant.cell.culture.in.a.controlled.environment.also.allows.better.batch-to-batch.product.repro-ducibility.and.the.ability.of.protein.production.under.cGMP..In.fact,.these.advantages.have.enabled.them.to.be.operated.similarly.to.conventional.bacterial.or.mammalian.cell.cultures.and.to.achieve.considerable.commercial.success.ahead.of.other.PMP.systems..These. successes. include. that. tobacco.suspension.cells.produced. the.first.FDA-approved.plant-made.poultry.vaccine.and.that.PMPs.produced.by.Lemna.or.carrot.cell.cultures.are.being.examined.in.Phase.2.or.Phase.3.human.clinical.trials.(http://www.biolex.com,.2011).

The.transient.expression.technology.based.on.“deconstructed”.viral.vectors.also.provides.advantages. in.biosafety.and.containment.because. it.does.not. involve. the.production.of.transgenic.plants..Since.high.level.of.PMP.product.can.be.obtained.easily.with.this.system,.the.amount.of.biomass.required.for.the.production.of.a.given.quantity.of.PMP.is.reduced,.which.provides.the.possibility.of.performing.the.pro-cesses.in.contained.facilities..This,.in.turn,.will.eliminate.the.need.for.field-grown.plants,.and.the.environmental.concerns.for.the.use.of.“deconstructed”.viral.vectors..Furthermore,.this.system.does.not.require.fermenters.or.containers.to.grow.biomass,.giving.it.an.easier.scalability.than.fermenter-based.duckweed.or.plant.tissue.cultures..This.transient.expression.technology,.therefore,.represents.one.of.the.most.promising.PMP.platforms.for.commercialization.

Biological ContainmentIt.is.necessary.in.certain.circumstances.to.use.biological.containment,.which.is.less.dependent.on.human.oversight.than.physical.containment.as.an.additional.measure.to.ensure.the.biosafety.of.PMP.production..Biological.containment.usually.employs.molecular.or.genetic. techniques. to.either.create.a.biological.barrier.against. trans-gene.flow.into.other.plants.or.organisms.or.to.prevent.the.production.of.transgenic.products.before.host.plants.being.harvested.or.moved.into.a.controlled.environment.

Transgenes.can.flow.from.PMP.transgenic.plants.in.two.major.forms—pollen.and. seeds. carried. by. various. vectors.. Correspondingly,. molecular. genetic. strat-egies. for. containing. transgene. outflow. have. been. focused. on. the. areas. of. male.sterility,.maternal.inheritance,.transgene.mitigation,.and.seed.sterility.(also.called.“terminator”.technology).253.Male.sterility.is.one.of.the.early.strategies.of.biologi-cal.containment.and.is.the.only.strategy.that.has.been.applied.to.commercial.trans-genic.plant.(rape.seeds).production.(Plant.Genetic.systems,.Ghent,.Belgium)..Male.sterile. plants. are. unable. to. produce. mature,. viable. pollen,. therefore. preventing.


transgene. flow. via. pollen.254. Examples. of. such. systems. include. pollen. sterility.in. tobacco. and. oilseed. rape. with. a. barnase. gene. driven. by. a. tapetum-specific.promoter255. and. male. sterility. in. tobacco. with. constitutive. expression. of. the.Agrobacterium rhizogenes.rolC.sterility.gene.256.These.systems.have.been.shown.to. be. successful. in. preventing. transgene. spread. from. GM. flowering. plants. into.other. related.species.254.Since.most.crop.chloroplasts.genes.are. inherited.mater-nally,.transgenes.inserted.into.the.plastid.genomes.are.unlikely.to.be.transmitted.by. pollen. transmission.35. As. discussed. earlier,. transplastomic. plants. have. been.extensively. used. for. PMP. expression. partially. due. to. their. advantage. in. ensur-ing.transgene.containment..To.further.reduce.the.risk.of. transgene.flow-through.pollination,.a.double.fail-safe.strategy.was.developed.using.combinations.of.both.male. sterility. and. transplastomic.genomes.257.One.of. the.handicaps.of. the.male.sterile.or.transplastomic.plants.is.that.they.can.still.be.pollinated.by.nontransgenic.varieties,.and.the.resulting.hybrids.can.serve.as.pollen.donors.to.spread.transgene.into. the. environment.. Transgene. mitigation. is. a. strategy. to. address. the. leakage.and.unidirectionality.handicap.of.male.sterility.and.maternal.inheritance..In.this.technology,.a.mitigation.gene,.which.encodes.a.trait.detrimental.to.wild.plants,.is.genetically.linked.to.the.transgene.258.If.the.transgene.is.escaped.and.transmitted.to.an.unintended.host.plant,.the.mitigation.gene.will.co-escape.with.the.transgene,.and.its.expression.would.prevent.the.hybrid.from.competing.with.wild.plants.and.setting.seed..For.example,.this.strategy.based.on.the.Arabidopsis.gibberellic.acid.insensitive. gene. (Δgai). was. able. to. induce. dwarfism. in. tobacco. plants.. When.Δgai.transgenic.plants.or.hybrids.are.grown.together.with.wild-type.tobacco,.the.dwarf.transgenic.or.hybrid.plants.cannot.compete.effectively.for.sunlight.with.the.taller.wild-type.plants.for.photosynthesis.and.rarely.survive.to.set.seed,.therefore,.preventing.the.establishment.of.transgene.in.the.population.258.Furthermore,.Δgai.gene.also.causes.male.sterility.in.tobacco.plants..The.combination.of.male.sterility.and.mitigation.effect.of.Δgai.makes.it.a.superb.gene.for.preventing.transgene.outflow.and.for.mitigating.the.flow.if.sterility.containment.failure.or.reverse.gene.influx.occurred.254. In.addition. to.pollens,.seeds.are.also.major. routes.of. transgene.and.transgenic. product. spreading. by. human. errors. or. animal. activities.. To. resolve. this.problem,.several.“genetic.use.restriction.technologies”.(GURT).including.the.“ter-minator”. (US.patent.5,723,765).and. its.alternatives.have.been.developed. to.pro-duce. “conditional-sterilized”. seeds.259. Seeds. from. plants. engineered. with. these.“terminator”-like.genes.are.usually.nonviable,.unless.the.plants.are.intentionally.exposed.to.specific.activating.molecules.or.environmental.stimulus..These.chemi-cal. or. environmental. conditions. will. induce. the. expression. of. a. repressor. gene,.which,.in.turn,.will.inactivate.the.terminator.gene..Several.such.technologies.have.been.shown.to.be.effective.260,261.For.example,.transgenic.plants.carrying.the.bar-nase.gene. routinely.produce.non-viable.seeds,.but.can.become.viable.only.after.an.exposure. to.a.heat. treatment.at.40°C.261.Such.seed.sterility. technologies.will.allow.effective.blocking.of.transgene.flowthrough.volunteer.seeds.and.will.prevent.recurrence.of.incidents.similar.to.the.ones.caused.by.ProdiGene..Inducible.expres-sion. systems. have. been. developed. to. prevent. human,. animal,. and. environmen-tal.exposure.to.plant.transgenic.protein.products..In.these.systems,.transgene.are.driven.by.an.inducible.promoter.instead.of.a.constitutive.one..As.a.result,.transgene.


transcription.and.translation.remain.being.switched.off.during.plant.growth.until.upon.the.application.of.the.specific.inducer.molecules.or.stimulus..Therefore,.no.transgenic.products.are.produced.in.the.field,.and.they.are.only.made.after.being.harvested.and.transported.into.a.controlled.environment..Such.inducible.systems.have.been.applied.to.both.nuclear.and.chloroplast.transgenic.plants.229,231.It.is.impor-tant.to.note.that.none.of.the.above.biological.containment.is.absolute.or.is.working.in. all. circumstances.. Novel. containment. strategies. with. high. level.of. reliability.and.simplicity.are.still.highly.desired.for.PMP.plants.

reGulatory polIcIeS reGardInG pmp

In.the.United.States,.the.regulatory.responsibilities.of.PMPs.are.distributed.among.three.relevant.agencies:.the.US.Department.of.Agriculture.(USDA),.the.Food.and.Drug.Administration.(FDA),.and.the.Environmental.Protection.Agency.(EPA).

The.regulatory.environment.has.evolved.tremendously.since.the.infancy.of. the.PMP.development.227,262.At.the.beginning,.the.novelty.and.complexity.of.this.tech-nology.caused.confusions.on.which.regulatory.agencies.should.regulate.it.or.where.the.PMP.would.fit.into.the.regulatory.agencies’.structured.framework.for.biotech-nology.pharmaceuticals..This.uncertain.regulatory.environment.contributed.to.the.inertia.of.large.pharmaceutical.companies.toward.PMPs.and.other.new.production.systems..With.the.great.stride.of.PMP.field.in.the.last.few.years,.however,.the.regula-tory.environment.has.also.evolved.from.a.chaotic.situation.in.which.the.regulatory.bodies.could.not.coordinate.with.each.other.to.a.more.welcoming.climate.where.the.regulatory. agencies. have. a. more. defined. role. in. regulating. PMPs. and. coordinate.with.each.other.and.begin.to.work.with.scientists.to.establish.more.clear.policies.and.guidelines.specifically.for.PMPs.262,264

CONCLUSION

Enormous.strides.have.been.made.in.the.past.decade.with.respect.to.PMP.expression.and.optimization..A.wide.spectrum.of.pharmaceutical.proteins.has.been.success-fully.produced.with.active.functionality.in.a.variety.of.crop.and.noncrop.plant.sys-tems..Plant.expression.strategies.based.on.the.combination.of.viral.vector.transient.expression.and.stable.transgenic.plants.have.the.greatest.potential.to.provide.a.tech-nology.with.the.speed.and.scalability.required.for.producing.affordable.high-quality.pharmaceutical.proteins.to.meet.the.increasing.global.demand..New.technologies.for.downstream.processing.have.received.increasing.attention.and.are.being.developed.to.realize.the.full.potential.of.the.PMP.platform..However,.like.any.biotechnology.innovation,. the.ultimate.commercial.success.of. this. technology.cannot.solely.rely.on. its. scientific. and. technical. merits.. Before. reaching. its. full. commercial. poten-tial,. extensive. work. has. to. be. done. to. overcome. challenges. in. areas. of. its. social.acceptance.and.in.attracting.the.interest.of.capital.investment.and.big.pharmaceuti-cal.companies..Ultimately.with.technical.optimization.and.careful.implementation.under.regulatory.guidelines,.plant.expression.technology.will.support.the.production.of.pharmaceutical.proteins.with.scalability,.speed,.efficiency,.cost-effectiveness,.and.safety.at.a.commercial.scale.


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125

5 Transgenic Fruit Crops in Europe

Henryk Flachowsky and Magda-Viola Hanke

IMPORTANCE OF FRUIT CROPS IN EUROPE

The.popular.adage.“an.apple.a.day.keeps.the.doctor.away”.is.only.one.example.that.illustrates.the.importance.of.fruits.for.human’s.diet..A.grain.of.truth.is.behind.the.adage.because.it.is.a.matter.of.common.knowledge.that.a.diet.rich.in.fruits.and.vegetables.and.low.in.saturated.fats.protects.against.a.number.of.diseases,.such.as.cardiovascular.dis-eases.and.certain.cancers.1–3.The.World.Health.Organization.(WHO).therefore.recom-mends.an.intake.of.more.than.400.g.of.vegetables.and.fruits.per.capita.per.day.(http://faostat.fao.org)..The.mean.daily.consumption.of.one.person.in.Europe.is.561.g.of.fruits.and.vegetables,.which.is.more.than.recommended.(http://faostat.fao.org)..However,.the.daily.intake.ranges.from.about.350.g.in.Slowakia.to.more.than.1100.g.in.Greece..In.southern.Europe.(Greece,.Italy,.Portugal,.Albania,.and.Spain),.more.fruits.and.veg-etables.are.consumed.(∼700–1100.g.per.capita.per.day).than.in.middle.and.northern.countries.(Finland,.Iceland,.Belarus,.Latvia,.Ukraine,.the.Czech.Republic,.Poland,.and.the.Russian.Federation).with.about.400–450.g.per.person.per.day.

CONTENTS

Importance.of.Fruit.Crops.in.Europe...................................................................... 125Field.Trials.with.GM.Fruit.Crops.in.Europe........................................................... 127

Apple.................................................................................................................. 127Citrus.................................................................................................................. 131Cherry................................................................................................................. 132Grapevine........................................................................................................... 133Kiwifruit............................................................................................................. 134Pear..................................................................................................................... 135Plum................................................................................................................... 135Raspberry........................................................................................................... 136Strawberry.......................................................................................................... 137

Focus.of.Research.on.GM.Fruit.Crops.in.Europe................................................... 137Improvement.of.Agronomically.Important.Traits.............................................. 137Use.of.Early.Flowering.GM.Plants.to.Accelerate.Breeding.Cycles.................. 137Development.of.a.Rapid.Assay.for.Gene.Function.Analysis............................. 138Development.of.New.Systems.for.GM.Fruit.Crop.Production.......................... 139

References............................................................................................................... 142


Fruit.crop.production.is.of.particular.economic.importance.in.Europe..In.2007,.a.total.of.77.5.million.metric.tons.of.fruit.crops.were.produced.in.Europe.on.approxi-mately.8.7.million.hectares.of.land.(http://faostat.fao.org)..Italy,.Spain,.and.France.are.the.main.producers.with.more.than.60%.of.the.overall.production..The.entire.spectrum.of.fruit.crops.produced.in.Europe.consists.of.more.than.30.different.fruit.species.with.grapes,.apples,.and.oranges.as.the.three.most.important..An.overview.of.the.cultivated.fruit.crops.is.given.in.Table.5.1.

TABLE 5.1European Fruit Crop Production in 2007

Fruit CropProduction Quantitya

Area Harvestedb

Number of Countries Leading Countriesc

Grapes 29 4140 28 IT.FR.SP.DE.PT.GR

Apples 14 1319 37 RU.IT.FR.PL.DE.UK.SP

Oranges 6.2 295 11 SP.IT.GR.PT

Watermelons 5.1 310 20 RU.SP.RO.GR.IT.UK

Peaches,.nectarines 4.3 301 24 IT.SP.GR.FR

Pears 3.1 209 35 IT.SP.NE.FR.BE.AU.PT

Tangerines,.mandarins,.clementines

3 177 9 SP.IT.GR

Plums,.sloes 2.5 580 35 SE.FR.RO.SP.IT.UK.BO.RU

Other.melons 2.3 110 14 SP.IT.FR.GR.U

Lemons,.limes 1.5 88 8 SP.IT.GR

Strawberries 1.5 177 36 RU.SP.PL.DE

Currants 0.9 144 27 RU.PL.UK

Cherries 0.8 191 34 IT.BE.FR.SP.RU.UK.GR

Apricots 0.8 118 23 IT.FR.GR.SP.RU.HU.UK

Sour.cherries 0.8 201 21 PL.RU.UK.SE.HU

Kiwifruit 0.6 35 8 IT.FR.GR

Bananas 0.4 12 4 SP.PT

Carobs 0.14 83 5 SP

Figs 0.12 130 12 SP.GR.IT.PT

Gooseberries 0.12 22 15 RU.PL.UK

Avocados 0.1 22 4 SP.PT

Quinces 0.08 11 22 SP.SE.RU.RO

Grapefruit 0.06 3 6 SP.PT.GR.IT

Persimmons 0.05 3 2 IT.SL

Dates 0.013 1 2 AL.SP

Pineapples 0.003 0.3 1 PT

Source:. http://faostat.fao.orga. Production.quantity.in.million.metric.tones.b. Area.harvested.in.thousand.hectaresc. AL—Albania,. AU—Austria,. BE—Belgium,. BO—Bosnia,. FR—France,. DE—Germany,. GR—

Greece,.HU—Hungary,.IT—Italy,.NE—the.Netherlands,.PL—Poland,.PT—Portugal,.RO—Romania,.RU—Russian.Federation,.SE—Serbia,.SL—Slovenia,.SP—Spain,.UK—Ukraine.

127Transgenic Fruit Crops in Europe

The.high.level.of.demand.for.fruit.and.fruit.products.requires.the.availability.of.highly.productive.cultivars.to.satisfy.such.a.demand..Therefore,.breeders.are.always.anxious.to.develop.new.cultivars.with.improved.traits..The.breeding.of.new.cultivars.of.woody.plants.is.time.consuming.and.very.expensive.4.Genetic.engineering.offers.an.exciting.tool.today.to.overcome.several.of.the.existing.problems..On.this.account,.many.European.scientists.have.started.to.develop.and.to.establish.methods.for.the.development.of.trans-,.intra-,.or.cisgenic.fruit.crops..Most.of.these.studies.have.been.performed.under.laboratory.or.glasshouse.conditions..Only.a.few.studies,.which.will.be.reviewed.here,.have.been.performed.in.the.open.field.

FIELD TRIALS WITH GM FRUIT CROPS IN EUROPE

In. Europe,. deliberate. releases. of. genetically. modified. (GM). plants. into. the. envi-ronment.require.notification.according.to.Directive.2001/18/EC.(http://gmoinfo.jrc.ec.europa.eu)..Directive.2001/18/EC.regulates.the.release.of.GM.plants.for.the.per-formance.of.field.trials.as.well.as.for.introduction.into.the.market..A.petition.for.a.deliberate.release.of.GM.plants.into.the.field.must.be.submitted.for.approval.by.the.national.authorities.of.the.member.state.in.which.the.release.will.be.conducted..At.that.time,.the.other.European.Union.(EU).member.states.are.informed.by.sending.them.a.SNIF.(summary.notification.information.format)..All.SNIFs.that.have.been.submitted.to.the.national.authorities.of.the.individual.EU.member.states.since.2001.are.available.at.the.European.Web.site,.http://gmoinfo.jrc.ec.europa.eu/..This.Web.site.is.managed.by.the.Joint.Research.Centre.of.the.European.Commission.on.behalf.of.the.Directorate.General.for.the.Environment.

However,.GM.fruit.crops.are.still.quite.far.away.from.commercial.use.in.Europe..In.recent.years,.a.total.of.44.SNIFs.for.a.deliberate.release.of.GM.fruit.crops.into.the.field.have.been.submitted..In.several.countries,.GM.plants.of.apple,.citrus,.pear,.plum,.strawberry,.grapevine,.raspberry,.and.kiwifruit.should.have.been.planted.in.the.field,.but.in.the.end,.only.a.few.field.trials.have.been.initiated.

apple

For. apple,. a. total. of. nine. summary. notifications. can. be. found:. four. from. the.Netherlands,. two. from. Belgium,. two. from. Sweden,. and. one. from. Germany.(Table 5.2)..All.notifications.had.the.goal.to.release.GM.plants.for.scientific.inves-tigations..The.intended.field.trials.were.focused.on.transgenic.plants.of.different.apple.scion.and.rootstock.cultivars.with.improved.resistance.to.fungal.or.bacte-rial.diseases.or.on.plants.with.a.better.rooting.ability.(Table.5.2)..Only.two.field.trials.with.GM.apples.have.been.performed.in.Europe.until.now..The others.were.refused.by.the.national.authorities.or.by.the.regional.minister.or.they.could.not.be.started.because.of.the.lack.of.consent.given.by.the.competent.authority.

One.field.trial.has.been.performed.in. the.Netherlands.(notification.numbers.B/NL/02/03,. B/NL/04/02).. This. field. trial. has. a. long. history. as. it. was. far. from.easy.to.obtain.the.planting.approval..The.field.trial.proposal.was.first.submitted.in.the Netherlands..After.rejection.of.the.notification.by.the.Dutch.minister.in.2001,.it.


TABLE 5.2Summary Notifications for the Deliberate Release of GM Fruit Crops in Europe

SpeciesLeading

CountriesNotification

Number Species/Cultivar Improved Trait

Apple NL B/NL/98/06 Gala,.Elstar Fungal,.bacterial.resistance

NL B/NL/00/04 Paradise.apple Fungal,.bacterial.resistance

NL B/NL/02/03 Gala,.Elstar Fungal,.bacterial.resistance

NL B/NL/04/02 Gala,.Elstar Fungal,.bacterial.resistance

BE B/BE/02/V1 Gala,.Elstar Fungal,.bacterial.resistance

BE B/BE/03/V1 Elstar Self-fertility

SW B/SE/99/1644 M26,.M9 Rooting.ability

SW B/SE/04/1227 M26,.M9 Rooting.ability

DE B/DE/03/140 Pinova,.Pilot,.Reka,.Remo,.Elstar,.Royal.Gala,.AU.56-83

Fungal,.bacterial.resistance

Carrizo.citrange

SP B/ES/06/43 C. sinensis.×.P. trifoliata Plant.architecture

SP B/ES/08/03 Carrizo.citrange Plant.architecture

SP B/ES/08/21 Carrizo.citrange Early.flowering

Cherry IT B/IT/98/27 P. avium Root.formation

IT B/IT/98/28 P. avium Root.formation

IT B/IT/98/29 P. avium Root.formation

Grapevine FR B/FR/99/03/10 V..×.berlandieri Virus.resistance

FR B/FR/96/03/14 V. vinifera Virus.resistance

FR B/FR/94/11/04-CON V. berlandieri.×.riparia,.V. berlandieri.×.rupestris

Virus.resistance

V. vinifera.×.berlandieri

FR B/FR/04/05/01 V. vinifera.×.berlandieri.41B Virus.resistance

IT B/IT/99/26 Thompson.seedless.(Sultana) Parthenocarpic.fruits

DE B/DE/98/100 Dornfelder,.Seyval,.Riesling Fungal.resistance,.gene.flow

Kiwifruit IT B/IT/98/24 A. deliciosa Root.formation

IT B/IT/98/25 A. deliciosa Root.formation

IT B/IT/98/26 A. deliciosa Fungal.resistance

Lemon IT B/IT/04/03 Citrus sp. Fungal.resistance

Pear SW B/SE/04/1227 BP10030 Rooting.ability

Plum SP B/ES/96/16 Stanley.C5 Virus.resistance

SP B/ES/05/14 Stanley.C5 Virus.resistance

CZ B/CZ/06/03 Stanley.C5 Virus.resistance

RO B/RO/07/04 Stanley.C5 Virus.resistance

PL DOPgmo.4301/02-4/2002

Stanley.C5 Virus.resistance


was.decided.to.try.to.obtain.the.permit.for.a.field.trial.in.Belgium..The.government.bodies.and.advisory.boards.to.the.Belgian.king.advised.in.favor.of.approving.the.permit,.but.the.Flemish.Minister.for.the.Environment.used.its.veto.right.to.refuse.the. trial.. Subsequently,. the. scientists. went. back. to. the. Netherlands. submitting. a.modified. version. of. the. notification,. and. after. a. change. of. government. the. new.minister.granted.the.permit..The.field.trial.(notification.number.B/NL/02/03).was.planted.in.2003,.but.the.permit.was.challenged.by.Greenpeace.and.revoked,.and,.subsequently,.a.temporary.permit.was.issued.by.the.minister..This.permit.was.later.replaced.by.a.new.permit.(B/NL/04/02)..The.new.permit.was.challenged.again,.but.remained.in.force.and.was.confirmed.by.the.High.Court.in.2005..This.field.trial.was.performed.with.GM.plants.of.the.apple.cultivars.‘Gala’.and.‘Elstar’.overexpress-ing.the.hth.gene.of.barley..The.hth.gene.encodes.for.a.type.1.alpha-hordothionin.and.the.GM.plants.were.tested.in.the.field.(Figure.5.1).for.their.resistance.to.apple.scab.(Venturia inaequalis),.powdery.mildew.(Podosphaera leucotricha),.and.fire.blight.(Erwinia amylovora)..This.field.trial.ended.in.2008.(Frans.Krens,.personal.communication).

TABLE 5.2 (continued)Summary Notifications for the Deliberate Release of GM Fruit Crops in Europe

SpeciesLeading

CountriesNotification

Number Species/Cultivar Improved Trait

Raspberry IT B/IT/99/25 R. idaeus Fruit.ripening

Strawberry IT B/IT/98/32 F..×.ananassa Fungal.resistance

IT B/IT/98/33 F..×.ananassa Root.formation

IT B/IT/99/23 F..×.ananassa Development

IT B/IT/99/24 F. vesca Fruit.ripening

IT B/IT/02/11 F..×.ananassa Parthenocarpic.fruits

SP B/ES/98/06 Fresa Fruit.development,.fruit.quality

SP B/ES/98/10 Andalucía Pollen.flow,.out-crossing

GB B/GB/95/R23/2 F. virginiana.×.chiloensis Insect.resistance

Sweet.orange

SP B/ES/96/15 Orange.cv..‘Pineapple’,.lime cv..‘Mexicana’,.citrange.cv..‘Carrizo’

Pollen.flow,.transgene.stability

SP B/ES/08/02 Navelina Modified.fruit.aroma

SP B/ES/08/04 Pineapple Resistance.to.Phytophthora

SP B/ES/08/05 Pineapple Flowering.time

Note:. NL—the.Netherlands,.BE—Belgium,.SW—Sweden,.DE—Germany,.IT—Italy,.FR—France,.CZ—Czech.Republic,.RO—Romania,.PL—Poland,.SP—Spain,.GB—United.Kingdom.


Another. field. trial. has. been. carried. out. in. Sweden. (notification. numbers.B/SE/99/1644. and. B/SE/04/1227).. This. field. trial. was. performed. with. GM.plants.of. the.apple.rootstocks.M26.and.M9/29. transformed.with. the.rolB.gene.of.Agrobacterium rhizogenes.to.improve.rooting.ability..The.GM.rootstocks.were.char-acterized,5–8.planted. into. the.field. in.2001,.and.budded.with.different.apple.culti-vars.including.‘Aroma’,.‘Jonagold’,.‘Elstar’,.‘Discovery’,.and.‘Elise’.in.2002.9.The.transformed.rootstocks.were.compared.to.non-GM.rootstocks.for.traits.such.as.tree.growth,. tree.anchorage,. tree. size,.branching,. leaf. form.flowering,. fruit. set,. fruit.quality,.transgene.stability,.and.systemic.transport.of.transgene.products.9,10

In.order.to.accelerate.classical.breeding,.molecular.techniques.can.be.applied.to.enrich.existing.commercial.apple.cultivars.with. functional.alleles. from.sexu-ally.compatible.plants,.preventing.genetic.drag.and.keeping. the.genetic.makeup.of. the. commercial. cultivar.. This. concept. is. termed. ‘cisgenesis’.11. Cisgenesis. is.defined.as.the.genetic.modification.of.a.recipient.plant.with.natural.gene(s).from.a.sexually.compatible.plant.12,13.In.cisgenesis,.no.foreign.genes.are.allowed.in.the.

FIGURE 5.1 (See color insert.).Dutch.field.trial.with.GM.apples.overexpressing.the.hth.gene.from.barley..GM.apple.plants.of.the.cultivars.‘Gala’.and.‘Elstar’.were.tested.for.their.resistance.to.apple.scab,.fire.blight,.and.powdery.mildew..The.field.trial.was.finished.in.2008..The.maximum.area.of.the.site.was.3850.m2..(The.figure.was.kindly.provided.by.Frans.Krens.from.PRI.Wageningen,.the.Netherlands.)


final.product..If.no.novel.traits.are.introduced.compared.to.classical.breeding,.it.can.be.argued.that.such.plants.are.as.safe.as.plants.from.classical.breeding,.and.should. be. exempted. from. the. GMO. regulations.12–15. In. the. near. future,. several.releases.of.cisgenic.apple.plants.are.expected.in.Europe..Scientific.groups.in.the.Netherlands,. Italy,. and.Switzerland.are.working.on. the.development.of. cisgenic.apple.plants.with.improved.resistance.to.apple.scab.using.the.HcrVf2.scab.resis-tance.gene.originating.from.the.crab-apple.Malus floribunda.821..The.HcrVf2.is.one.of.four.genes.clustered.at.the.Vf-region.in.the.M. floribunda.821.genome..All.four.genes.encode.for.receptor-like.proteins.with.similarity.to.the.Cladosporium fulvum.resistance.gene.family.of.tomato.and.were.therefore.named.HcrVfs.(homo-logues.of.C. fulvum.resistance.genes.of.the.Vf-region).16.Later.Barbieri.et.al.17.and.Belfanti.et.al.18.confirmed.that.the.HcrVf2.gene.induces.scab.resistance.in.trans-genic. plants. of. susceptible. apple. cultivars. (for. review. see. Gessler. et. al.19).. This.gene.has.become.one.of.the.most.promising.genes.for.biotechnological.approaches.on. apple. in. the. world.. As. cisgenesis. allows. stacking. of. desired. genes,. multiple.traits.can.be.brought.together..The.HcrVf2.gene.is.race.specific,.and.there.is.much.interest.for.stacking.with.other.scab.resistance.genes,.providing.resistance.to.other.isolates.of.the.pathogen..There.are.also.several.new.resistance.genes.for.apple.scab.that.are.nearly.isolated.and.functionally.analyzed,.like.Vr2.and.Vm..By.stacking.these.genes,.a.broad.spectrum.of.resistance.to.apple.scab.can.be.realized.leading.to.an.increase.in.durability.of.resistance.11

The.Plant.Research.International.(PRI).in.Wageningen,.the.Netherlands,.the.pri-vate.fruit.breeding.company.Inova.Fruit.BV,.and.two.other.partners.have.set.their.joint.goal.to.release.the.first.cisgenic.scab-resistant.apple.cultivar.in.Europe.in.2012.20

Further.objectives.of.ongoing.projects.are.the.development.of.low.allergenic.GM.apple. cultivars.by. silencing. the.major. apple. allergen. Mal d121. and. cisgenic. apple.cultivars.with.an.increased.amount.of.health-promoting.compounds.by.overexpress-ing.the.recently.identified.MYB.transcription.factors.(MdMYB1.and/or.MdMYB10),.which.are.known.to.upregulate.genes.of.the.flavonoid.biosynthesis.22–24

cItruS

Eight. summary. notifications. can. be. found. for. the. release. of. GM. citrus. plants. in.Europe. (Table. 5.2):. four. for. sweet. orange. (B/ES/96/15,. B/ES/08/02,. B/ES/08/04,.B/ES/08/05),.three.for.citrange.(B/ES/06/43,.B/ES/08/03,.B/ES/08/21),.and.one.for.lemon.(B/IT/04/03)..All.summary.notifications.for.GM.citrus.plants.have.been.sub-mitted.in.Spain,.except.for.lemon.which.was.submitted.in.Italy.

The. first. field. trial. with. GM. citrus. plants. in. Europe. (notification. number.B/ES/96/15). was. planted. in. 1997.. This. field. trial. was. notified. by. the. Instituto.Valenciano. de. Investigaciones. Agrarias. (IVIA). in. Spain. and. spanned. an. area. of.1638.m2..A.total.of.48.GM.trees,.16.orange.(cv..‘Pineapple’),.16.lime.(cv..‘Mexicana’),.and.16.citrange.(cv..‘Carrizo’),.containing.the.nptII.and.the.uidA.gene.were.planted..These.plants.were.used.to.study.transgene.stability,.the.inheritance.of.the.transgenic.traits,.and.the.rate.of.gene.flow.over.a.number.of.years.

The.second.field.trial.with.GM.citrus.plants. in.Europe.(notification.number.B/ES/06/43).was.initiated.3.years.ago.by.the.same.notifier..A.semidwarf.transgenic.


citrange.rootstock.expressing.a.GA20.oxidase.gene.from.citrange.in.antisense.orien-tation.was.planted..The.notifiers.are.investigating.the.effect.of.GM.rootstock.plants.on.nontransgenic.scions..The.main.question.is.whether.the.roostock.confers.compact.size.to.the.non-GM.scion.without.negative.effects.on.yield.and.fruit.quality.

In.2008,.IVIA.submitted.five.additional.summary.notifications.for.the.release.of.GM.citrus.plants..These.field.trials.will.be.performed.in.a.1.ha.area.at.the.experi-mental.fields.of.IVIA.in.the.STA.of.Villareal.(Castellón,.Spain).with.a.total.of.more.than.800.plants..The.first.notification.(B/ES/08/02).was.submitted.for.a.field.trial.with.GM.sweet.orange.plants.of.the.cultivar.‘Navelina’.expressing.a.limonene.syn-thase.gene. from.citrus. in.sense.and.antisense.orientation..The.GM.plants.will.be.tested.for.the.effect.of.the.altered.endogenous.content.of.limonene.on.fruit.aroma,.on.phenological. and.morphological.characteristics.of. the. transgenic. trees,.and.on.possible. new. interactions. of. transgenic. fruits. with. herbivore. insects,. as. Ceratitis capitata,. and. predators. of. herbivores.. Furthermore,. a. new. field. trial. (notification.number.B/ES/08/03).for.GM.citrange.plants.expressing.a.GA20.oxidase.gene.from.citrange.in.antisense.orientation.was.initiated..This.field.trial.has.the.same.goals.as.the.second.(notification.number.B/ES/06/43).described.above..Another.notification.(B/ES/08/04).was.submitted.for.GM.sweet.orange.plants.of.the.cultivar.‘Pineapple’..The.GM.plants.overexpress.the.PR P23.gene.of.tomato..The.PR P23.gene.encodes.for.an.osmotin-like.protein,.which.led.to.a.higher.tolerance.to.Phytophthora citroph-thora..The. last. two.field. trials. (notification.numbers.B/ES/08/05.and.B/ES/08/21).were.initiated.for.sweet.orange.and.citrange.plants.overexpressing.the.flowering.genes.apetala1.(AP1),.suppressor of overexpression of constans 1.(SOC1),.and.flowering locus t. (FT).. The. plants. will. be. evaluated. for. early. flowering,. as. shown. for. AP1.plants.in.Figure.5.2,.and.for.effects.on.tree.growth,.flower.and.fruit.development,.and.fruit.quality.characteristics..The.notifiers.are.currently.planting.the.five.notified.new.field.trials.(Leandro.Pena,.personal.communication).

In.Italy,.one.summary.notification.has.been.submitted.for.GM.lemon.plants.of.the.commercial.cultivar.‘Femminello.siracusano’.(B/IT/04/03)..This.notification.is.still.in.progress.25.The.GM.lemon.plants.were.transformed.with.the.chit42.gene.(encod-ing. for. an. endochitinase). of. the. microparasite. Trichoderma harzianum. CECT 2413..The transgenic.clones.overexpressing.the.chit42.gene.showed.an.increased.resistance.to.the.phytopathogenic.fungi.Phoma tracheiphila.and.Botrytis cinerea.25.This.proj-ect.is.promising.because.the.cultivar.‘Femminello.siracusano’.is.the.most.important.lemon.cultivar. in.Italy,.and. the.transgenic.clones.could.theoretically.be.cultivated.after.the.field.evaluation..However,.whether.GM.lemon.plants.will.be.cultivated.in.Europe.within.the.next.few.years.or.not.cannot.be.stated.at.the.moment.due.to.strict.GMO.regulations.and.low.consumer.acceptance.

cherry

There.are.three.summary.notifications.for.the.release.of.GM.cherries.in.Italy.(Table.5.2)..The.GM.plants.of. the.cherry. rootstock. ‘Colt’. carrying. the. transferred.DNA.(T-DNA).of.Agrobacterium rhizogenes.were.planted.in.1999.26.The.aim.of.this.study.was.to.improve.the.rooting.ability.of.the.rootstock..However,.GM.cherries.are.still.quite.a.long.way.from.commercial.use.in.Europe.


GrapevIne

A. total. of. six. summary. notifications. (B/FR/94/11/04-CON,. B/FR/96/03/14,.B/FR/99/03/10,. B/FR/04/05/01,. B/IT/99/26,. B/DE/98/100). can. be. found. for. the.deliberate.release.of.GM.grapevine.plants.in.Europe..Petitions.for.the.release.of.GM.grapevine.plants.were.submitted.in.France,.Italy,.and.Germany.(Table.5.2)..However,.GM.grapevine.plants.are.still.quite.a.long.way.from.commercial.use.in.Europe.

The.first.field.trial.with.GM.grapevine.plants.(notification.number.B/FR/94/11/04-CON).was.carried.out.in.a.vineyard.in.the.Champagne.region.of.France..Plants.of.18. transgenic.grapevine. lines.of. the. rootstocks.41B. (V. vinifera.×.V. berlandieri).and.SO4.(V. berlandieri.×.V. riparia).overexpressing. the.coat.protein.gene.of. the.Grapevine fanleaf virus.(GFLV).were.established.in.spring.2006.27.Nontransformed.scions.of.V. vinifera cv..Chardonnay.were.subsequently.grafted.onto.these.rootstocks.and.tested.for.GFLV-type.symptoms.for.several.years..Furthermore,.the.plants.were.used.to.study.the.occurrence.of.recombination.events.between.viral.transgene.tran-scripts.and.ribonucleic.acids.(RNAs).from.indigenous.virus.populations..The.study.published.by.Vigne.et.al.27.provides.evidence.that.the.transgenic.grapevines.did.not.promote.the.emergence.of.viable.GFLV.recombinants.

Another. grapevine. field. trial. (notification. number. B/IT/99/24). has. been. per-formed.in.Italy.(Figure.5.3)..Transgenic.plants.of.the.grapevine.cultivars.‘Thompson.Seedless’. and. ‘Silorca’. overexpressing. the. chimeric. gene. construct. DefH9-iaaM.were.established.in.the.field.at.the.Experimental.Farm.of.the.Marche.Polytechnic.University. in. Agugliano. in. March. 2001.28. These. plants. were. evaluated. for. the.

FIGURE 5.2 (See color insert.).Juvenile.control.(left).and.overexpressing.APETALA1.juve-nile.Carrizo.citrange.plants.showing.flowering.8.months.after.transferring.to.the.greenhouse..The.juvenile.period.of.this.genotype.is.usually.5–7.years..(The.figure.was.kindly.provided.by.Leandro.Pena.from.the.Department.of.Plant.Protection.and.Biotechnology.at.the.Instituto.Valenciano.de.Investigaciones.Agrarias.[IVIA].in.Spain.)


effects.of.DefH9-iaaM.on.plant.vegetative.development,.fecundity,.and.fruit.nutri-tional.quality.from.the.third.to.the.fifth.production.cycles.(2004,.2005,.and.2006)..Costantini.et.al.28.found.that.the.expression.of.DefH9-iaaM.in.grapevine.resulted.in.enhanced.fecundity..The.berry.number.per.bunch.was.increased.in.both.transgenic.cultivars.whereas.the.quality.and.the.nutritional.value.of.the.GM.berries.were.com-parable.to.non-GM.control.fruits.

In. Germany,. one. field. trial. (notification. number. B/DE/98/100). has. been. per-formed.at.the.Institute.for.Grapevine.Breeding.Geilweilerhof.of.the.Federal.Research.Centre.for.Cultivated.Plants.in.Siebeldingen..GM.grapevine.plants.of.the.cultivars.‘Dornfelder’,.‘Seyval’,.and.‘Riesling’.overexpressing.glucanase,.chitinase,.or.ribo-somal. inactivating. protein. encoding. genes. were. planted. in. the. field. and. used. to.investigate.the.distance.of.pollen.(gene).flow.

kIwIfruIt

A. total. of. three. summary. notifications. (B/IT/98/24,. B/IT/98/25,. and. B/IT/98/26).can.be.found.for.GM.kiwifruit.plants.(Table.5.2)..These.notifications.were.submit-ted.by.the.Università.degli.Studi.della.Tuscia.Dipartimento.di.Produzione.Vegetale.in.Italy.for.a.period.from.April.1998.to.April.2007..The.field.trials.were.performed.in.the.province.Viterbo.on.areas.of.650.m2.(B/IT/98/24),.150.m2.(B/IT/98/25),.and.

FIGURE 5.3 (See color insert.).DefH9-iaaM.transgenic.grapevines.grown.in.a.field.trial. at. the. Experimental. Farm. of. the. Marche. Polytechnic. University. in. Agugliano.(Italy).. The. GM. grapevine. plants. were. planted. in. March. 2001. and. tested. for. plant.vegetative. development,. fecundity,. and. fruit. nutritional. quality. from. 2003. to 2005..(The figure. was. kindly. provided. by. Bruno. Mezzetti. from. the. Marche. Polytechnic.University Ancona,.Italy.)


900.m2. (B/IT/98/26).. The. GM. kiwifruit. plants. expressed. either. the. rol. genes. of.Agrobacterium rhizogenes.or.an.osmotin.gene.expected.to.confer.resistance.to.fun-gal. pathogens.. The. field. trials. were. focused. on. the. effect. of. transferred. genes. to.fungal.diseases’.resistance.or.to.the.rooting.ability.of.the.transformed.plants..The.commercial.use.of.GM.kiwifruits.in.Europe.is.not.to.be.expected.in.the.near.future.

pear

In. Europe,. only. one. summary. notification. was. found. for. a. pear. rootstock.. The.intended.field.trial.(B/SE/04/1227).was.carried.out.in.Sweden..The.GM.plants.con-taining.the.rolB.gene.of.Agrobacterium rhizogenes.were.planted.at.the.same.place.as.the.transgenic.apples.

The.GM.pear.plants.have.an.improved.rooting.ability..The.purposes.of.the.field.trial.were.the.evaluation.of.the.effects.of.the.transgenic.rootstocks.on.growth,.flower-ing,.fruit.set.and.fruit.quality.of.grafted.cultivars,.the.stability.of.the.modified.trait.(rooting.ability),.and.the.rooting.ability.of.the.transgenic.rootstocks.themselves.by.stooling..GM.pears.are.still.quite.a.long.way.from.commercial.use.

plum

The.most.important.agronomic.trait.in.plum.is.virus.resistance,.especially.to.Plum pox virus.(PPV),.the.etiological.agent.of.sharka.disease,.which.is.one.of.the.most.devastating. pathogens.. Sharka. disease. can. cause. extensive. economic. losses.29,30.Furthermore,.the.virus.has.quarantine.status.in.many.countries.31.In.Europe,.there.are.about.100.million.stone.fruit.trees.currently.infected.with.the.virus.32.Breeders.are.always.anxious.to.develop.resistant.cultivars,.but.breeding.of.sharka-resistant.plums.is.difficult..PPV.resistance.is.often.polygenic.and.in.addition.may.be.strain.specific..The.long.juvenile.period.of.seedlings.hampers.the.success.of. traditional.breeding. programs. considerably.. Genetic. engineering. offers. an. exciting. tool. to.overcome. some.of. the. existing.problems..Several. recent. studies.have. focused. on.overexpression. of. coat. proteins. of. the. PPV.33,34. The. transgenic. line. C5. (named.‘HoneySweet’).was.selected.because.of.its.high.level.of.resistance..This.line.con-tains.a.multicopy.insert.of.the.cpPPV.gene.that.acts.as.a.single.locus..The.expres-sion.level.of.this.gene.is.reduced.in.HoneySweet,.a.result.of.posttranscriptional.gene.silencing.(PTGS).(reviewed. in.Scorza.and.Ravelonandro31)..Based.on. inoculation.studies,.it.was.found.that.HoneySweet.is.highly.resistant.to.the.major.serotypes.of.PPV..The.stability.and.durability.of.the.PTGS-based.PPV.resistance.of.HoneySweet.was.tested.in.field.trials.in.different.countries.for.a.number.of.years.35–37.Since.the.early.1990s,.field.trials.with.HoneySweet.have.been.performed.in.Europe.and.the.United. States.. In. Europe,. a. total. of. five. summary. notifications. can. be. found. for.GM. plums. (http://bgmo.jrc.ec.europa.eu/deliberate/dbplants.asp).. Field. trials. with.HoneySweet.were.performed.in.Spain.(two),.in.Poland.(one),.in.the.Czech.Republic.(one),.and.in.Romania.(one).

Based. on. the. results. obtained. with. HoneySweet,. a. petition. for. deregulation.was.approved.in.the.United.States.in.June.2007.38.The.Animal.and.Plant.Health.Inspection. Service. (APHIS). of. the. U.S.. Department. of. Agriculture. (USDA).


excluded.the.GM.plum.line.C5.HoneySweet.from.the.regulations.at.7.CFR.part.340.(http://www.isb.vt.edu/cfdocs/fieldtests1.cfm).. It. has. also. been. cleared. by. the.U.S..Food.and.Drug.Administration.(FDA)..Approval.by.the.U.S..Environmental.Protection. Agency. was. granted. in. 2010,. and. subsequent. commercialization. of.HoneySweet.in.the.United.States.is.expected.within.the.next.few.years..However,.GM.plums.are.still.quite.a.long.way.from.commercial.use.in.Europe,.although.the.results.obtained.on.HoneySweet.are.quite.promising.

raSpberry

Only. one. summary. notification. (B/IT/99/23). can. be. found. for. the. release.of. GM. raspberry. plants. in. Europe.. The. field. trial. has. been. performed. at. the.Experimental. Farm. of. the. Marche. Polytechnic. University. in. Agugliano,. Italy.(Figure.5.4)..Forty.transgenic.plants.expressing.the.chimeric.DefH9-iaaM.gene.construct.and.40.control.plants.were.planted.in.four.plots.and.evaluated.for.dif-ferent.fruit. traits.34.The. transgenic.plants.showed.a.significant. increase.in. fruit.number,.fruit.size,.and.total.yield..Furthermore,.they.had.more.flowers.per.inflo-rescence. and. an. increased.number.of. inflorescences.per.plant.. It. is. interesting.to.note.that.the.DefH9-iaaM.gene.construct.has.led.to.parthenocarpic.fruits.on.emasculated.flowers.34

FIGURE 5.4 (See color insert.).DefH9-iaaM.transgenic.raspberry.plants.grown.in.a.field.trial.at. the.Experimental.Farm.of.the.Marche.Polytechnic.University. in.Agugliano.(Italy)..The.GM.raspberry.plants.were.planted.in.2001.and.tested.on.different.fruit.parameters.in.the.field.for.two.consecutive.years.(2002.and.2003)..(The.figure.was.kindly.provided.by.Bruno.Mezzetti.from.the.Marche.Polytechnic.University.Ancona,.Italy.)


Strawberry

Eight.summary.notifications.can.be.found.for.GM.strawberries..Field.trials.with.GM.strawberry.plants.have.been.performed.in.Spain,.Great.Britain,.and.Italy.(Table.5.2)..In.Great.Britain,.transgenic.plants.with.improved.resistance.to.insects.were.planted.whereas.the.GM.plants.planted.in.Spain.were.evaluated.on.fruit.development,.fruit.quality,. pollen. flow,. and. out-crossing.. Unfortunately,. information. on. the. results.of. these. studies. is. scarce..More. information. is.available. for. the. Italian.field. trial..This.field.trial.was.performed.at.the.Experimental.Farm.of.the.Marche.Polytechnic.University. in. Agugliano.. Transgenic. plants. of. Fragaria vesca. cv.. ‘Alpina. W..Original’.and.Fragaria.×.ananassa.breeding.selection.AN93.231.53.expressing.the.chimeric.DefH9-iaaM.gene.construct.and.nontransgenic.control.plants.of.each.were.planted. in. the.field.and.evaluated. for. fruit. traits.34.The. results.obtained.on.straw-berry.were.nearly.identical.to.that.of.DefH9iaaM.transgenic.raspberries..Transgenic.plants.showed.a.significant.increase.in.fruit.number,.fruit.size,.and.yield..They.had.more.flowers.per.inflorescence,.an.increased.number.of.inflorescences.per.plant,.and.parthenocarpic.fruits.on.emasculated.flowers.39.However,.GM.strawberries.are.still.quite.a.long.way.from.commercial.use.in.Europe.

FOCUS OF RESEARCH ON GM FRUIT CROPS IN EUROPE

Improvement of aGronomIcally Important traItS

In.recent.times,.the.European.focus.of.research.on.GM.plants.has.switched.from.the.development.of.efficient.regeneration.and.transformation.protocols,.including.the.selection.of.suitable.marker.genes.to.the.improvement.of.selected.agronomi-cally.important. traits..Whereas.earlier.studies.were.mostly.aimed.at. the.method.of.gene.transfer.itself,.newer.studies.are.focused.on.the.development.of.products.with. improved. traits. for. introduction. into. the.market..For. apple. and.pear,. traits.like.resistance.to.insects,.bacterial.(fire.blight.caused.by.Erwinia amylovora.is.the.main.disease.in.Europe).and.fungal.diseases.(scab.and.mildew),.stress. tolerance.(cold,.heat),.precocity,.color.and.health.properties,.and.reduced.allergenic.potential.are.of.particular.importance..For.Prunus.species.the.improvement.of.resistance.to.the.PPV,.the.most.dangerous.pathogen.in.Europe,.is.the.primary.goal..The.studies.on.small.fruits.like.strawberry.are.mainly.aimed.at.the.improvement.of.resistance.to.insects,.viruses,.and.fungal.diseases.(e.g.,.Verticillium dahliae,.Botrytis cinerea,.and. Colletotrichum acutatum).. Other. traits. such. as. salt. or. freezing. tolerance,.reduced.softening,.sugar.content,.fruit.color,.flavor,.ripening,.and.yield.are.also.of.importance.

uSe of early flowerInG Gm plantS to accelerate breedInG cycleS

Recently,.a.study.was.published.by.a.German.group.which.describes. the.use.of.transgenic.apple.plants.with.a. reduced. juvenile.phase. for.applied.breeding..The.transgenic.plants.were.used.to.accelerate.the.breeding.process.because.fruit.tree.breeding. is. time. consuming,. requires. substantial. space,. and. is. therefore. very.


expensive.4.The.production.of.a.new.apple.cultivar.takes.at.least.15–20.years.and.can.cost.in.the.range.of.€400,000.(Ken.Tobutt,.personal.communication.cited.in.Fenning.and.Gershenzon40)..The.breeding.effort.is.mostly.hampered.by.the.long.juvenile.phase.in.which.plants.are.not.able.to.flower.and.fruit..In.contrast.to.annual.plants.which.flower.within.a.few.months,.juvenility.in.tree.species.can.last.5–40.or.more.years.4.A.full.evaluation.of.a.progeny,.the.selection.of.the.best.seedlings.and.therefore.all.further.breeding.activities.must.wait.until.the.plants.complete.the.juvenile. phase..Therefore,. shortening. the. juvenile.phase. is. always. an. important.breeding.objective.for.fruit.crops..In.recent.years.much.effort.has.been.made.using.biotechnological.methods..Leandro.Pena.and.coworkers.were.the.first.to.describe.the.successful.induction.of.early.flowering.in.a.fruit.tree.species.after.overexpres-sion.of.the.flower.inducing.genes.LFY.and.AP1.from.Arabidopsis thaliana.41.Since.that.time.several.studies.on.different.fruit.crop.species.using.different.genes.have.been. published. which. were. focused. on. the. reduction. of. the. juvenile. phase. (for.review.see.Hanke.et.al.42)..However,.recently.Flachowsky.et.al.4.were.the.first.to.report.that.transgenic.early.flowering.apple.plants.were.successfully.used.for.prac-tical.breeding.(Figure.5.5).

Transgenic.apple.plants.overexpressing.the.BpMADS4.gene.of.silver.birch43.were.pollinated. with. pollen. of. the. apple. wild. species. M. fusca.. Transgenic. seedlings.flowered.within. the.first.season.(Figure.5.6)..They.were.pollinated.with.pollen.of.the.apple.cultivar.‘Topaz’.and.the.first.fruits.were.harvested.in.the.end.of.the.first.growing.season.4.This.work.demonstrated.that.one.crossbred.generation.per.year.is.feasible.and.represents.a.dream.come.true.for.breeders..Several.scientific.groups.in.Europe.are.currently.evaluating.this.system..However,.the.GM.apple.plants.used.in.this.study.were.not.optimal..The.transgenic.plants.constitutively.overexpressing.the.BpMADS4.gene.are.often.malformed.and.the.fruit.yield.and.seed.set.is.very.low.4.Therefore,.new.plants.expressing.the.gene.driven.by.an.inducible.promoter.are.under.development.

development of a rapId aSSay for Gene functIon analySIS

Functional.genome.analysis. in. fruit.crops,.especially. for.genes.expressed. in. the.fruit,. is. often. very. laborious.. Genes. to. be. characterized. must. either. be. overex-pressed. or. silenced. in. transgenic. plants.. The. production. of. stably. transformed.plants.is.time.consuming.and.expensive..A.full.evaluation.of.the.effects.coming.from.the.transgene.is.not.possible.before.the.plant.enters.the.adult.phase,.and.this.can. sometimes. take. up. several. years.. Much. more. complicated. is. the. character-ization. of. genes,. which. are. members. of. a. multigene. family.. Several. transgenic.lines. for.each.gene.are.necessary..This. requires. time,. space,.and. the.associated.expenses..Recently.an.ingenious.method.was.described.by.Hoffmann.et.al.44.on.strawberry,. which. is. based. on. transient. gene. silencing. through. the. infiltration.of. Agrobacterium tumefaciens. carrying. an. engineered. plasmid. into. ripening.fruits..Young.fruits,.which.were.still.attached.to.the.plants,.were.injected.with.an.Agrobacterium.suspension.by.using.a.sterile.1.mL.hypodermic.syringe..The.bac-teria.containing.a.hairpin.gene.construct.were.evenly.distributed.throughout. the.entire.fruit..Using.this.method.it.was.possible.to.silence.the.FaCHS.gene.in.nearly.


the. entire. fruit. (Figure. 5.7)..The. functionality.of. the.Agrobacterium. infiltration.silencing.method.was.confirmed.by.Griesser.et.al.,45.who.silenced.a.glycosyltrans-ferase.encoding.gene.(FaGT1).in.strawberry.fruits.in.the.same.way..The.method.published.by.Thomas.Hoffmann.and.colleagues.provides.a.powerful.tool.for.func-tional.gene.analysis.in.strawberry..The.knowledge.obtained.from.strawberry.can.then.be.easily.transferred.to.other.Rosaceae.crops,.so.that.strawberry.can.be.used.as.a.model.species.in.the.future.

development of new SyStemS for Gm fruIt crop productIon

The.development.of.GM.fruit.crops.in.Europe.is.primarily.based.on.Agrobacterium tumefaciens. mediated. transformation.. Different. tissues,. such. as. leaf. blades,.stem internodes,.axillary.shoot-meristems,.cotyledons,.hypocotyl.slices,.or.shoot.

t

F1

t

P ×

goi

BCx́

goi

t+goi

t goi t+goi

×

FIGURE 5.5 (See color insert.).Schema.of.the.breeding.program.using.BpMADS4.trans-genic.early.flowering.apple.plants.(in.accordance. to.Flachowsky.et.al.4).. t—transgene.(red.mark),.induces.early.flowering;.goi—gene.of.interest.(yellow.mark),.for.example,.resistance.gene. for. scab.or.fire. blight. resistance,. green.color—number.of.positive. traits. is.high. (top.cultivar,.e.g.,.‘Pinova’,.‘Elstar’,.‘Gala’);.brown.color—number.of.negative.unwanted.traits.is.high.(apple.wild.species.used.as.resistance.donor,.e.g.,.Malus.×.robusta.5.for.fire.blight.resis-tance);. blue. arrow—genotype. useful. for. further. breeding. steps,. white. arrow—genetically.improved,. but. nontransgenic. (transgene.and.gene.of. interested.must. be. unlinked. to.obtain.such. genotypes);. P—parental. generation;. F1—first. filial. generation,. seedlings. contain. 50%.genome.(genes/alleles).of.each.parent;.BC′x—generation.after.numerous.pseudo-backcrosses.of.‘t+goi’.genotypes.by.high-quality.cultivars,. the.percentage.of.wild.species.genome.was.reduced.by.backcross.breeding.


apices.have.been.tested.to.determine.the.best.starting.material.for.each.species..However,.the.most.effective.and.reproducible.method.for.plant.regeneration.has.remained. through. adventitious. shoot. formation. and. in.most. cases. leaves. from.in.vitro.shoots.are.used.for.transformation..The.selection.of.transgenic.regener-ants.was.and.is.still.done.by.using.selectable.marker.genes.conferring.resistance.to.selective.chemical.agents,.such.as.antibiotics.or.herbicides..The.presence.of.marker.gene.sequences.in.GM.crops.has.been.critically.discussed.in.recent.years..Public.opinion. in.Europe.demands. that.genetically. engineered.plants. released.into.the.environment.be.marker-free.or.at.least.with.marker.genes.not.conferring.resistance.to.antibiotics.used.for.medical.or.veterinary.purposes..This.and.other.demands.are.considered.in.the.2001.passed.European.Union.directive.2001/18/EC..The.directive.2001/18/EC.forbids.the.release.of.GM.crops.containing.antibiotic.marker.genes.“which.may.have.adverse.effects.on.human.health.and. the.envi-ronment”. beginning. in. December. 2008.. Although. the. European. Food. Safety.Authority. (EFSA). stated. in.2004.and.again. in.2007. that. “the.use.of. the.nptII.gene.as.selectable.marker.in.GM.plants.does.not.pose.a.risk.to.human.or.animal.

FIGURE 5.6 (See color insert.).BpMADS4. transgenic.apple.seedling..First.flowers.were.obtained.approximately.4.months.after.seed.planting..The.seedling.was.obtained.after.cross-ing.a.F1.plant.of.the.cross.T1190.(BpMADS4.transgenic.line.of.the.apple.cv..‘Pinova’,.pub-lished.by.Flachowsky.et.al.43).by.M. fusca.(fire.blight-resistant.apple.wild.species).and.the.scab-resistant.apple.cv..‘Topaz’.


health. or. to. the. environment,”. there. remains. a. reluctance. to. use. this. impor-tant. tool. for.GM.plant. selection..Therefore,. it. is.considered.essential.by.many.research.groups.to.establish.methods.which.allow.the.production.of.marker-free.plants..Clean.vector.technologies.are.the.most.promising.strategy.because.other.methods,. which. are. based. on. sexual. outcrossing. of. the. marker. gene,. are. not.usable. for. fruit. crops.. Self-incompatibility. and. a. high. degree. of. heterozygoz-ity. make. such. methods. often. unsuitable.. The. first. successes. in. the. establish-ment.of.clean.vector.technologies.have.recently.been.reported.for.apple,46.sweet.oranges,47. strawberry,48. and. apricot.49,50. However,. the. development. of. highly.effective. strategies. for. the. production. of. marker-free. plants. is. still. one. of. the.most.important.goals.in.many.countries..For.apple,.there.is.a.large.program.in.progress.at.the.moment..Scientists.of.several.countries.have.joined.into.Working.Group.4.“Biotechnological.Approaches.for.Pome.Fruit.Trees”.of.the.European.COST-Action. 864. “PomeFruitHealth.”. They. evaluate. various. vector. systems.(an. example. is. shown. in. Figure. 5.8). to. find. the. most. usable. system. for. apple.transformation.

Other.technologies.such.as.transformation.without.the.use.of.marker.genes.have.also.been.tested.on.apple.12,13.While.the.results.are.promising,.no.molecular.evidence.exists.to.date.confirming.these.results.

All.of.these.technologies.offer.the.possibility.to.transfer.a.gene.of.interest.from.one.to.another.genotype.from.the.same.or.a.crossable.species.without.DNA.from.noncrossable.organisms..In.Europe.the.first.commercial.GM.plant.will.likely.contain.

ihp- CHS-RNAi Control

FIGURE 5.7 (See color insert.).Transient.silencing.of.the.chalcone.synthase.(CHS).gene.in.strawberry.fruits.(left).after.agroinfiltration.using.the.ihp-CHS-RNAi.gene.construct.(accord-ing.to.Hoffmann.et.al.44)..(The.figure.was.kindly.provided.by.Wilfried.Schwab,.Technical.University.Munich,.Biomolecular.Food.Technology,.Germany.)


a. gene. transferred. from. a. crossable. species. with. its. native. introns. and. flanking.regions,.such.as.native.promoter.and.terminator.in.a.sense.orientation..Such.“cisgenic”.plants11.will.probably.be.sooner.accepted.than.GM.plants.containing.marker.genes.and.genes.of.noncrossable.species.

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FIGURE 5.8 (See color insert.).Heat.stress.induced.removal.of.the.nptII.marker.gene.on.apple..Transgenic.apple.plants.containing.the.nptII.marker.gene.and.an.Flp.recombinase.gene.between. two.FRT.recombination.sites.were.produced..The.expression.of. the.Flp. recombi-nase.is.controlled.by.a.heat-stress.inducible.promoter..Both.genes.(nptII.and.Flp).are.located.between.the.CaMV.35S.promoter.and.the.uidA.marker.gene..In.transgenic.plants.containing.nptII.and.Flp.no.GUS.gene.expression.is.possible.(left),.because.the.CaMV.35S::uidA.reading.frame.is.interrupted..The.reading.frame.is.restored.by.excision.of.nptII.and.Flp..GUS.expres-sion.is.only.possible.in.cells.in.which.both.genes.have.successfully.been.removed.(right)..(The.figure.was.kindly.provided.by.Katja.Herzog,.Julius.Kühn-Institut,.Federal.Research.Centre.for.Cultivated.Plants.(JKI),.Institute.for.Breeding.Research.on.Horticultural.and.Fruit.Crops.Dresden,.Germany;.unpublished.)


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147

6 Transgenic Horticultural Crops on the African Continent

Idah Sithole-Niang

INTRODUCTION

Globally,.the.area.planted.to.genetically.modified.crops.has.continued.to.increase..In.2008,.the.area.planted.to.genetically.modified.organisms.(GMOs).was.125.mil-lion. hectares,. representing. 13.3 million. farmers. in. 25. countries. made. up. of. 15.developing.countries.and.10.developed.countries.1.Of. these.countries,.only. three.African.countries.have.commercialized.GMOs,.namely,.South.Africa,.Egypt,.and.Burkina. Faso,. with. the. latter. two. being. in. their. first. year. of. commercialization..South.Africa.first.commercialized.Bacillus thuringiensis.(Bt).cotton.in.1997,.and.in.subsequent.years,.six.other.crop/trait.combinations.would.be.commercialized:.Bt.maize.in.1998,.round-up.ready.(RR).soybean.in.2000,.RR.cotton.also.in.2000,.herbicide.tolerant.(HT).maize.in.2002,.the.stacked.traits.HT.and.Bt.cotton.in.2005,.

CONTENTS

Introduction............................................................................................................. 147South.Africa............................................................................................................ 149

Bt.Potato............................................................................................................. 149Fruits.................................................................................................................. 150

Egypt....................................................................................................................... 150Squash,.Melon,.Watermelon,.and.Cucumber..................................................... 150

Kenya...................................................................................................................... 150Sweet.Potato....................................................................................................... 150

Uganda.................................................................................................................... 151Transgenic.Banana............................................................................................. 151

Ongoing.Projects..................................................................................................... 151Status.of.Biosafety.Regulation.in.Selected.African.Countries............................... 152Expected.Economic.Effects.of.Introducing.Transgenic Horticultural.Crops.in Ghana.................................................................................................................. 152Way.Forward:.Synergies.and.Lessons.Learned...................................................... 153References............................................................................................................... 153


and.HT.and.Bt.maize.in.2007.2.Three countries.are.still.a.small.number.of.coun-tries.in.Africa.but.nevertheless.a.welcome.improvement.on.previous.years,.as.the.spread.now.covers.all.regional.blocks.on.the.continent. that.could.result.in.viable.synergies..For.the.first.time,.each.block.has.a.regional.lead.country,.Egypt.to.the.north,.Burkina.Faso.to.the.west,.and.South.Africa.to.the.east.and.south..The.signing.into.law.of.the.draft.Biosafety.bill.in.Kenya.in.January.2009.as.well.as.the.fact.that.Kenya.already.has.a.number.of.GM.crops.in.the.pipeline.clearly.places.Kenya.in.the.driving.seat.to.take.the.lead.in.the.east.

In.2004,. the. International.Food.Policy. Research. Institute. (IFPRI).published. a.study.on.the.development.of.transgenic.crop.varieties.in.Africa.3.The.study.showed.that.there.were.37.events.being.developed.on.the.continent,.half.of.which.were.virus.and.insect.resistance.and.the.rest.representing.agronomic.performance,.fungal.resis-tance,.product.quality,.herbicide. tolerance,.and.bacterial. resistance.4.South.Africa.had.the.widest.diversity.of.crops.under.study,.and.the.events.spanned.seven.traits..Twenty-one.out.of.the.28.events.had.been.developed.independent.of.public.private.partnerships.using.local.germplasm..Up.until.2007,.South.Africa.was.the.only.coun-try.to.have.commercialized.GM.crops.on.the.continent.5.These.were.seven.in.all,.maize.with. insect. resistance,.both.white.and.yellow,.HT.soybean,. insect. resistant.cotton,.and.cotton.with.stacked.gene.traits,.made.up.of.insect.resistance.and herbi-cide.tolerance..To.date,.both.Egypt.and.Burkina.Faso.have.commercialized.Bt.maize.and.Bt.cotton,.respectively.1

Globally,. the. transgenic. horticultural. crops. commercialized. to. date. include.tomato,. sweet. corn,. potato,. squash,. papaya,. sweet. pepper,. and. biotech. carnation,.with.the.United.States.and.China.leading.in.research.and.development.(R&D).6.In.Africa,.while.having.a.wide.range.of.horticultural.crops.(vegetable,.fruits,.nuts,.and.ornamentals),. there.are.a. few. transgenic.horticultural.crops. that.have.been.devel-oped,.and. these. include.squash,.melon,.watermelon,.grapes,.apples,.potato,. sweet.potato,.groundnut,.and.banana..These.crops.are.limited.to.four.countries.only:.South.Africa,.Egypt,.Kenya,.and.Uganda.(Table.6.1).

Some.of.the.opportunities.for.developing.transgenic.horticultural.products.desired.by.consumers.and.producers.include7

•. Novel.genetic.methods.for.disease.and.insect.protection•. Weed.control•. Longer-lived.flowers•. Slower-growing.grass

In.terms.of.transgenic.horticultural.crops,.the.limited.market.distribution.and.R&D.efforts.are.not.unique.to.Africa..The.first.transgenic.horticultural.crop,.Flavr.Savr,.developed.to.have.an.extended.shelf.life,.and.another.version.with.higher.viscosity.intended.for.easier.processing.was.commercialized.in.1994.in.the.United.States.but.has. since. been. withdrawn.. Subsequently,. sweet. corn,. potato,. squash,. and. papaya.varieties.made.to.resist.viruses.were.also.developed.but.failed.to.garner.a.significant.market. share..To.date,.only. the.virus-resistant.papaya.enjoys.a. significant.market.share.with.70%.of.the.Hawaiian.crop.shipped.to.mainland.United.States.annually.being.from.this.product.7

149Transgenic Horticultural Crops on the African Continent

The.major.challenges,.on.the.other.hand,.have.been.summarized.by.Bradford.and.Alson7.as.encompassing.technical,.economic,.regulatory,.and.market-related.factors.

While. a. number. of. products. have. been. developed. and. tested,. unfortunately.they.have.not. reached.commercialization.8.Consequently,. there.has.been.a.drastic.decrease.in.R&D.efforts.going.into.horticultural.products.with.investors.choosing.to.invest.in.breeding.and.the.use.of.molecular.markers.9,10

SOUTH AFRICA

bt potato

The.case.studies.on.Bt.potato.in.both.Egypt11.and.South.Africa.have.been.extensively.covered12.and.will.not.be.described.in.detail.here..South.Africa.has.had.11.years.of.experience.working.with.Bt.potato,.and.five.of.those.years.have.been.spent.on.con-fined.field.trials.(CFTs)..The.Agricultural.Research.Council.(ARC).has.now.applied.for.approval.of.larger-scale.field.trials.in.a.number.of.selected.areas.and.that.approval.is.still.awaited..Just.as.was.found.in.Egypt,.the.Bt.technology.for.resistance.to.potato.tuber.moth.works.extremely.well..The.outstanding.safety.concerns.have.to.do.with.stewardship.issues,.seeing.that.South.Africa’s.neighbors,.except.for.Zimbabwe,.do.not.have.functioning.biosafety.regulations.in.place.to.safeguard.the.transboundary.movement.of.potato,.a.vegetatively.propagated.crop.

TABLE 6.1Transgenic Horticultural Crops and Traits under Commercialization and Status of NBFs in Four African Countries

Country Transgenic Hort Crops and Traits Biosafety Law CPB Commercialization

Egypt Watermelon-VR Yes ✓ Bt.maize.2008

Squash-VR

Melon-VR

Cucumber-VR

Potato-IR

Kenya Sweet.potato-VR 2009 ✓ None

South.Africa Grapes-FRPotato-IRSweet.potato-VRStrawberries-RR

1997,.2003 ✓ Bt.maize-yellowBt.maize-whiteBt.cottonRR-soybean

Stacked.gene.traits

Uganda Banana-FR Draft.bill ✓ None

Banana-AP

Banana-BR

Banana-NE

Note:. AP,. agronomic.performance;.BR,.bacterial. resistance;.CPB,.Cartagena.Protocol.on.Biosafety;.FR,.fungal.resistance;.IR,.insect.resistance;.NE,.nutritional.enhancement;.RR,.round-up.ready;.and.VR,.virus.resistance;.NBFs,.national.biosafety.frameworks.


fruItS

The.ARC-Infruitec.has.requested.approval.for.a.CFT.of.fungal.resistant.grapes.to.see.if.the.reporter.gene.works.well..Similarly,.transgenic.apples.have.undergone.CFTs.for.the.last.5–7.years.to.see.if.the.novel.genes.are.expressed..The.first.GM.straw-berry.with.tolerance.to.glyphosate.was.developed.locally.and.tested.in.a.field.trial.in.the.early.1990s..The.RR.gene.worked.well,.but.the.project.did.not.go.commercial..Meanwhile,.the.landscape.for.transgenic.horticultural.crops.might.still.change.given.that.Monsanto.Company.acquired.Seminis.(a.seed.company),.a.year.ago.

EGYPT

SQuaSh, melon, watermelon, and cucumber

Egypt. produces. an. estimated. 46,000. hectares. (ha). of. watermelon,. 28,000.ha. of.squash,.pumpkin.and.gourds,.20,000.ha.cantaloupes.and.other.melons,.and.18,000.ha.of.cucumber.annually..Production.constraints. are.due. to.both.biotic. (viruses).and.abiotic. (drought. and. salinity). stresses.. The. most. economically. important. viruses.being.zucchini.yellow.mosaic.virus.(ZYMV),.watermelon.mosaic.virus.(WMV),.the.watermelon.strain.of.papaya.ringspot.virus.(PRSV-W),.and.cucumber.mosaic.virus.(CMV)..The.ZYMV.coat.protein.gene.was.cloned.and.sequenced.at.Michigan.State.University.and.used.to.develop.transgenic.cucurbits..In.collaboration.with.Egyptian.scientists.at.the.Agricultural.Genetic.Engineering.Research.Institute.(AGERI),.trans-genic.cucurbits/melon.and.squash.were.produced..The.transformation.technology.for.melon.together.with.the.coat.protein.gene.were.transferred.to.AGERI.and.used.to.produce.local.transgenic.melons.and.squash..The.technology.also.resulted.in.local.scientists.developing.transformation.and.regeneration.protocols.for.local.varieties.of.both.melon.and.watermelon.11

While.all. these.efforts.have.worked.well,. the.actual.challenge.for.Egypt. is. the.limited.biosafety.data.that.are.available.to.support.commercialization.of.these.prod-ucts..Furthermore,.the.Egyptian.government.might.have.been.reluctant.to.tackle.the.anti-GM.activism.that.surrounded.the.Bt.potato,.although.it.is.interesting.to.note.that.in.2008,.Egypt.did.indeed.commercialize.Bt.maize,.a.food.crop.1

KENYA

Sweet potato

The.average.yield.of.sweet.potato.in.Kenya.is.6.ton/ha,.whereas.in.China.it.is.18.ton/ha..While.sweet.potato.is.widely.grown.in.Kenya,.the.yields.continue.to.decline.due. to. nematode. and. viral. diseases. with. the. sweet. potato. feathery. mottle. virus.(SPFMV).being.the.major.virus..Transgenic.sweet.potato.carrying.the.coat.protein.gene.from.SPFMV.was.developed.by.Kenyan.and.Monsanto.scientists.using.mate-rials. from. Kenya.. When. technical. problems. developed. with. this. material,. the.material.from.Papua.New.Guinea.was.used.instead..When.transgenic.sweet.potatoes.were.field.tested.in.Kenya,.the.resistance.broke.down.and.a.whole.negative.publicity.ensued.13.The.development. of. virus-resistant. sweet.potato. still. continues. and. this.


time.in.collaboration.with.scientists.at.the.Vegetable.and.Ornamental.Plant.Institute.of.the.Agricultural.Research.Council.(VOPI-ARC).in.South.Africa..While.negative.publicity.surrounded.the.Kenyan.VR-sweet.potato,.a.lot.of.the.effort.in.developing.a.biosafety.regulatory.framework.in.Kenya.came.as.result.of.this.project..It.was.used.at.every.step.to.fine.tune.and.streamline.the.process..The.fact.that.certain.experiments.fail.is.precisely.the.reason.why.field.trials.are.required.to.see.whether.the.material.holds. up.under.field. conditions. and. that.practice.must. be. encouraged.. It.must. be.encouraged.as.long.as.the.right.questions.are.asked.at.the.right.stage.of.development,.that.is,.not.asking.for.food.safety.data.when.all.an.application.is.seeking.to.address.is.a.performance.under.field.conditions..In.January.2009,.President.Kibaki.signed.the.Biosafety.Bill.into.law..This.heralds.a.new.era.for.Kenya,.which.already.boosts.a.number.of.GM-products.in.the.pipeline.and.two.crops.under.CFTs,.Bt.cotton,.and.VR-cassava.

UGANDA

tranSGenIc banana

Bananas. are. a.major. staple. food. in. the.Great.Lakes. regions.with.per. capita. con-sumption.estimated.at.450.kg/year..Major.production.constraints.are.due.to.weevils.(Cosmopilities sordidud),. nematodes. (Pratylenchus. sp.,. and. Helicotylenchus. sp.).and.the.fungal.(Black.Sigatoka,.fusarium.wilt),.viral.(Banana.buchy-top.virus.and.Banana.streak.virus),.and.bacterial.(banana.bacterial.wilt).diseases.14.The.national.banana.research.program.has.designed.both.short-.(germplasm.evaluation.for.resis-tance,.importation.of.hybrids,.and.propagation.of.clean.planting.materials).and.long-term. (development. of. transgenic. materials). strategies. to. address. these. problems..Through. an. Agricultural. Biotechnology. Support. Project. II. (ABSPII). partnership.with,.the.Catholic.University.of.Leuven.(KUL),.the.University.of.Leeds,.the.United.Kingdom,.and.the.Ugandan.National.Agricultural.Research.Organization.(NARO).transgenic.banana.carrying.the.rice.antifungal.chitinase.resistance.gene,.RCG3,.to.control.Black.Sigatoka,.and.a.maize.cystatin.gene.and.other.nematicidal.genes.have.also.been.developed.and.are.being.evaluated..To.date,.a.CFT.for.banana.with.fun-gal. resistance. to. Black. Sigatoka. is. being.conducted. at. the. Kawanda. Agricultural.Research.Institute,.National.Agricultural.Research.Laboratory.(NARL)..The.NARL.also.has.other.transgenic.bananas.in.the.pipeline,.namely,.banana.biofortified.with.provitamin. A,. zinc,. and. iron,. banana. with. a. cell. cycle. regulatory. gene. for. rapid.growth,.and.banana.with.bacterial.wilt-.or.weevil-resistance.

ONGOING PROJECTS

The. ABSPII. located. at. Cornell. University,. United. States,. in. collaboration. with.Malian.scientists.engineered.a.gene.for.resistance.to.potyviruses.from.pepper.(Pvr1).into.tomato.(http://www.absp2.cornell.edu/projects)..Furthermore,.they.are.breeding.tomato.with.resistance.to.the.tomato.yellow.leaf.curl.virus.(TYLCV),.with.the.goal.of.backcrossing. the. resistance.genes. into. locally.preferred.varieties.with. the.final.product.containing.both.conventionally.bred.resistance.to.TYLCV.and.genetically.engineered.resistance.to.the.potyvirus.


The.African.Agricultural.Technology.Foundation.(AATF).has.acquired.a.royalty-free.technology.license.agreement.with.Academia.Sinica.of.Taiwan.for.their.plant.ferredoxin-like.protein.(pflp).from.sweet.peppers.for.use.in.conferring.bacterial.wilt.resistance.in.banana..The.AATF.has.in.turn.granted.a.sublicense.to.the.International.Institute.of.Tropical.Agriculture.(IITA).for.this.purpose..The.pflp.gene.has.already.been.used.successfully.in.rice,.tobacco,.potato,.tomato,.and.orchids..Current.efforts.on. transgenic. banana. have. already. gone. beyond. the. proof-of-concept. stage. and.shown.to.be.resistant.when.tested.against.inoculum.of.Xanthomonas campestris.pv.musacearum.

The.International.Crops.Research.Institute.for.the.Semi-Arid.Tropics.(ICRISAT).in. India.has.developed.groundnuts. resistant. to.groundnut. rosette.virus,. and. these.plants.are.currently.being.evaluated.in.South.Africa..A.gene.for.enhanced.proline.production. to. confer.drought. tolerance.was. incorporated. into.groundnut. in.South.Africa.and.is.currently.being.evaluated.

STATUS OF BIOSAFETY REGULATION IN SELECTED AFRICAN COUNTRIES

To.date,.there.are.11.African.countries.with.fully.developed.national.biosafety.frame-works.(NBFs)..Some.laws.are.explicit,.such.as.the.ones.in.South.Africa.and.Zimbabwe,.whereas.others.such.as.those.for.Egypt.and.Tanzania.are.implicit.and.embedded.in.other.national.laws..There.are.13.countries.with.Interim.NBFs,.15.that.are.described.as.a.work.in.progress,.and.16.that.have.no.NBFs..Of.these.countries,.only.three.have.commercialized. transgenic.crops:.South.Africa,.Egypt,.and.Burkina.Faso..Uganda.launched.two.Bt.cotton.CFTs.in.2009:.one.at.Serere.and.the.other.at.Kasese..Kenya.has.conducted.a.number.of.CFTs.while.Nigeria.has.given.approval.for.two.CFTs.on.Bt-cowpea.and.VR-cassava.

EXPECTED ECONOMIC EFFECTS OF INTRODUCING TRANSGENIC HORTICULTURAL CROPS IN GHANA

In.2007,.IFPRI15–18.conducted.an.economic.impact.assessment.study.on.the.potential.of.introducing.transgenic.tomato,.cabbage,.garden.egg,.and.cassava.in.Ghana..The.study.looked.at.the.feasibility,.cost-effectiveness,.and.long-term.impact.on.produc-tivity.and.yield.stability..The.study.showed.that.the.benefits.of.using.these.transgenic.crops.would.accrue. to.both. the. individual.as.well.as. the.entire.Ghanaian.society..Since.the.cost.of.pesticides.for.use.on.tomato,.cassava,.and.garden.egg.is.minimal,.the.investments.in.pesticides.as.a.percentage.of.total.production.costs.would.be.low;.however,. in.the.case.of.cabbage,.the.costs.would.be.significant.and.were.likely.to.influence.adoption..In.the.long.term,.any.technology.that.was.likely.to.reduce.yield.variability.was.also. likely. to.contribute.positively. to.poverty. reduction..Since. the.export.levels.to.Europe,.of.the.crops.under.study,.are.low,.the.benefits.at.the.farm.level.were.likely.to.be.higher.than.the.potential.losses.in.trade.

These.findings.have.policy.implications.and.highlight.the.role.that.government,.the.public,. and.private. sectors. could.play. if. the. farmers. are. to. realize. maximum.


benefits.from.the.technology..The.public.sector.could.be.involved.in.the.development.of.the.GM.varieties,.and.those.varieties.should.meet.both.consumer.and.producer.demand.and.preference..The.private.sector.could. lead.in.developing.a.viable.veg-etable.seed.multiplication.and.distribution.sector,.while.the.government.could.play.a.role.in.the.provision.of.an.effective.agricultural.extension.service.

WAY FORWARD: SYNERGIES AND LESSONS LEARNED

Biosafety. regulatory. frameworks. are. still. highly. fragmented.. Even. the. CPB. has.not.managed.to.get.countries. to.work.around.the.regional.economic.communities.(RECs)..Approaches.are.not.aligned.regionally.nor.are.the.limited.skills.ever.shared.giving.rise.to.even.more.stringent.regulations.2.Because.some.multinational.compa-nies.have.set.such.a.high.regulatory.bar,.it.will.be.difficult.for.African.countries,.most.of.them.having.limited.investments.in.the.area.to.fulfill.such.a.regulatory.package.

The.first.Bt. cotton. that.was.developed.by. China. is. already.being.marketed. in.India..This.is.an.interesting.case.of.south–south.collaboration.that.African.countries.could.emulate..The.notion.that.biotechnology.is.not.for.the.small-scale.farmer.is.far.from.the.truth..Indian.farmers.are.already.reaping.massive.welfare.benefits.from.the.technology.1.Meanwhile,.African.countries.still.operate.under.fear.of.losing.markets.even. when. the. evidence. points. to. a. nonexistent. threat.. The. Common. Market. for.Eastern.and.Southern.Africa.(COMESA).commissioned.a.study.that.analyzed.the.value.and.volume.of.agricultural.food.and.feed.exports.by.six.African.countries.to.various.regions.of.the.world.including.the.EU..Of.the.countries.under.study,.espe-cially.for.Kenya.and.Uganda.only.1.1%.and.6.5%.of.total.export.value.was.at.risk.of.being.rejected,.respectively.19.Meanwhile,.the.emergence.of.an.enabling.environment.such.as.the.Open.Forum.on.Agricultural.Biotechnology.(OFAB).represents.a.pub-lic.platform.that.is.fast.gaining.political.mileage..Under.the.auspices.of.the.AATF.and.with.patronage.of.the.Kenyan.Minister.for.Science.and.Technology,.OFAB.was.formed.in.2006.to.provide.a.monthly.platform.for.the.exchange.of.factual.informa-tion.amongst.stakeholders.in.agricultural.biotechnology..The.Uganda.chapter,.which.is.equally.active,.was.formed.in.2008,.while.the.Nigerian.chapter.was.launched.in.April.2009,.and.the.Tanzanian.chapter.was.launched.in.May.2009.20.With.six.coun-tries.outside.South.Africa.that.have.conducted.CFTs.for.a.number.of.crops,.perhaps,.the.time.has.come.when.African.countries.can.do.away.with.ambivalence.toward.the.technology.and.begin.to.reap.the.benefits.

REFERENCES

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155

7 Transgenic Horticultural Crops in Asia

Desiree M. Hautea, Von Mark Cruz, Randy A. Hautea, and Vijay Vijayaraghavan

INTRODUCTION

Asia.is.home.to.more.than.half.of.the.world’s.population.and.many.of.the.countries.situated. in. the. region.are.developing.countries..According. to. the.United.Nations,.the.world’s.population.reached.6.82.billion.in.2009.with.the.population.in.Asia.at.approximately. 4.12. billion.. By. the. year. 2050,. this. is. projected. to. increase. to.6.01.billion,.which.is.about.54%.of.the.projected.world.total.of.11.03.billion.1.The.Asian.population.growth.coupled.with. increases. in.per.capita. incomes. caused.by.economic.growth.and.urbanization.has.rapidly.expanded.the.demand.for.food.and.other.resources.2.Most.notably,.the.change.in.consumption.patterns.and.diet.due.to.increasing.incomes.in.Asia.can.be.seen.as.a.shift.from.basic.staples.to.a.more.diver-sified.diet.of.meat,.vegetables,.fruits,.and.processed.foods.3.On.the.other.hand,.Asia.continues.to.have.the.highest.absolute.incidence.of.hunger,.accounting.for.about.two-thirds.of.the.world’s.hungry.people.4

The.large.food.market.and.demand.in.Asia.create.challenges.on.ways.to.ensure.increased. and. sustainable. food.production.. In. the.past,. the.Green.Revolution. has.helped.avert.widespread. food. shortages. in. the. region,.most.notably. in. the. Indian.subcontinent.and.countries.in.Southeast.Asia.5,6.The.continuing.challenge.to.provide.

CONTENTS

Introduction............................................................................................................. 155Horticulture.Crop.Supply.and.Demand.in.Asia...................................................... 156Overview.of.Current.Adoption.of.Transgenic.Crops.in.Asia.................................. 159Public.Acceptance.and.Perception.of.Biotech.Crops.in.Asia................................. 161Transgenic.Horticultural.Crops.in.the.Pipeline.in.Asia.......................................... 162Development.and.Delivery.of.Transgenic.Horticultural.Crops:.Experiences.and.Lessons.Learned.from.South.and.Southeast.Asia............................................ 165

Development.of.Transgenic.PRSV-Resistant.Papaya.for.Southeast.Asia.......... 166Development.and.Commercialization.of.Fruit.and.Shoot.Borer–Resistant.Eggplant.in.South.and.Southeast.Asia............................................................... 168

Conclusion.............................................................................................................. 170Acknowledgments................................................................................................... 171References............................................................................................................... 171


adequate.and.affordable.food.in.Asia.is.daunting.as.the.key.production.resources.of. available. arable. land. and. water. are. more. constrained. today. than. in. the. past..National. and. regional. research. and. development. programs. for. crop. improvement.and.management.remain.essential.to.meet.the.demands.for.staples.and.horticultural.products.such.as.vegetables.and.fruits..The.food.price.hikes.of.2008.provided.addi-tional.impetus.not.just.on.ensuring.food.security.but.also.food.self-sufficiency.

Numerous.published.studies.and.experiences.have.asserted.that.biotechnology.applications,.specifically.genetic.engineering.and.transgenesis,.could.make.a.sig-nificant.contribution.in.helping.conventional.systems.increase.yields.while.provid-ing. more. efficient. use. of. resources. and. products. that. benefit. human. health. and.the.environment.7.Studies.have.also.suggested.that.increasing.crop.yields.through.applications.of.agricultural.biotechnology.is.essential. to. increasing.food.produc-tion. to.meet. future.needs.8.Since.biotech.crops.were.first.commercialized,. there.has.been.a.steady. increase.in.adoption.of. the.four.major. transgenic.crops. in.the.market. with. around. 134. million. ha. planted. in. 25. countries. in. 2009.9. However,.from.the.time.the.first.transgenic.vegetable.(the.Flavr.Savr.tomato).was.put.out.to.market.in.1994,.there.has.been.only.a.handful.of.transgenic.horticultural.crops.that.followed.10.Among.these.are.the.virus.resistant.squash.and.papaya.in.the.United.States.and.the.virus-resistant.sweet.pepper.and.papaya.in.China..Several.reasons.on.the.slow.development.and.commercialization.of.products.of.horticultural.biotech-nology.include.the.technical.challenge.in.developing.genotype-independent.trans-formation.procedures.despite. the.fact. that.protocols. for.genetic. transfor.mation.have. been. developed. with. success. and. reported. in. horticultural. crops. such. as.Brassica,.cucumber,.chilli.pepper,.eggplant,.lettuce,.apple,.and.tomato.11–14.Where.Agrobacterium. transformation. method. is. used,. technical. challenges. remain. on.designing.strategies.to.avoid.its.persistence.in.plant.issues.especially.those.on.veg-etatively.propagated.fruit.trees.12.Other.than.the.technical.constraints,.economic,.regulatory,.and.market.factors.pose.additional.hurdles.to.developing.commercial.biotech.horticultural.crops.compared.to.biotech.field.crops.such.as.corn.or.soybean..For.example,.the.relatively.small.market.and.the.limited.hectarage.of.horticultural.crops. in.general. have.been. identified.as.major. factors. that. limit. the. recovery.of.product.development.costs.15–18.These.hurdles.limit.the.application.and.deployment.of.biotech.traits.and.products.that.can.potentially.provide.broader.direct.benefits.to.consumers.such.as.those.related.to.improving.nutritional.quality.or.enhancing.aesthetic.quality..Moreover,.the.fact.that.most.horticultural.crops.are.consumed.as.food,. in. raw.or.semi-raw.form,.apparently.elicits.a.more.extensive.and.demand-ing.food.safety.and.regulatory.requirements. to.ensure.consumer.confidence.and.market.acceptance.

HORTICULTURE CROP SUPPLY AND DEMAND IN ASIA

In.Asia,.horticulture.crops.mean.food,.income,.nutrition,.and.well-being,.particularly.for.women..Asia.contributes.a.substantial.proportion.to.the.global.horticultural.pro-duction,.particularly.vegetables.and.fruits..According.to.the.FAO,.the.area.harvested.in.the.region.in.2007.is.equivalent.to.about.73%.and.48%.of.the.world’s.vegetable.and.fruit.production.areas,.respectively..The.cultivation.area.of.vegetables.is.about.

157Transgenic Horticultural Crops in Asia

1.47. times. larger. than. that. of. fruits.. China. alone. accounts. for. about. 60%. of. the.world’s.production.and.50%.of.the.world’s.vegetable.production.area..India.is.second.to.China. in.vegetable.production. in. the. region. primarily.because.majority.of. the.population.are.vegetarian.19.About.14.9%.of.the.country’s.total.farm.area.in.India.is.being.utilized.for.vegetable.growing.20

Figure.7.1.shows.an.increasing.trend.in.the.area.harvested,.volume.of.production,.consumption,.and.yield.of.vegetables.and.fruits.in.Asia,.as.well.as.globally..The.yield.of.vegetables.in.Asia.remains.higher.than.the.world’s.average,.while.the.yield.of.fruit.species.though.lower.in.the.past.decade.has.also.surpassed.the.world’s.average.in.2007..Northeast.Asian.countries. like.China,.Japan,.North.Korea,.South.Korea,.and.Taiwan. in.particular.have.higher.per.capita.consumption.and.production.of.horticultural.crops.than.other.countries.in.the.region.

Vegetables

Are

a har

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ed (h

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0102030405060

1997

Mill

ions

Cons

umpt

ion

(ton)

0100200300400500600700800

1993

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Prod

uctio

n (to

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0100200300400500600700800900

1000

1997 1999 2001 2003 2005 2007

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ions

Yiel

d (h

g/ha

)Th

ousa

nds

155

160

165

170

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180

1997 1999 2001 2003 2005 2007

2005 2007200320011999

20032001199919971995

Fruits

0102030405060

1997 1999 2001 2003 2005 2007

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ions

050

100150200250300350400450

1993 1995 1997 1999 2001 2003

Mill

ions

0100200300400500600

1997 1999 2001 2003 2005 2007

Mill

ions

800

850

900

950

1000

1050

1997 1999 2001 2003 2005 2007

Hun

dred

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Asia World

FIGURE 7.1 Selected.indicators.on.production.and.consumption.of.vegetables.and.fruits.in.Asia.and.the.World,.1997–2007..(Data.from.Food.and.Agriculture.Organization.of.the.United.Nations,.Rome,.Italy,.FAOSTAT.database,.http://faostat.fao.org,.accessed.October.28,.2009.)


The.substantial.growth.in.both.volume.and.variety.of.fruits.and.vegetables.traded.globally.has.been.noted.due.to.rising.incomes,.falling.transportation.costs,.improved.technology,.and.evolving.international.agreements.21.However,.within.Asia,.trading.of.horticultural.products.still.occurs.mostly.among.neighboring.countries.or.those.within.the.region.22,23.In.2007,.the.total.import.value.of.fruits.and.vegetables.in.Asia.was.more. than.$26.billion.with.banana. and.apple. contributing. about.$1.3.and.$1.0.billion,.respectively..The.total.export.value.of.fruits.and.vegetables.on.the.other.hand.was.higher,.at.about.$30.billion..The.trade.difference.(export–import).in.many.developing.Asian.countries.(Table.7.1).has.remained.relatively.low,.an.observation.also.reported.by.Lee.in.1998,22.which.he.attributed.to.a.well-established.self-supply.system. in. these. countries.. The. top. export. vegetables. and. fruits. from. the. region.include.onions,.tomatoes,.bananas,.garlic,.and.apples..China,.Thailand,.India,.and.

TABLE 7.1Trade Statistics of Fruits and Vegetables in Selected Asian Countries, 2007

Import Export 2007 Difference (Export − Import)

Bangladesh 285,214 70,487 −214,727

Brunei 31,957 29 −31,928

China 2,928,422 11,529,843 8,601,421

Hong.Kong,.SAR 1,743,593 586,395 −1,157,198

India 2,283,659 1,741,764 −541,895

Indonesia 738,506 496,539 −241,967

Iran 100,679 918,254 817,575

Israel 372,776 1,154,544 781,768

Japan 6,853,672 182,395 −6,671,277

Korea,.DPR 14,036 3,557 −10,479

Korea,.Republic.of 1,639,434 339,802 −1,299,632

Malaysia 782,266 270,666 −511,600

Myanmar 15,186 355,102 339,916

Nepal 84,517 4,338 −80,179

Pakistan 440,813 194,341 −246,472

Philippines 402,062 1,540,489 1,138,427

Singapore 848,485 210,626 −637,859

Sri.Lanka 189,687 84,078 −105,609

Thailand 496,378 2,542,711 2,046,333

Turkey 453,776 3,558,332 3,104,556

Vietnam 181,908 1,017,798 835,890

United.Kingdom 12,643,526 1,195,303 −11,448,223

United.States 17,633,830 13,373,194 −4,260,636

Source:. FAOSTAT.database,.http://faostat.fao.org,.accessed.October.28,.2009.Note:. Data. from. the. United. Kingdom. and. the. United. States. are. included. for. comparison.

(in thousands.of.dollars).


the.Philippines.are.the.largest.exporter.of.fruits.and.vegetables.while.Japan,.India,.Hong.Kong,.South.Korea,.and.Singapore.are.the.top.importers.24

It.is.evident.that.horticultural.crops.in.Asia.represent.an.enormous.opportunity.for.appropriate.agricultural.biotechnology.applications.that.can.help.develop.and.com-mercially.deploy.a.broad.range.of.improved.horticultural.products.that.meet.national.and.regional.needs..This.mostly.explains.the.extensive.level.of.biotech.research.and.development. on. horticultural. crops. in. the. region. as. elaborated. in. the. succeeding.sections.

OVERVIEW OF CURRENT ADOPTION OF TRANSGENIC CROPS IN ASIA

Of. the.25.countries. that. reportedly.planted. transgenic. (or.biotech).crops. in.2009,.three. mega-biotech. countries. (countries. that. are. growing. 50,000.ha. or. more). are.developing.countries.in.Asia..These.are.India.(8.4.million.ha),.China.(3.7.million.ha),.and.the.Philippines.(0.5.million.ha)..Their.combined.areas.accounted.for.9.4%.of.the.total.global.area.planted.to.biotech.crops.in.2009.9.While.China,.India,.and.the.Philippines.are.the.only.countries.in.Asia.cultivating.biotech.crops,.other.countries.in.the.region.have.granted.regulatory.approvals.for.their.importation.and.direct.use.for. food,. feed. or. processing. only.. However,. there. are. still. many. countries. in. the.region. that.have.no.official. record.of.granting. approval. for.both.planting. and. for.direct. use. of. any. transgenic. crops. although. trade. of. commodities. between. these.Asian.countries.and.countries.growing.transgenic.crops.are.on.going.realities..Many.of.these.countries.in.Asia.are.parties.to.the.Cartagena.Protocol.but.are.still.in.vari-ous.stages.of.approval.or.in.very.early.phases.of.implementation.of. their.national.biosafety.framework,.laws,.and.guidelines.

Table.7.2.presents.a.summary.of.number.of.regulatory.approvals.of.the.various.transgenic.crops.and.traits.in.the.region.as.of.2009..Asian.countries.with.most.num-ber.of.regulatory.approvals.are.Japan,.South.Korea,.Philippines,.and.China..Only.China.and.the.Philippines.have.planted.biotech.food.crops.in.the.region..Japan.has.the.most.number.of.approvals,.second.only.to.the.United.States.but.does.not.have.any.commercial.planting.of.any.of.the.approved.transgenic.crops.due.to.market.factors.and.consumer.acceptance..Interestingly,.India,.which.ranks.fourth.in.global.produc-tion.of.transgenic.crops,.has.only.approved.Bt.cotton..It.is.also.evident.in.Table.7.2.that.the.majority.of.regulatory.approvals.granted.are.for.agronomic.or.field.crops,.which.was.not.surprising..Of.the.total.number.of.approvals.granted,.those.given.for.horticultural.crops.accounted.for.only.18%.of.planting.and.12%.for.direct.use.for.food.and.feed..A.closer.inspection.of.Table.7.2.shows.that.the.only.horticultural.food.crops.approved.for.planting.are.all.grown.in.China..Japan.has.given.approval. for.ornamentals.but.these.are.not.planted.in.the.field..The.Philippines.has.only.approved.transgenic.horticultural.food.crop.for.direct.use.for.food.and.feed.such.as.potato..In.terms.of.traits,.single.event.“input.traits”.led.by.herbicide.tolerance,.dominate.the.approvals..Overall,.Asia’s.total.number.of.approvals.for.commercial.cultivation.of.transgenic.crops.is.still.quite.few..This.scenario.is.expected.to.change.dramatically.within.the.next.5.years.in.light.of.recent.developments.in.crop.biotechnology.in.the.


TABLE 7.2Regulatory Approvals Granted for Transgenic Crops and Traits for Planting and Direct Use for Food, Feed, or Processing (FFP) in Selected Countries in Asia through 2009

Country Crop Group (Crops) Traita

Number of Eventsb Approved

Planting FFP

China Vegetables.(tomato,.green.and.sweet.pepper)

DR,.VR 4 4

Fruit.(papaya) VR 1 1

Ornamental.(petunia) FC 1

Others.(canola,.cotton,.maize,.poplar,.rice,.soybean)

HT,.IR,.HT.+.IR,.high phytase

4 21

India Others.(cotton) IR 6 6

Indonesia Others.(cotton) IR 0 1

Japan Ornamental.(carnation,.rose) FC,.HT,.flav.path,.FC 9

Vegetables.(tomato,.potato) DR,.IR,.IR.+.VR 0 21

Others.(alfalfa,.canola,.cotton,.maize,.poplar,.rice,.soybean,.sugar.beets)

HT,.IR,.HT.+.F,.HT.+.IR,.Lys,.Lys.+.IR,.CPP,.OC,.OC.+.HT,.high.cellulose

52 101

Malaysia Others.(soybean) HT 0 1

Pakistan Others.(cotton) IR,.IR/IR 0 2

Philippines Vegetable/root.(potato) IR,.IR.+.VR 0 8

Others.(alfalfa,.canola,.cotton,.maize,.soybean,.sugar.beet)

HT,.IR,.HT.+.IR,.Lys,.Lys + IR,.plant.qual,.IR/HT

5 46

Singapore Others.(cotton,.maize,.sugar.beet) IR,.HT 0 4

South.Korea Vegetable/root.(potato) IR,.IR.+.VR 0 4

Others.(canola,.cotton,.maize,.soybean,.sugar.beet)

HT,.IR,.HT.+.IR,.IR/HT 0 53

Taiwan Others.(maize,.soybean) HT,.IR,.HT.+.IR,.IR/HT 0 26

Thailand Others.(maize,.soybean) HT 0 2

Horticultural.crops 15 38

Agronomic/field/other.crops 67 263

Total 82 301

Source:. James,. C.,. Global Status of Commercialized Biotech/GM Crops: 2009,. ISAAA. Briefs. 41,.International.Service.for.the.Acquisition.of.Agri-biotech.Applications,.Ithaca,.New.York,.2009.

Trait legend:. .HT,.herbicide.tolerance;.IR,.insect.resistance;.DR,.delayed.ripening/altered.shelf.life;.VR,.virus. resistance;.FC,.modified.flower.color;.Lys,.enhanced. lysine.content;.OC,.modified.oil.content;.F,.fertility.restored;.CPP,.cedar.pollen.peptide;.plant.qual,.modified.amylase;.flav.path,.flavonoid.pathway.

a. A.transgenic.event.refers.to.a.unique.product.resulting.from.stable.transformation—incorporation.of.foreign.DNA.into.a.living.cell.

b. Approvals.for.planting.have.been.granted,.but.no.actual.planting.in.the.country.has.been.done.


region..The most.significant.of. these.developments.are.China’s.approval.of. trans-genic. rice. and. maize,. India’s. Genetic. Engineering. Approval. Committee. (GEAC).recommendation. for.commercial. release.of.Bt.brinjal. (eggplant),. Japan’s.approval.for.commercial.planting.of.transgenic.blue.rose,.and.the.significant.number.of.trans-genic.food.crops.that.are.already.in.advanced.stages.of.development.and.commer-cialization.in.India.and.China.

PUBLIC ACCEPTANCE AND PERCEPTION OF BIOTECH CROPS IN ASIA

Public. acceptance. of. biotech. crops. and. products. ultimately. decides. whether. the.products. are. commercially. deployed. and. can. deliver. on. their. potential. benefits..Stakeholders.in.Asian.developing.countries.particularly.in.the.Philippines,.Malaysia,.India,.Vietnam,.and.China.were.reported.to.have,. in.general,.positive.perceptions.on.biotechnology.applications.25–27.Consumers. in.the.industrial.Asian.countries.of.Japan.and.South.Korea,.on.the.other.hand,.were.reported.to.be.more.conservative.in. outlook. or. negative. in. perception. toward. agricultural. biotechnology. compared.to.others.in.the.region.26,28.The.underlying.differences.among.these.country.groups.were.partly. attributed. to. the. relative. importance.of. agriculture. in. these. societies..Japan.and.South.Korea.are. food. importing.countries.where. the.generally.affluent.consumers.are.rather.unsure.about.the.potential.contributions.of.the.technology.to.their.individual.or.household.food.status.28.However,.if.made.aware.about.potential.contributions.of.the.technology.to.sustainability,.consumers.in.both.countries.show.a.marked.positive.shift. in.their.perception.and.acceptance.of. the.technology,.sug-gesting.that.they.easily.identify.themselves.with.the.higher.value.and.goal.of.sus-tainability..The.availability.of.the.biotech.blue.rose.recently.unveiled.in.Japan.and.the.apparent.market.acceptance.and.excitement.about.the.product.further.suggest.a.positive.consumer.response.to.a.product.with.perceived.added.value.

The.rising.hectarage.planted.to.biotech.crops.that.are.commercialized.in.some.Asian.countries.and.the.increase.in.the.number.of.regulatory.approvals.for.import-ing. countries. suggest. a. growing. confidence. and. acceptance. of. biotech. crops. and.products..In.Malaysia,.Bangladesh,.and.the.Philippines,.surveys.indicated.that.bio-technology.is.perceived.to.be.highly.important.and.would.be.instrumental.in.future.economic.development..Farmers.generally.accept.biotech.crops.if. they.can.derive.benefits.from.its.production,.as.the.preliminary.survey.on.biotech.rice.in.Bangladesh.revealed.29. Similar. perspectives. of. farmers. and. consumers. were. also. reported. in.Malaysia.and.the.Philippines.25,30,31.In.India,.Krishna.and.Qaim32.reported.that.60%.of.urban.consumers.will.likely.adopt.Bt.eggplant.when.commercialized.even.when.marketed.at.the.regular.vegetable.prices,.indicating.a.relatively.high.acceptance.level.of.the.eggplant.technology.

Among. the.stakeholders,. farmers’.perception.on.biotech.crops.and.biotechnol-ogy. in.general. is.very. important. since. they.are. the.direct. principal.beneficiaries..In.the.Philippines,.the.positive.experience.of.farmers.on.Bt.corn.since.it.was.com-mercialized.starting. in.2003.averted.what.could.have.been.continuing.opposition.from.sectors.that.were.initially.against.the.technology.31.In.most.cases,.ambivalence.


to.biotech.crops.and.biotechnology.were.attributed.to.critically.low.awareness.and.knowledge. on. the. subject. by. the. public.33–35. This. knowledge. gap. has. also. been.stressed. in.Taiwan.where.there.is.public.disconnect.on. the.topic. though.there.are.many.ongoing.research.on.biotechnology.in.the.country.34

The.effect.of.increasing.the.public’s.knowledge.of.biotechnology.is.a.decrease.in.their.perceived.risks.of.various.gene.technology.applications.in.food.production.and.in.agriculture..Collectively,.enhancing. the.awareness.of. the.public.will.help. them.make.informed.decisions.and.hopefully.create.a.favorable.attitude.toward.products.of. the. technology..These. factors. are. important. in. spelling. success. or. failure.dur-ing.the.commercialization.process.on.biotech.crops.36,37.There.were.recommenda-tions.that.early.consultation.and.dialogs.with.various.stakeholder.groups.should.be.considered.during.product.development,.regulatory.review.of.those.in.the.pipeline,.and.prior.to.final.genetically.modified.(GM).crop.approvals.to.ensure.product.suc-cess.36,38. Overall,. the. huge. economic. benefits. and. positive. experience. on. biotech.products.already.being.grown.and/or.used.in.Asia.is.expected.to.help.pave.the.way.for.future.success.and.consumer.uptake.of.other.biotech.horticultural.crops.

TRANSGENIC HORTICULTURAL CROPS IN THE PIPELINE IN ASIA

Since.biotech.crops.were.first.commercialized,.there.has.been.a.steady.increase.in.adop-tion.of.the.four.major.transgenic.crops.in.the.market.with.more.than.130.million ha.planted.in.2009.9.However,.despite.current.and.potential.benefits.offered.by.the.tech-nology,.development.and.commercialization.of.transgenic.horticultural.crops.in.the.United.States.has.almost.ground. to.a.halt.17.This. is. in.marked.contrast. to.what. is.happening.in.Asia.where.transgenic.horticultural.crops.have.taken.center.stage.par-ticularly.in.India.and.China.

Led.by.China,.Asia.has.significantly.increased.its. investments. in.agricultural.biotechnology.. In. 1999,. China’s. investment. was. estimated. to. be. $112. million39.and.in.2008,.the.government.announced.to.spend.up.to.$3.5.billion.for.a.special.project. to.develop.new.transgenic.crop.varieties.40.Other.countries. in. the.region.that. identified.agricultural.biotechnology.as.important.for. their.future.economic.development.have.also.allocated.part.of.their.national.agricultural.Research.and.Development.(R&D).budget.to.investments.in.biotechnology.including.about.$10.2.billion.in.South.Korea.and.$115.million.annually.in.India..Even.the.small.devel-oping.country. like. the.Philippines. allocated.5%.of. its. national. agriculture.bud-get.(Agriculture.and.Fisheries.Modernization.Act).to.biotechnology..Overall,.the.investment. of. the. public. sector. to. respective. national. agricultural. R&D. ranges.from.90.7%.to.100%.in.various.Asian.countries.41

As.a.result.of. this. favorable. investment.climate,. the.development.of. transgenic.crops. has. begun. in. several. countries. in. Asia.. FAO. database. shows. that. about.603.transgenic.crop.events.are.under.various.phases.of.development.and.commer-cialization.pipeline.in.the.region.(Figure.7.2).although.majority.of.these.are.in.the.experimental.phase..India.has.the.highest.number.of.transgenic.horticultural.crops.in.the.research.pipeline.and.many.of.these.are.horticultural.crops..In.2009,.a.signifi-cant.number.of.transgenic.horticultural.crops.have.reached.advanced.R&D.pipeline,.led.not.surprisingly.by.India.(Table.7.3)..Advanced.R&D.pipeline.refers.to.the.stage.


when. a. transgenic. event. is. not. yet. in. the. regulatory. process. but. at. late. stages. of.development,.that.is,.in.large.scale,.multi-location.field.trials,.and.generation.of.data.for.the.review.process.42.Table.7.3.and.Figure.7.3.present.the.summary.of.the.current.(2009).and.possible.numbers.of.expected.transgenic.horticultural.crops.and.traits.in.Asia.by.2015..The.numbers.presented.were.obtained.from.the.worldwide.survey.of.the.transgenic.crop.pipeline42.and.other.sources.9,39,43–45.Figure.7.3.shows.the.relative.proportion.of.GM.crops.by.trait..Table.7.3.also.shows.that.at.least.30.transgenic.horticultural.events.(approximately.60%.of.Asia’s.total).are.predicted.to.reach.the.market.by.2015..From.2009. to.2015,. the. total. number.of. transgenic.horticultural.crops. and. traits. grown. in. the. region. is. expected. to. increase. by. almost. fivefold.(from.7.to.33);.approximately.70%.(22.out.of.33).of.these.are.transgenic.vegetables.from.India.and.China..The.most.dramatic.increase.(from.0.to.16.events).is.expected.to.happen.in.India.if.the.current.situation.on.the.pending.market.release.of.Bt.brinjal.(eggplant).in.India.will.be.resolved.soon.

It.is.also.evident.in.Table.7.3.that.majority.of. the.efforts.have.been.focused.on.crops. and. traits. identified. to.be. important. to.national. and. regional.needs.. India’s.main.focus.on.vegetables.and.“input”.traits,.particularly.pest.and.disease.resistance,.is.understandable.given.the.vegetarian.diet.of.its.people,.the.high.incidence.of.pest.and.diseases.that.limits.production,.and.problems.with.abiotic.stresses..What.is.not.shown.in.the.table,.but.available.from.the.sources.listed,.is.the.high.proportion.(16/39.or.41%).of.private.(local.and.multinational).companies.involved.in.the.development.of.transgenic.crops.in.India.9,42.This.is.in.sharp.contrast.with.the.situation.in.China,.where.most.of.the.developers.are.public.institutions.funded.by.the.Chinese.govern-ment.39,42.It.remains.to.be.seen.which.between.China’s.public-sector-dominated.or.India’s.public–private.investment.strategy.will.prove.to.be.more.effective.and.sus-tainable. in. the. long. term..Notwithstanding. the.difference. in. their.strategies,.what.

BangladeshChinaIndia

IndonesiaMalaysiaPakistan

PhilippinesSouth Korea

Sri LankaThailandVietnam

0 10 20 30 40 50Number

60 70 80

Experimental phaseField trialCommercialization

90

FIGURE 7.2 Relative.number.of.transgenic.horticultural.crops.among.Asian.countries.at.different. stages.of. product. development.. (Data. from. FAO.Bio-Dec,.Food.and.Agriculture.Organization.Biotechnology.for.Developing.Countries.database.(FAO-BioDeC),.2009,.http://www.fao.org/biotech/inventory_admin/dep/default.asp)


TABLE 7.3Number of Current and Possible Transgenic Horticultural Crops and Traits in Selected Asian Countries through 2009 and Expected to Reach the Market by 2015

CountryHorticulture Crop Group Crops Trait(s)

Commercial in 2009

Advanced R&D

Total by 2015

China Vegetables.and.melons

Cabbage,.chili,.melon,.potato,.sweet.pepper,.tomato

Virus.resistance,.fungal.resistance,.quality.improvement,.abiotic.stress.tolerance,.shelf.life

3 7 10

Fruits Papaya Virus.resistance 1 0 1

Ornamental Petunia Modified.flower.color

1 0 1

Others Pogostemon Bacterial.resistance

0 1 1a

India Vegetables.and.melons

Cabbage,.cauliflower,.eggplant,.okra,.tomato,.watermelon

Insect.resistance,.abiotic.stress.tolerance,.fungal.resistance,.plant.stature,.quality.improvement,.virus.resistance

0 15 15

Others Mustard Male.fertility 0 1 1

Japan Ornamental Rose,.carnation

Modified.flower.color

2a 0 2

Philippines Fruits Papaya Shelf.life,.virus.resistance

0 2

Vegetables Eggplant Insect.resistance 0 1 (1)b

Bangladesh Vegetables Eggplant Insect.resistance 0 1 (1)b

Thailand Fruits Papaya Virus.resistance 0 1

Total 7 29 30–33

Sources:. James,. C.,. Global Status of Commercialized Biotech/GM Crops: 2009,. ISAAA. Briefs. 41,.International.Service.for.the.Acquisition.of.Agri-biotech.Applications,.Ithaca,.New.York,.2009,.Annex.1;.Stein,.A..and.Rodriguez-Cerezo,.E.,.Nat. Biotechnol.,.28,.23,.2010,.Supplementary.data;. Huang,. J.. et. al.. Science,. 295,. 674,. 2002;. Huang,. J.. and. Rozelle,. S.,. Calif. Agric.,.58, 112,.2004;.Authors’.firsthand.knowledge.

a. A.shrub.used.to.extract.oil.for.fragrance.or.medicinal.purposes.45

b. Same.transformation.event.used.in.India.but.bred.into.local.varieties.in.Bangladesh.and.the.Philippines.


seems.to.be.certain. is. that. transgenic.horticultural.crops.will.become.widespread.in.China.and.India,.with.high.potential.to.be.spread.in.their.neighboring.countries.considering. their. physical. proximity. and. the. nature. and. size. of. trade. among. the.countries.in.the.region.

DEVELOPMENT AND DELIVERY OF TRANSGENIC HORTICULTURAL CROPS: EXPERIENCES AND LESSONS LEARNED FROM SOUTH AND SOUTHEAST ASIA

The.development.pipeline.of.transgenic.crops.from.discovery.to.commercialization.could.be.a.long,.demanding,.and.expensive.undertaking..Depending.on.the.crop.and/or.trait,.it.has.been.estimated.that.it.would.take.between.7.and.15.years.at.an.esti-mated.cost.ranging.from.hundreds.of.thousand.to.millions.of.dollars.to.successfully.bring.to.market.a.transgenic.crop..A.number.of.published.papers.have.identified.the.possible. barriers. which. limit. the. successful. development. and. commercialization.of. transgenic.crops,. including.horticultural.crops. in. the.United.States16,17,31,46. and.in.developing.countries.36,47.These.potential.barriers.fall.mainly.into.the.following.categories:. (1). technical/technology,. (2). intellectual. property. rights. and. freedom.to.operate,.(3).regulation,.(4).market.considerations,.and.(5) .consumer.acceptance..Technical/technology.barriers. include. challenges. in.developing.efficient. transfor-mation. procedures. and. designing. vector. constructs,. choosing. the. crop. and. trait,.designing.critical.product.concept,.and.trait.efficacy.in.the.field..Intellectual.property.protection.and.lack.of.freedom.to.operate.present.hurdles.in.accessing.proprietary.technologies. that.are.owned.mostly.by.the.private.sector.and.concerns.on.poten-tial.for.liability.claims.associated.with.unwanted.transgenes.(low.level.presence).in.local.crops.42.The.high.cost.of.compliance.and.uncertainties.in.regulatory.approval.

IR 20%

OO 6%

DR 11%

AB 9%

FR 6%QI 11%

SL 6%

VR 31%

FIGURE 7.3 (See color insert.).Total.events.distributed.by.traits..AB, abiotic.stresses;.DR,.disease. resistance. including.bacterial. resistance;.FR,. fungal. resistance;. IR,. insect. resistance;.OO,.others;.QI,.quality.improvement;.SL,.shelf.life;.VR,.virus.resistance.


process.are.major.hurdles.particularly.for.horticultural.crops.because.of.small.mar-ket.and.limited.hectarage.that.could.limit.the.potential.for.recovery.of.production.costs. or. developing. countries. could. simply. not. afford.. Market. reluctance. partic-ularly. by. actors. in. the. supply. chain. (food. manufacturers,. processors,. and. retail-ers).also.presents.an.enormous.challenge.even.in.transgenic.crops.that.have.been.approved.for.market.release.due.to.worries.about.consumer.acceptance.and.higher.cost. and. potential. for. liability. associated. with. identity. preservation. and. product.stewardship.46

In.this.section,.we.discuss.our.own.experiences.and.the.lessons.we.learned.in.dealing.with.many.of.the.barriers.similar.to.the.ones.described.above,.in.the.course.of.pursuing.the.development.and.commercialization.of.two.important.horticultural.crops. in. the. region—the. transgenic. fruit. and. shoot. borer–resistant. eggplant.(Bt.eggplant).in.South.and.Southeast.Asia.and.the.papaya.ringspot.virus.(PRSV).resistant.papaya.in.Southeast.Asia..We.hope.that.by.sharing.these.experiences,.we.can.contribute.by.sharing.knowledge.on.and.insights.into.some.approaches.adopted.by. a. developing. country’s. national. R&D. programs. to. address. or. overcome. the.identified.constraints.to.biotech.crop.development.and.deployment..Table.7.4.sum-marizes.the.identified.principal.barriers.to.biotech.crop.development.and.deploy-ment.of.Bt.eggplant.and.PRSV-R.papaya.and.some.of.the.approaches.that.the.two.regional. projects. adopted. to. address. the. specific. constraints. as. discussed. in. the.succeeding.sections.below.

development of tranSGenIc prSv-reSIStant papaya for SoutheaSt aSIa

The.development.of.the.transgenic.PRSV.resistant.papaya.was.undertaken.through.the.Papaya.Biotechnology.Network.of.Southeast.Asia.(the.Network)..The.Network.was. established. in. 1998. with. the. assistance. of. the. International. Service. for. the.Acquisition.of.Agri-biotech.Applications.(ISAAA).with.funding.support.from.the.governments.of.the.five.member.countries.and.other.public.and.private.sector.donors.

An.early.consultation.and.planning.process.among.the.five.countries.of.Southeast.Asia—Indonesia,.Malaysia,.Philippines,.Thailand,.and.Vietnam—made.possible.the.exchange.of.research.updates.and.strategies.among.the.five.countries.and.identified.common.constraints. and.opportunities.. It.became.evident.during. the. consultation.process.that.it.would.be.highly.beneficial.to.establish.partnerships.among.the.countries.to.enable.a.regional.collaboration.that.would.capitalize.on.their.respective.strengths.and. build. a. regional. critical. mass. of. expertise. that. would. otherwise. take. time. to.develop.in.each.individual.country.

The. network. requested. and. designated. Malaysia. Agricultural. Research. and.Development.Institute.and.Thailand.(National.Center.for.Genetic.Engineering.and.Biotechnology.and.Kasetsart.University).to.serve.as.the.two.regional.hubs..Malaysia.and.Thailand,.which.were.the.furthest.advanced.in.the.development.of. transgenic.papaya,.provide.advanced.technical.assistance.and.training.support.to.other.mem-bers.of.the.network.in.molecular.virology,.tissue.culture.and.transformation,.molecu-lar.analyses,.among.others.

Among.the.early.identified.common.constraints.were:.(1).intellectual.property.restrictions. to. research. and. development. tools,. (2). technical. skill. limitations,.


TABLE 7.4Barriers to Biotech Crop Development and Deployment and Examples of Some Approaches from Two Regional Projects in Asia

Barrier

Strategic Approach

Bt Eggplant PRSV-Resistant Papaya

Technical/technology:.event.generation/selection

Access.and.use.a.common,.single.near-market.transformation.event.and transfer.by.conventional.breeding.to local.varieties

Country-specific.transformation.using.local.virus.strain.to.address.virus.diversity.and.trait.efficacy

“Shuttle.breeding”.through.internship.of scientists.from.partner.institutions.at Mahyco’s.Research.Center

Internship.of.scientists.from.partner.institutions.at.Monsanto.Laboratory

Intellectual.property.(IP).and.freedom.to.operate

Royalty-free.sublicensing.agreement.through.public–private.partnership.(PPP)

Royalty-free.licensing.agreement.through.public–private.partnership.(PPP)

Capacity.building.on.IP.management.thru.workshops.and.trainings

Capacity.building.on.IP.management.thru.workshops.and.trainings

Regulation Build.and.share.food.and.safety.data.package.of.the.common.transformation.event.to.spread.the.cost.of.food.safety.package

Capacity.building.for.scientists.and.regulators

Design.and.generate.an.insect.resistance.management.plan

Workshop.to.harmonize.regional.regulatory.needs.for.papaya

Early.discussion.with.regulators.on.regulatory.data.requirements

Technical.assistance.on.DNA.detection

Market Design.product.stewardship,.monitoring.and.seed.distribution.plan

Market.analyses.and.design.of.seed.distribution.plan

Capacity.building.of.partners.on.product.stewardship.and.monitoring

Outreach.and.communications.to.actors.in the.supply.chain

Public.acceptance Proactive.and.continuous.information,.communication,.and.education.(IEC).activities.for.various.sectors.of.society.implemented.through.a.network.of.partners;.activities.include

Workshops,.seminars,.public.forums

Publications

Regular.conduct.of.public.perception.surveys

Study.visits.to.project.sites

Interaction.with.the.media.thru.interviews;.news.sources,.guest.in.radio.and.TV broadcast,.television.features


(3) anticipated.regulatory.compliance.requirements.coupled.with.lack.of.regula-tions.and.guidelines.in.most.countries.at.the.time.the.network.was.established,.and.(4).anticipated.public/consumer.acceptance.issues.

The.network. facilitated.a. technology.donation.agreement.with.a.private. sector.technology. provider,. Monsanto,. for. all. virus. resistance. technologies. over. which.Monsanto. has. effective. intellectual. and. tangible. proprietary. rights.. Beyond. the.technology.donation,.Monsanto.also.hosted.and.provided.key.technical.training.to.papaya.scientists.from.all.five.countries.on.genetic.engineering.for.virus.resistance.and.provided.continuing.technical.assistance.in.product.development.and.regulatory.data.development.

In.addition.to.the.public–private.technology.transfer,.Thailand.and.Malaysia.pro-vided.key. technical. training.and.assistance. to.other.member.countries.of. the.net-work,.and.Malaysia.actually.served.as.a.research.laboratory.for.the.Philippines.for.the.initial.transformation.work.at.the.time.that.existing.Philippine.regulations.did.not.allow.Agrobacterium-mediated.transformation.of.papaya.

Recognizing. the.need.at. the. time. to.simultaneously.facilitate.product.develop-ment.with.the.evolving.regulations.and.guidelines,.the.regional.network.also.broad-ened. the.capacity.building.activities. to. include. intellectual.property.management,.biosafety,.and.food.safety,.to.include.not.just.the.national.technology.developers.but.the.emerging.corps.of.national.regulators..Undertaking.the.capacity.building.activi-ties.at.a. regional.basis.enabled.a. level.of.harmonization. in. the.science-based.risk.assessment.among.countries.without.intruding.into.the.sovereign.rights.of.member.countries.to.formulate.their.respective.regulations.

Finally,.the.network.has.been.proactive.in.education.and.information.activities.to.reach.out.to.various.sectors.and.the.general.public.by.raising.awareness.and.under-standing.of.modern.biotechnology.including.transgenic.crops.

The.network.is.considered.as.among.the.first.of.regional.initiatives.in.Asia.that.served.as.a.model.of.North–South.and.South–South.technology.transfer.partnership.in.agricultural.biotechnology..It.facilitated.the.transfer.of.proprietary.technologies,.enhanced.regional.and.national.capacity.in.biotechnology.including.the.associated.policy.and.regulatory.areas,.and.helped.shift.the.focus.of.national.R&D.from.mostly.academic. work. toward. developing. tangible,. commercializable. products. that. meet.farmers’.needs.and.compliant.to.best.practices.and.standards.

development and commercIalIzatIon of fruIt and Shoot borer–reSIStant eGGplant In South and SoutheaSt aSIa

The.development.and.commercialization.of.transgenic.fruit.and.shoot.borer–resis-tant.eggplant.containing.cry1Ac.gene.from.Bacillus thuringiensis.(Bt.eggplant).is.a.public–private.partnership.project.initiated.through.the.initiative.of.the.Agricultural.Biotechnology.Support.Project.II.(ABSPII),.a.consortium.of.public.and.private.sector.institutions.led.by.Cornell.University.and.funded.by.the.U.S..Agency.for.International.Development.(USAID)..Bt.eggplant.was.chosen.through.a.two-stage.priority.setting.process.. In. the.first.stage,.a.consultation.meeting.was.conducted.with. the.various.stakeholders.(scientists,.policy.makers,.regulators,.industry.representatives,.and.con-sumers).in.our.focus.countries.of.India,.Bangladesh,.and.the.Philippines..Bt.eggplant.


was.given. high.priority. in. each. country.based. on. SWOT. (strengths,.weaknesses,.opportunities,.and.threats).analyses.performed..In.the.second.step,.ex-ante.analysis.on.the.socioeconomic.impacts.of.Bt.eggplant.was.conducted.by.a.team.of.local.and.international.economists.working.in.the.project..The.results.showed.that.eggplant.is.a.very.important.food.for.the.people.of.India,.Bangladesh,.and.the.Philippines.and.a.highly.profitable.cash.crop.for.small.farmers.in.the.three.countries..Farmers.who.grow.eggplant. in.both. regions.share. the.same.problem—need.for.a.cost-effective.and.safe.method.to.control.the.most.damaging.insect.pest.of.eggplant—the.eggplant.fruit.and.shoot.borer.(EFSB)..Eggplant.yields.and.incomes.are.drastically.reduced.by.the.damage.caused.by.EFSB..Farmers.resort.to.frequent.application.and.exces-sive.amounts.of.pesticide.to.control.the.pest,.raising.the.level.of.pesticide.residue.in.the.environment.and.the.eggplant.consumed.by.the.public..Conventional.breeding.of.insect-resistant.varieties.has.failed.because.no.resistant.eggplant.varieties.have.been.found.that.can.effectively.control.EFSB..The.results.of.the.ex-ante.analyses. revealed.that.substantial.economic.and.environmental.benefits.are.projected.if.Bt.eggplant.is.commercialized.in.all.three.countries.48,49.Other.important.considerations.such.as.freedom.to.operate,.favorable.regulatory.approval.and.likelihood.of.gaining.market.acceptance.and.public.support.were.deemed.feasible.

Considering. the. cost. of. product. development. and. regulation,. ABSPII. made. a.strategic.decision.to.use.one.common.transformation.event.owned.by.Maharashtra.Hybrid.Seed.Company.(Mahyco),.Mahyco.eggplant.event.EE-1,.in.all.three.geogra-phies..The.strategy.presented.opportunities.to.spread.the.regulatory.cost.and.contrib-ute.to.the.regional.harmonization.of.regulatory.systems.by.building.a.common.safety.package.for.the.event,.which.was.proposed.to.be.submitted.to.the.regulators.in.each.partner.country..In.addition,.the.trait.stability.and.efficacy.of.EE-1.has.already.been.proven.and.was.determined.to.be.technologically.a.near-market.product..The.choice.of.EE-1.also.presented.an.opportunity.to.explore.the.opportunities.of.a.public–private.partnership.

From. the. very. outset. of. the. Bt. eggplant. project,. ABSPII. in. cooperation. with.Hyderabad-based.Sathguru.Management.Consultants.Pvt..Ltd..worked.with.Mahyco.to. devise. a. system. whereby. all. farmers. in. India. as. well. as. Bangladesh. and. the.Philippines. could.gain.access. to. the.Bt. eggplant. technology..The.dialogue. led. to.a. mutually. beneficial. arrangement. that. addressed. freedom-to-operate. issues. via.appropriate.licensing.arrangements..The.Bt.eggplant.transformation.event.developed.by.Mahyco.with.support.from.Monsanto.(which.licensed.the.use.of.Cry1Ac.expres-sion.vector).was.thus.cleared.of.potential.intellectual.property.(IP).barriers,.paving.the.way. for. further. development. and.commercialization..Mahyco. sublicensed. the.technology.on.a.royalty-free.basis.to.the.public.sector.partner.institutions.in.India,.Bangladesh,.and.the.Phiilippines.50.A.specific.product.stewardship.and.monitoring.strategy.to.be.implemented.by.the.licensee.to.safeguard.the.interest.of.the.licensors.was.also.devised.during.the.sublicensing.process..The.issue.of.market.share.among.the. different. Bt. varieties. in. India. was. also. addressed. by. segmenting. the. market.between.the.Bt.eggplant.hybrid.of.Mahyco.and.the.Bt.varieties.of.the.public.partners.

The.regulatory.file.development.of.Bt.eggplant.benefited.tremendously.from.the.expertise.and.experience.of.Mahyco,.the.first.Indian.company.to.commercialize.Bt.cotton.hybrids.in.India..In.addition.to.access.to.data.for.the.cry1Ac.gene.construct.


used.in.both.Bt.cotton.and.Bt.eggplant,.Mahyco.led.the.partners.in.streamlining.the.development.of.a.comprehensive.environmental.and.food.safety.regulatory.package.for.Bt.eggplant.event.EE-1..The.regulatory.file.complied.by.Mahyco.for.event.EE-1.was.made.available.in.the.Ministry.of.Environment.website.(http://www.envfor.nic.in/divisions/csurv/geac/information.brinjal.htm)..The.same.dossier.was.provided.to.all.partners. for.submission.and.review.of. relevant. information.by. their. respective.regulatory. bodies,. thus. accelerating. the. approval. process. in. these. two. countries..Proactive.and.frequent.interactions.with.the.regulatory.bodies.in.the.three.countries.also.helped.in.accelerating.the.approval.process.

A.proactive.information,.communication,.and.education.strategy.was.also.devel-oped. very. early. in. the. project. implementation. to. address. anticipated. public. and.market.perception.issues..The.communication.design.followed.the.product.develop-ment.pathway.so.that.the.communication.and.outreach.activities.and.messages.were.focused.on.the.needs.of.specific.stakeholders.as.the.product.moved.from.the.develop-ment.stage.toward.regulatory.clearance,.precommercial.stage,.and.eventually.toward.the.commercial.stage..An. important. feature.of. the.communication.strategy.was.a.regular.survey.of.perception,.attitude,.and.level.of.knowledge.and.information.needs.of.stakeholders.about.biotechnology.in.general.and.the.Bt.eggplant.in.particular.

CONCLUSION

Asia.is.home.to.more.than.half.of.the.world’s.population.as.well.as.to.the.highest.num-ber.of.poor.people.suffering.from.hunger.and.malnutrition..Ensuring.the.food.security.of.Asia.is.a.formidable.challenge.not.only.because.of.the.enormous.demand.brought.about.by.a.growing.population.with.rising.incomes,.but.because.the.additional.food.will.have.to.be.produced.amidst.decreasing.land.and.water.resources.and.increasingly.severe.and.frequent.biotic.and.abiotic.stresses.attendant.to.climate.change.

The.region.accounts.for.about.75%.and.50%,.respectively,.of.the.global.hectarage.growing.vegetables.and.fruits..Horticultural.crops.thus.comprise.a.major.part.of.the.region’s.and.the.world’s.agricultural.production.and,.being.high.value.cash.crops,.contribute. substantially. to. the. income. and. well-being. of. millions. of. farmers. and.consumers.especially.in.the.developing.countries.

Modern.biotechnology.applications,.including.genetic.engineering,.are.a.power-ful. tool. to.complement. the.conventional.methods.of.crop. improvement..Asia.cur-rently.has. three.countries.cultivating.biotech/transgenic.crops—China,. India,.and.the.Philippines,.but.only.China.commercially.grows.a.transgenic.fruit.crop,.a.papaya.engineered. for. virus. resistance.. Biotech-improved. blue. rose. is. also. cultivated. in.Japan.and.as.an.ornamental.crop.would.qualify.as.a.new.commercial.transgenic.hor-ticultural.product.in.the.region..Genetic.engineering.of.various.horticultural.crops.is.extensively.pursued.by.most.countries.in.the.region.that.have.developed.the.basic.capacity.to.undertake.modern.biotechnology.

Timely.development.and.commercial.deployment.of.biotech.horticultural.crops.in. the. region.are.hampered.by. several. constraints,. including. (1).access. to.propri-etary.biotechnology.applications,.(2).regulations.(or.the.absence.thereof).and.costs.of.regulatory.compliance,.(3).technical.capacity,.and.(4).market.and.public.acceptance..Overcoming.these.constraints.are.key.to.effective.development.and.use.of.a.broader.


range.of.biotech.crop.products.that.meet.national/local.needs..Innovative.approaches.such.as.public–private.and.multicountry.partnerships.and.networks,.product-focused.capacity. building. activities. and. proactive. and. dynamic. engagement. with. various.stakeholders.may.facilitate.overcoming.some.of.these.hurdles.and.expedite.commer-cial.adoption.of.new,.improved.products..Two.examples.of.such.regional.initiatives,.working.on.biotech.vegetable.(eggplant).and.fruit.(papaya).products,.are.cited.in.this.chapter.to.draw.some.lessons.and.insights.

The.biotech.pipeline.for.horticultural.crops.in.the.Asian.region,.coming.mostly.from.public.sector.institutions,.is.a.rich.array.of.key.crops.and.traits.that.meet.the.identified.priority.needs.of.producers.and.consumers..It.is.essential,.for.the.present.and. future. well-being. of. Asian. farmers. and. consumers,. for. these. products. to. be.commercially.deployed.in.the.most.appropriate.and.timely.manner.for.their.benefit.

ACKNOWLEDGMENTS

The.authors.gratefully.acknowledge.ABSPII,.a.United.States.Agency.for.International.Development.(USAID)-funded.consortium.led.by.Cornell.University.and.the.Papaya.Biotechnology.of.Southeast.Asia. facilitated.by.ISAAA.for.allowing.us. the.use.of.their.project.strategies.and.experiences.in.Asia.as.examples.of.regional.biotech.initiatives..We.wish.to.thank.Dr..Lourdes.D..Taylo.and.Ms..Zabrina.J..Bugnosen.for.their.valuable.assistance.in.proofreading.and.formatting.of.the.manuscript.

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175

8 The Economic and Marketing Challenges of Horticultural Biotechnology

Steven Sexton and David Zilberman

INTRODUCTION

In.the.13.years.that.followed.the.commercial.introduction.of.genetically.engineered.field. crops. in. 1996,. agricultural. biotechnology. became. the. most. quickly. adopted.crop.technology.in.history..More.than.14.million.farmers.in.25.different.countries.planted. a. combined. 134. million. hectares. of. genetically. modified. (GM). crops. in.2009.1.Virtually,.none.of.this.land,.however,.was.planted.to.horticultural.crops,.even.though. agricultural. biotechnology. was. born. with. the. Flavr. Savr. tomato. in. 1994..While.the.introduction.of.agricultural.biotechnology.in.major.field.crops.has.been.a.success.and.marked.by.a.tremendous.acceptance.by.farmers.and.a.strong.empirical.record.of.productivity.growth,.the.development.of.horticultural.biotechnology.has.been.slow..The.Flavr.Savr.tomato,.intended.to.resist.damage.in.shipping,.is.no.longer.produced,.nor.is.a.strawberry.engineered.to.resist.frost.damage.or.an.insect-resistant.potato..Only.the.GM.papaya.has.achieved.any.degree.of.commercial.success,.with.70%.of.the.U.S.-bound.Hawaiian.crop.planted.with.GM.seeds..No.GM.horticultural.crop.has.been.deregulated.since.1999,.and.the.number.of.field.trials.has.declined.in.recent.years.2

CONTENTS

Introduction............................................................................................................. 175High.Fixed.Costs.of.R&D....................................................................................... 176Consumer.Acceptance............................................................................................. 178Regulation............................................................................................................... 180

Bans.on.GMOs................................................................................................... 181Identity.Preservation,.Segregation,.and.Labeling.............................................. 182

Directed.R&D.and.Role.for.Public.Sector:.Public–Private.Partnerships............... 183Predictions.and.Policy.Recommendations.............................................................. 186References............................................................................................................... 187


In. spite. of. the. commercial. failure. of. horticultural. biotechnology,. there. exists.ample. opportunity. for. welfare-improving. innovation. in. specialty. crops.. In. fact,.given.the.agronomic.demands.of.specialty.crops.and.the.quality.demands.of.those.who. consume. them,. the. capacity. for. genetic. improvements. in. horticultural. crops.may.exceed.that.of.major.field.crops..Farmers.of.horticultural.crops.could.benefit.not.just.from.the.herbicide-tolerant.and.insect-resistant.traits.introduced.into.major.field.crops.like.cotton,.maize,.soybean,.and.canola,.but.also.from.traits.to.protect.against.plant.disease.3–5.Traits. that. extend. the. shelf. life.of.produce.or.boost. their.nutrient.content.would.also.be.valuable,.as.would.traits.to.control.growth.in.orna-mental.plants.and.make.flowers.last.longer.2.In.addition,.because.plant.breeding.is.particularly.slow.and.cumbersome.for.specialty.crops,.the.capacity.to.introduce.an.advantageous.trait.into.an.elite.cultivar.without.backcrossing.is.highly.beneficial.2

In. spite. of. the. serious. potential. for. gains. to. producers. and. consumers,. horti-cultural. crops.have.been. largely.overlooked.by. the.biotechnology. revolution,. and.research.and.development.(R&D).for.these.crops.is.declining..The.failure.of.horti-cultural.biotechnology.innovation.to.proceed.at.the.same.pace.as.agricultural.bio-technology.is.largely.a.matter.of.economics..In.this.chapter,.we.explain.how.high.fixed.costs.for.biotechnology.research,.diversity.in.horticultural.crops,.market.size,.consumer.doubts.about.GM.foods,.consolidation.in.food.processing.and.marketing,.and.government.regulation.of.genetic.plant.engineering.constrain.the.development.of.new.specialty.crops..We.further.articulate.policies.to.help.spur.socially.beneficial.innovation.in.horticultural.biotechnology.and.offer.predictions.for.where.horticul-tural.biotechnology.may.succeed.

HIGH FIXED COSTS OF R&D

In.Schmookler’s.seminal.book,.Invention and Economic Growth,6.he.observed.that.“invention. is. largely. an. economic. activity,. which,. like. other. economic. activities,.is.pursued. for.gain.”.The. role.of.profits. in.determining. the.pace.and.direction.of.technological. change. is. a. subject. that. has. motivated. vast. theoretical. and. empiri-cal.literatures..Profits.are.central.not.just.to.endogenous.growth.models.but.also.to.theories.of.induced.innovation.and.directed.technical.change.7–10.In.his.assessment.of.the.adoption.of.hybrid.corn.seed.in.the.United.States,.Griliches.provided.early.evi-dence.that.profitability.and.market.size.are.closely.tied.to.innovation.and.technology.adoption.11.More.recently,.Kremer.argued.that.investment.in.cures.to.Third.World.diseases.is.too.low.because.markets.for.such.cures.are.too.small.12.The.literature.on.adoption.and.innovation.finds.that.adoption.and.innovation.activities.require.making.risky.investments.13,14.The.likelihood,.timing,.and.scale.of.adoption.and.innovations.are.affected.by.risk.considerations..Increased.variability.and.riskiness.tend.to.reduce.economic.activity.15,16

The.challenges. to. innovation. in.horticultural. biotechnology.are. fundamentally.economic,.and.they.are.derived.from.the.profitability.and.risk.management.decisions.made.by.firms.throughout.the.supply.chain,.which.affect.the.rate.and.direction.of.innovation..They.are.not.altogether.different.from.the.challenges.the.medical.world.faces.in.overcoming.Third.World.diseases:.The.potential.for.welfare.gains.is.tremen-dous,.and.yet.the.relevant.R&D.is.substantially.lacking..As.we.will.see.in.subsequent.

177The Economic and Marketing Challenges of Horticultural Biotechnology

sections.of.this.chapter,. the.potential.to.recoup.costly.investments. in.horticultural.R&D.is.limited.by.consumer.acceptance.and.regulation..Against.these.expectations.of.low.benefits,.firms.must.weigh.the.costs.of.innovation,.which.are.mostly.fixed..While. the.costs.of.commercializing.GM.crops.are. independent.of. the. size.of. the.crop,.the.benefits.are.directly.proportional.to.the.size.of.crops,.which.means.that.the.development.of.GM.horticulture.is.less.likely.to.be.profitable.than.the.development.of.GM.field.crops.

Apart.from.the.costs.of.research,.the.regulatory.approval.process.for.new.bio-tech.crop.varieties. is. slow.and.expensive,. imposing.barriers. to. the.development.and. commercialization. of. new. technologies. and. creating. orphan. crops.17,18. The.cost. of. commercializing. a. single. transgenic. variety. can. exceed. $100. million.18.Once.a.new.trait.has.been.developed,.it.must.undergo.extensive.testing.for.envi-ronmental.and.human.safety.in.order.to.be.approved.for.production..In.the.United.States,.no.fewer.than.three.federal.agencies.are.responsible.for.approving.various.aspects.of.GM.technologies—the.U.S..Environmental.Protection.Agency,.the.U.S..Department.of.Agriculture.(USDA),.and.the.U.S..Food.and.Drug.Administration.(FDA).. The. registration. process. relies. on. ex. ante. testing. of. new. technologies,.followed.by.continual.monitoring.and.inspection.in.the.field,.with.the.technology.being. subject. to. cancellation. if. adverse. events.occur..The.outcomes.of. research.efforts,. the. regulatory. costs,. and. time. of. approval. are. uncertain,. and. investors.require.extra.gains.to.take.these.risks.

Each. transgenic. event. must. undergo. the. same. rigors. of. testing,. regardless. of.whether.the.same.trait.was.previously.approved.for.a.different.plant.species.or.variety.19.Registration. of. each. GM. variety. is. estimated. to. cost. as. much. as. $15. million..Testing.at.the.varietal.level.can.be.costly.and.causes.firms.to.introduce.new.traits.into.a.smaller.number.of.varieties.than.would.be.optimal.absent.the.costly.testing.20.Evidence.suggests. that.crop.biotechnology.innovation.has.slowed.because.of.high.costs.of.regulatory.compliance.17,21,22

The.diversity.of.horticultural.crops.causes.biotechnology.regulatory.costs.to.be.disproportionately.burdensome.relative.to.field.crops..Horticulture.comprises.hun-dreds.of.species.and.thousands.of.cultivars.each.produced.in.relatively.small.quanti-ties..It.can.be.costly.to.introduce.a.GM.trait.into.any.specific.crop.and.cultivar,.even.if.the.trait.has.previously.been.used.in.other.species.2.In.many.horticultural.crops,.several.different.varieties.are.commercially.important..If.introgression.of.the.new.trait.via.backcrossing.is.not.an.option,.such.as.may.be.the.case.for.clonally.propa-gated.varieties.that.do.not.breed.true,.each.variety.must.be.separately.transformed.in.the.lab,.and.each.must.be.separately.tested.and.approved..Regulatory.costs.would.add.up,.but. they. could.not.be. spread.out.over.nearly. as. large. a.market. as. could.row.crops.19.A.regulatory.regime.based.on.testing.at.the.trait.level.rather.than.the.event.or.varietal.level.would.reduce.compliance.costs.and.encourage.development.of. traits. that.could.be. introduced. into.closely.related.specialty.crops,. like.water-melon.and.cantaloupe.19

Another.cost.associated.with.the.development.of.horticultural.biotechnology.is.the.cost.of.gaining.access.to.complimentary.intellectual.property.rights.(IPRs)..The.transaction.costs.for.gaining.the.freedom.to.operate.(FTO).in.relevant.IP.space.are.considerable—as.high.as.$100,000.per.contract..And. these.costs.are. independent.


of.market.size..The.costs.also.increase.as.firms.seek.to.export. to.foreign.markets.because.additional.FTO.agreements.must.be.negotiated..A.large.share.of.horticul-tural.crop.output.is.exports,.making.these.costs.an.important.consideration.in.invest-ment.decisions..The.costs.of.FTOs.are.higher.for.horticultural.crops.than.field.crops.because.the.relevant.IP.is.more.secured.and.distributed.across.a.greater.number.of.IP.holders.23

Due. to. IP. congestion. and. the. difficulty. in. managing. IPRs. and. navigating. IP.spaces,.several.leading.U.S..public-sector.agricultural.research.organizations.estab-lished. the. Public. Intellectual. Property. Resource. for. Agriculture. (PIPRA),. which.provides.IP.management.solutions.for.public.sector.and.small.private.sector.players.in.horticulture..PIPRA.seeks. to.coordinate. the.disparate.portfolios.of. its.member.organizations.to.support.specialty.crop.applications..For.instance,.PIPRA.attempts.to. better. specify. licensing. agreements. to. distinguish. the. “fields. of.use”. so. that. a.technology.could.at.once.be.licensed.for.use.on.major.row.crops.as.well.as.for.uses.in.smaller.markets,.including.those.in.developing.countries..PIPRA.also.is.developing.an.IP.clearinghouse.and.working.to.pool.patents.to.be.licensed.as.a.bundle.for.uses.in.specific.crops.or.specific.regions..Such.efforts.can.help.to.mitigate.some.of.the.barriers.to.entry.in.horticultural.biotechnology.

In.spite.of.efforts.like.PIPRA,.the.high.fixed.costs.of.biotechnology.R&D,.par-ticularly,.the.regulatory.burden,.require.a.substantial.market.in.order.for.innovating.firms.to.recoup.their.investments..Many.of.California’s.specialty.crops.have.become.technological.orphans.because.agricultural.chemical.companies.have.concluded.that.the.potential.market.is.too.small.to.warrant.high.fixed.costs.to.develop.pesticides.for.specialty.crops..By.a.number.of.measures,.it.appears.that.seed.companies.share.the.sentiment.of.the.chemical.companies,.preferring.to.invest.in.conventional.breeding.of.horticultural.crops,.rather.than.genetic.engineering..Bradford.et.al..argue.that.the.marked.decline.in.R&D.intensity.for.lower.market.crops.is.a.consequence.of.high.registration.costs.2

CONSUMER ACCEPTANCE

In. the.11,000.years. since. the.domestication.of.plants,. crop. improvement.has.been.widely.hailed.as.a.necessity.for.overcoming.human.suffering.and.promoting.economic.growth..Plant.breeding.has.advanced.considerably.since.the.days.of.weak.selection..It.was.not.until.genetic.plant.engineering.was.introduced.that.sophisticated.breeding.techniques.elicited.consumer.opposition.24.The.application.of.the.same.genetic.pro-cesses.to.pharmaceutical.production.has.been.widely.accepted.by.consumers,.whereas.there. remain. considerable.health. and.environmental. concerns.associated.with.bio-technology.in.food.crops..In.the.13.years.since.the.commercial.introduction.of.GM.field.crops,.the.technology.has.largely.proven.to.be.safe.to.human.health.and.the.envi-ronment.and.to.provide.considerable.benefits.in.terms.of.increased.food.output.and.reduced.use.of.chemical.pesticides.and.tilling.operations.25.Shelton.et.al..reviewed.the.risks.of.insect-resistant.GM.crops.and.determined.the.risks.to.humans.and.the.envi-ronment.to.be.lower.than.the.risks.posed.by.alternative.pest-control.technologies.26.Nevertheless,.surveys.of.public.attitudes.routinely.find.large.portions.of.the.popula-tion.are.skeptical.of.biotechnology.in.general.and.food.applications.in.particular.27,28


Moschini.traced.current.doubts.about.biotechnology.to.the.introduction.of.recom-binant. DNA. and. the. surrounding. controversy. in. the. 1970s.27. Twenty. years. later,.and.with.accumulated.evidence.from.millions.of.experiments,.the.debate.about.the.safety.of.recombinant.DNA.was.settled—at.least.to.the.satisfaction.of.the.scientific.community.29.By.2003,.following.a.comprehensive.review.of.existing.evidence,.the.International.Council.for.Science.had.also.declared.consumption.of.foods.contain-ing.GM.ingredients.to.be.safe..The.safety.of.commercialized.GM.food.products.was.also.certified.by.a.number.of.national.science.academies.and.governmental.agen-cies,.including.the.USDA.

Environmental.concerns.center.on.the.impact.of.biotechnology.on.nontarget.spe-cies,.including.the.risk.of.gene.flow.beyond.the.farm.gate..While.such.risks.surely.exist.and.some.isolated.cases.of.gene.flow.between.GM.crops.and.proximal.non-GM.crops.have.been.documented,.governments.have.guarded.against.contamination.of.conventional.crops.by.requiring.buffer.crops.between.transgenic.and.conventional.crops..Against.these.risks,.including.the.yet.undocumented.risk.of.gene.flow.to.wild.flora.and.fauna,.weigh.environmental.benefits.associated.with.the.adoption.of.agri-cultural.biotechnology,.including.reduced.use.of.chemical.pesticides,.substitution.to.less.toxic.and.less.persistent.herbicides,.and.adoption.of.low-till.and.no-till.farming.practices.that.reduce.soil.erosion.and.carbon.emissions.

Not.withstanding. these.assurances.by.scientific.and.regulatory.agencies,.bio-technology. in. food. crops. remains. controversial,. perhaps. owing. to. heterogene-ity.with. regard. to. scientific. literacy.among. the.public.and.misperceptions.about.how. inserted. traits. alter. plant. development. and. food. production.27. Miranowski.et.al..and.Josling.and.Tangermann.reported.differing.opinions.on.biotechnology.between.scientists.and.the.public.at.large.30,31.Despite.a.spirited.debate.about.the.safety.of.biotechnology.in.academic.and.policy.circles,.surveys.consistently.show.that.consumers.have.little.information.about.the.risks.and.benefits.associated.with.GM.foods.32,33

Much.of.the.controversy.surrounding.consumption.of.GM.foods.may.stem.from.the.fact.that.the.first.commercialized.transgenic.crops.contained.input.traits.designed.to. improve.agronomic.performance.and.food.production,. rather. than.output. traits.intended.to.boost.food.quality.34.The.former.presumably.benefits.consumers.in.the.form.of.lower.food.prices,.though.these.benefits.may.be.less.discernable.to.consumers.than.products.with.new.attributes,.like.additional.nutrients.or.extended.shelf.lives..Agronomic.traits.can.be.perceived.as.benefiting.farmers.and.processors.rather.than.consumers.. Based. on. a. survey. of. more. than. 2000. consumers,. however,. Bredahl.found.broad.rejection.of.GM.technology.overall,.regardless.of.trait.35

A.number.of.studies.have.found.that.consumers.are.willing.to.pay.a.premium.for.food.products.that.are.free.from.GM.ingredients.36–39.Opposition.to.GM.foods.declines,. however,. when. consumers. are. provided. information. about. the. health.or. environmental. benefits. of. genetically. engineered. traits.37,38,40,41. Frewer. et. al..and.Mucci.and.Hough.found.evidence.that.consumers.are.more.willing.to.accept.genetic.modification.that.provides.nutritional.or.health.benefits.but.not.traits.that.reduce.cost. or. extend. shelf. life.42,43.Chern. and.Rickertsen. reported. results. from.a. cross-national. survey. that. showed. acceptance. of. GM. technology. was. broader.among. consumers. in. the. United. States. than. in. Norway.44. As. many. as. 70%. of.


Americans.consumed.products.with.GM.traits.that.either.reduced.chemical.appli-cations.on.crops.or.increased.the.nutritional.value.of.foods..Golden.rice—fortified.with. beta-carotene—elicits. a. positive. willingness. to. pay. (WTP).. Any. WTP. for.reduced.pesticide.residual.in.food,.however,.is.offset.by.the.perceived.risks.associ-ated.with.GM.technology.

Low. consumer. acceptance. of. crop. biotechnology. reverberates. up. the. supply.chain,.providing.a.disincentive.for.innovation..Food.processors.have.difficulty.in.marketing.products.made.with.GM.ingredients,.so.they.contract.with.farmers.for.non-GM.crops..Without.contracts.for.GM.crop.output,.farmers.are.disinclined.to.plant.GM.seeds..And.if.farmers.will.not.purchase.the.seed,.seed.companies.and.biotechnology.firms.will.not.invest.in.the.substantial.R&D.costs.necessary.to.pro-duce.the.seeds.

The.backlash.against.the.GM.potato.serves.as.a.cautionary.tale.about.the.limits.of.consumer.willingness.to.accept.GM.foods..McDonald’s,.the.largest.consumer.of.potatoes. in. the.United.States,.began.using.an. insect-resistant.potato. in. its.French.fries.in.order.to.reduce.costs..Consumer.opposition.was.intense,.however,.and.the.company.and.other.major.food.processors.stopped.sourcing.GM.potatoes.in.early.2000..One.year.later,.Monsanto,.which.had.developed.the.insect-resistant.potato.6.years.before,.stopped.marketing.it.altogether.

The.extent.to.which.the.experience.of.the.GM.potato.slowed.innovation.in.horti-cultural.biotechnology.is.not.clear,.but.it.is.likely.no.coincidence.that.R&D.directed.toward.food.crops.declined.sharply.after.Monsanto.withdrew.its.GM.potato..In.1999,.374.field.test.notifications.were.submitted.for.GM.horticultural.crops..By.2003,.that.number.had.fallen.to.97.2.In.contrast,.the.number.of.field-test.notifications.for.major.GM.field.crops.rose.from.506. in.1999. to.520. in.2003..Consumers.have.generally.been.more.tolerant.of.GM.crops.destined.for.animal.feed.rather.than.human.con-sumption,. which. may. explain. why. the. most. rapid. innovation. is. occurring. in. the.development.of.drought-tolerant.varieties.for.major.crops.

REGULATION

Consumer.acceptance.of.GM.foods.certainly.limits.the.returns.to.agricultural.and.horticultural. R&D.. But. government. regulation. further. constrains. the. market. and.deters. commercialization. of. GM. horticultural. crops. that. would. be. demanded,. at.least.in.niche.markets..Ex.ante.testing.and.registration.requirements.for.new.trans-genic.events.increase.the.fixed.costs.of.commercialization.and.create.a.disincentive.for.investment.in.horticultural.biotechnology.research,.as.was.discussed.previously..However,. two. other. elements. of. government. policy. also. limit. the. profitability. of.GM.horticulture.throughout.the.supply.chain..First,.some.governments.ban.imports.of.foods.containing.GM.ingredients.and.deny.registrations.for.domestic.GM.crop.production..These.policies.not.only.restrict.market.access.for.GM.output.but.also.limit. demand. for. seed. technologies. themselves.. Second,. where. GM. products. are.commercialized,.processors.often.face.strict.rules.for.segregating.and.labeling.GM.foods.and.derivatives.of.GM.ingredients..Liability.associated.with.adventitious.com-ingling.of.GM.and.non-GM.ingredients.creates.a.deterrent.to.GM-food.provision.by.food.processors.


banS on GmoS

The.United.States.was.an.early.and.aggressive.adopter.of.agricultural.biotechnol-ogy;.the.E.U..was.not..In.fact,.it.adopted.a.precautionary.approach.that.emphasized.a.zero-risk.tolerance.and.resulted.in.a.de.facto.ban.on.GM.technologies.for.a.decade,.ending.only. in. 2008..The. typical. explanation. for. the. stark.divergence. in.policies.is. that. European. consumers. are. more. risk. averse. than. American. consumers,. in.part. because. of. their. history. with. major. food. scares.45–48. Graff. and. Zilberman,.however,.rejected.this.conventional.wisdom.because.risk.preferences.for.agricultural.chemicals.and.intra-E.U..trade.reveal.a.level.of.acceptance.that.is.inconsistent.with.the.zero-risk.tolerance.that.defined.GM.food.policy.49.They.instead.offered.an.expla-nation. for. policy. divergence. based. on. political. economy. considerations. in. which.Europeans.sought.to.protect.the.agrochemical.industry.in.which.they.had.a.compara-tive.advantage.from.the.agricultural.biotechnology.industry.dominated.by.American.firms..Graff.et.al..formalized.this.hypothesis.in.a.model.in.which.interested.parties,.like.chemical.companies,.environmental.activists,.and.some.farming.cohorts,.per-suaded.consumers.to.be.wary.of.GM.foods.50.Consumer.sentiment.then.influenced.policy,.resulting.in.regulations.that.may.not.serve.the.interests.of.consumers.in.the.E.U..or.elsewhere.

The. European. ban. on. GM. foods. has. influenced. policies. in. Africa,. which. has.largely. banned. agricultural. biotechnology. in. spite. of. the. potential. for. substantial.gains.51. African. countries. largely. rely.on.European. countries. for. export.markets,.so. they.are.unlikely. to.deviate. from.European.policy..Still,. there. is.evidence. that.existing.technologies.could.generate.cost.savings.in.agricultural.production.of.10%.and.that.yields.would.climb.52.Subsequent.generations.of.agricultural.biotechnology.could.yield.even.greater.benefits.for.Africa,.providing.drought-tolerant.varieties.and.boosting.the.nutrient.content.of.staple.crops.

The.European.ban.on.GM.food. imports.has. impacted. the.adoption.decisions.of.farmers.beyond.Africa.48.For. instance,.Europe. is.a.major.destination. for.U.S.. farm.production.and.vice.versa..Following.the.institution.of. the.GM.food.moratorium.in.Europe,.U.S..corn.exports.to.the.region.essentially.halted.out.of.fear.of.comingling.GM.and.non-GM.corn.53.A.consequence.of.GMO.bans.around.the.world—and.especially.in.Europe—is.that.the.market.for.GM.traits.is.artificially.constrained..Even.though.the.adoption.of.agricultural.biotechnology.has.been.rapid.by.historical.standards,.only.about.one-fourth.of.the.world’s.countries.planted.GM.seeds.in.2009—and.half.of.it.was.planted.in.the.United.States.1.Farmers.in.nonadopting.countries.cannot.use.the.new.technologies.even.if.they.want.to.use.them,.and.those.in.adopting.countries.may.not.want.to.use.them.if.adoption.would.jeopardize.export.markets.

Without.a.robust.market.for.new.seed.technologies,.the.R&D.pipeline.shrinks,.stranding. traits. that.have.already.been.created.and.slowing. the.creation.of.new.traits..Graff.and.Zilberman.show.the.fall.in.patent.applications,.investment.dol-lars,.and.firm.start-ups.that.occurred.during.the.European.ban.49.While.stringent.regulation.may.have.been.intended.to.protect.against.risks.associated.with.damage-controlling.GM.traits,.they.also.surely.delayed.the.introduction.of.quality-enhancing.traits.. The. introduction.of. the. second-generation. GM.crops. is. long. overdue.. The.European. moratorium,. then,. may. prove. to. have. significant. and. lasting. social.


welfare costs. in. terms.of.delayed. or. foregone. innovation. in.nutrition,. production.efficiency, and.environmental.mitigation.54

The.E.U..lifted.its.moratorium.on.GM.products.in.2008.and.by.2010.had.deregu-lated.its.first.transgenic.event.in.more.than.a.decade..Nevertheless,.the.adoption.of.GM.crops. in.Europe.declined. from.2008. to.2009,. and.Germany. instituted. a.ban.on.the.production.of.GM.crops..Spain.remains.the.only.consistent.and.significant.adopter.of.GM.technologies.in.Europe,.though.the.land.it.plants.to.GM.seed.con-stitutes.a.little.more.than.a.rounding.error.in.the.total.GM.land.base..Adoption.in.Africa.continues.to.lag.1

IdentIty preServatIon, SeGreGatIon, and labelInG

Despite.broad.consumer.opposition.to.GM.foods,.there.are.niche.markets.that.can.be.served.by.particular.genetically.engineered.traits..These.niches.may.be.under-served,. however,. because. of. demands. from. consumers. and. regulators. that. foods.derived.from.GM.crops.be.segregated..The.provision.of.GM.foods,.then,.may.require.separate.transportation,.shipping,.and.labeling.facilities..Identity.preservation.of.GM.and.non-GM.food.is.consistent.with.broader.trends.in.agriculture.and.food.produc-tion..Agricultural. commodities. are. increasingly.becoming.differentiated.products.in.response.to.wealthier.and.more.sophisticated.consumers.who.demand.foods.that.offer.environmental.and.health.benefits.relative.to.conventional.production..In.order.to.serve.niche.markets.for.organic,.free-range,.hormone-free,.and.local.foods,.pro-cessors.increasingly.contract.for.farm.output.in.order.to.ensure.quality.attributes.and.traceability.

Identity.preservation.of.GM.foods.is.likely.to.be.more.difficult.than.in.other.con-texts.for.a.number.of.reasons..First,.policies.on.GM.foods.vary.by.country..Some.GM.varieties,.produced.in.the.United.States,.for.instance,.have.not.been.approved.in.the.E.U..Heterogeneous.policies.require.processors.and.distributors.to.maintain.a.high.level.of.food.purity.or.risk.access.to.foreign.markets..More.than.40.countries.have.adopted. labeling. regulations.. The. United. States. is. most. lax,. providing. voluntary.labeling.guidelines. for.GM-free.food,.but. imposing.no.requirement..Only.foods.that. are. substantially. different. from. their. conventional. counterparts,. with. respect.to.nutritional.content,.for.instance,.require.labels..The.E.U..is.most.strict,.requiring.labeling.of.GM.foods.with.a.0.9%.tolerance.for.adventitious.presence.of.GM.crops.55.Australia,.Japan,.South.Korea,.and.China.have.adopted.stringent.regulations,.while.Canada,.like.the.United.States,.has.no.labeling.requirement.

Strict. mandatory. labeling. creates. a. disincentive. for. use. of. GM. ingredients..Following.the.adoption.of.labeling.requirements,.GM.food.disappeared.from.store.shelves.in.the.E.U..and.Japan.56.As.Lapan.and.Moschini.noted,.the.European.regula-tion.requires.food.produced.from.a.GM.ingredient.to.carry.a.GM.label.regardless.of.whether.the.final.product.contains.DNA.or.protein.of.GM.origin.57.Against.such.a.standard,.the.cost.of.avoiding.commingling.throughout.the.supply.chain.is.likely.to.be.enormous.and.prohibitive,.prompting.firms.to.source.from.suppliers.who.do.not.handle.any.GM.products..Such.regulations,.with.low.tolerance.thresholds.while.intended. to. improve.consumer.welfare.by.allowing.consumers. to.choose.between.GM.and.conventional.products,.practically.kill.the.market.for.GM.foods.


To.avoid.comingling,.GM.and.non-GM.crops.must.be.separated.on.the.farm.by.a.sufficient.distance.to.prevent.cross-pollination..They.must.also.be.kept.separate.in.transportation.to.grain.elevators.and.from.elevators.to.processors..The.challenge.of.isolating.crop.varieties.within.the.grain.marketing.system.and.preventing.unwanted.comingling.is.illustrated.by.the.2000.discovery.in.the.human.food.supply.of.a.GM.corn.variety.approved.only.for.animal.and.industrial.uses..The.StarLink.corn.was.discovered.first.in.taco.shells.marketed.at.retail.grocery.outlets,.including.the.Taco.Bell.brand.marketed.by.Kraft..The.discovery.ultimately.led.to.the.recall.of.200.food.products.and.a.temporary.ban.on.U.S..corn.imports.in.Japan.until.corn.purity.could.be.verified.

The.incident.was.estimated.to.cost.U.S..producers.as.much.as.$290.million.58.Aventis,. the. maker. of. StarLink. corn,. reportedly. budgeted. $1. billion. for. costs.associated.with.recalling.contaminated.corn.and.food.and.for.compensating.farmers.59.A. number. of. lawsuits. were. also. filed. against. Aventis,. which. finally. settled. for.$125.million.. It. took.nearly.half. a.decade. for. the. contaminated. corn. to. exit. the.food.supply.chain.despite.efforts. to.destroy. it. quickly..The.contamination. likely.occurred.via.gene.flow.across.buffer.crops.to.conventional.crops.destined.for.human.consumption.

The.StarLink.episode.demonstrates.that.without.reasonable.thresholds.in.label-ing.and.trade.import.regulations.for.the.adventitious.presence.of.GM.DNA.or.pro-teins,.the.risk.to.food.processors.is.high.while.the.benefits.are.low,.particularly.in.small.markets..Given.low.consumer.acceptance.and.strict.and.heterogeneous.label-ing.requirements,.firms.face.considerable.risk.to.serve.available.markets..Adverse.events.could.not.only.impose.significant.liability.but.also.jeopardize.brand.reputa-tion,.which.could.spread.to.non-GM.operations.of.the.firm..Niche.markets.for.GM.food.will.be.underserved.by.risk-averse.firms,.further.constraining.the.market.for.biotechnology.in.horticultural.crops.

DIRECTED R&D AND ROLE FOR PUBLIC SECTOR: PUBLIC–PRIVATE PARTNERSHIPS

Funding. for. horticultural. biotechnology. research. has. lagged. investment. in. trans-genic.research.for.major.field.crops,.even.controlling.for.relative.market.size.23.By.2004,.investment.in.horticultural.biotechnology.had.all.but.died.off,.with.leading.innovators.focusing.their.horticultural.R&D.efforts.on.conventional.plant.breeding.instead.of.genetic.engineering..Theory.predicts.that.the.investment.in.R&D.is.sub-optimally.low.because.of.free-rider.problems,.except.where.strong.IPRs.are.vigor-ously.enforced..Where.IPRs.are.strong,.however,.innovating.firms.are.rewarded.with.monopoly.power.for.the.terms.of.their.patents..Monopoly.power.permits.patent.hold-ers.to.earn.supra-normal.profits,.but.high.technology.prices.lead.to.adoption.rates.that.are.suboptimally.low.from.a.social.welfare.perspective..The.traditional.response.to.underprovision.of.agricultural.innovation.is.to.invest.public.dollars.in.research..Historically,.the.government.has.been.a.leader.in.agricultural.research,.and.horticul-ture.has.been.more.dependent.on.public.support.than.major.crops..Since.the.1990s,.however,.public.research.has.begun.to.decline.as.new.R&D.paradigms.emerge.


Since.the.mid-nineteenth.century,.governments.around.the.world.have.supported.agricultural.research,.which.has.historically.produced.publicly.available.crop.vari-ety.improvements.that.left.little.room.for.private.returns.60.The.rise.of.genetic.plant.engineering.at.the.close.of.the.twentieth.century,.however,.dramatically.changed.the.agricultural.R&D.landscape..Major.advances.in.crop.science.are.no.longer.mostly.in.the.public.domain..Instead,.due.to.the.nature.of.the.technologies.and.the.devel-opment.of.strong.IPRs,.the.benefits.of.innovation.are.excludable..Since.the.1980s,.governments.began.adopting.policies,.like.IPRs,.to.motivate.a.greater.share.of.agri-cultural.research.investment.from.the.private.sector..Consequently,.most.agricultural.biotechnology.R&D.is.undertaken.by.biotechnology.start-ups.and.established.seed.and.agrochemical.companies.

Because.the.costs.of.horticultural.biotechnology.are.immense,.including.the.fixed.costs.of.commercialization,.the.costs.of.identity.preservation,.and.the.risks.associ-ated.with. consumer. acceptance. and.comingling,. the.private. sector. is. expected. to.invest.scarce.R&D.dollars.elsewhere,.like.GM.applications.for.large.market.crops.that.do.not.directly.enter.the.food.supply..The.declining.investment.in.horticultural.biotechnology.has.been.documented.by.a.number.of.authors.54,61

Horticultural.research.has.typically.been.undertaken.in.the.public.sector,.but.bio-technology.research.has.been.dominated.by.the.private.sector..While.there.are.some.examples.of.public–private.partnerships.for.research.in.biotechnology,.none.relates.directly.to.horticultural.biotechnology.62.Horticultural.biotechnology.research.would.benefit. from. public–private. partnerships. because. the. traditional. paradigm. of.one-way.information.flows.from.public.research.institutions.to.private.firms.is.out-dated..Policy.changes.designed.to.push.research.expenses.toward.the.private.sector,.the.consequent. stagnation.of.public. funding. for.crop.science.research,. the. rise.of.large. life. sciences. firms,. and. the. alignment.of.public. and.private. incentives. for.long-term.R&D.created.an.imperative.for.a.new.formulation.of.the.R&D.pipeline.that.involved.closer.collaboration.between.industry.and.university.62,63

Universities.are.considered.to.have.a.comparative.advantage.in.basic.research.and.industry.in.applied.research.64–66.The.public-good.attributes.of.basic.research.make.it. an.appropriate.fit. for. the.university.environment.67–70.Applied. research,.with. its.focus.on.solving.particular.problems,.can.be.better.motivated.by.a.profit.incentive,.making.it.a.better.fit.for.industry..The.division.of.research.labor.between.industry.and.academia,.therefore,.allows.both.the.university.to.pursue.its.traditional.mission.of.providing.an.intellectual.commons.and.the.firm.to.pursue.its.profit.motive..The.complementarities.between.university.and.industry.research.have.been.fully.appre-ciated.only.in.recent.decades.66,71–73.In.line.with.their.mission.of.maintaining.“intel-lectual. commons,”. universities. have. historically. placed. innovations. in. the. public.domain.74.Administrators.now.recognize,.however,.that.they.can.increase.the.benefit.to.society.and.enhance.university.prestige.and.revenue.if.they.license.knowledge.to.firms.that,.by.virtue.of.having.rights.to.technologies,.will.invest.in.their.development.and.deployment.75.Firms.have.little.incentive.to.commercialize.a.technology.in.the.public.domain.because.competing.firms.can. freely.capitalize.on. the.R&D.invest-ments.of.others.76

While.the.outlook.for.investment.in.new.horticultural.biotechnologies.appears.dim. based. on. trends. in. public. funding. and. the. diversion. of. private. funds. to.


higher.valued.investments,.the.induced.innovation.hypothesis.offers.a.glimmer.of.hope.that.new.traits.in.horticultural.crops.can.be.introduced..A.considerable.literature. on. agricultural. R&D. has. subjected. the. induced. innovation. hypoth-esis. proposed.by.Hicks77. and. formalized.by.Hayami. and.Ruttan78. to. consider-able.scrutiny..It.has.emerged.as.both.an.intuitive.explanation.for. innovation.in.the. industry.and.one. that.explains.observed.phenomena.of. the.past.century.or.more.9,79–82.As.first.proposed.by.Hicks,.it.holds.that.“a.change.in.relative.prices.of. the. factors.of.production. is. itself. a. spur. to. invention,. and. to. invention. of. a.particular.kind—directed.to.economizing.the.use.of.a.factor.which.has.become.relatively.expensive.”.More.generally,.induced.innovation.holds.that.firms’.R&D.effort.is.directed.toward.technical.changes,.for.which.there.is.demand,.typically.because.of.scarcity.

More.generally,.the.theory.predicts.that.innovation.responds.to.scarcity.and.crisis..It.is.not.surprising.that.one.of.the.few.GM.horticultural.crops.on.the.market.today.was.developed.at.a. time.when.the.industry.was.severely.threatened..By.1994,. the.papaya. ringspot.virus,.which.had. forced. the. relocation.of.Hawaii’s.papaya. indus-try.from.two.other.islands,.reached.the.final.refuge.of.papaya.farmers.in.the.Puna.district.of.Hawaii. Island..The. industry.was. in.crisis..Fortunately,.as.crop.damage.spread,. an. effort. to. commercialize. a. papaya. variety. with. genetically. engineered.resistance.moved.forward..GM.papaya.was.commercialized.in.1998,.effectively.sav-ing.the.industry.83.Today,.more.than.70%.of.Hawaiian.papaya.is.produced.from.the.GM.seed.

The. underprovision. of. biotechnology. solutions. for. horticulture. is. analogous.to. the. problem. of. provision. for. developing. countries. and. mirrors. problems. in.medicine.regarding.the.development.of.treatments.and.cures.for.rare.diseases.and.those.that.plague.the.poor..Strong.patent.protections.in.medicine.have.motivated.R&D.in.global.illnesses.like.AIDS.and.cardiovascular.disease,.but.relatively.little.public.or.private.research.is.directed.at.diseases.specific.to.developing.countries,.like.tuberculosis.and.malaria,.even.though.millions.are.afflicted.by.these.diseases.every.year.84

The.incentive.problems.in.these.areas.of.research.are.the.same.as.that.which.causes.the.orphan.drug.problem—too.small.markets.for.firms.to.undertake.risky.R&D.investments..But. the.1983.Orphan.Drug.Act. in. the.United.States.has.pro-vided.a. jolt. to. research.on. rare. illnesses. like.Wilson’s.disease.and.Huntington’s.disease..The.Act.provided.tax.incentives.for.research.into.illnesses.that.caused.less.than.200,000.deaths.per.year,. guaranteed.exclusivity. for.7.years. (in.addition. to.patent.protection),.provided.expedited.reviews.by.the.FDA,.and.introduced.grants.for.qualifying.research..Twelve.times.as.many.biotechnology-based.treatments.for.rare.diseases.were.introduced.annually.in.the.decade.following.the.enactment.of.the.legislation.compared.to.the.decade.before..Mortality.from.rare.illnesses.also.declined.after. the.bill,.both.in.absolute.terms.and.relative.to.more.common.dis-eases..Deaths.from.rare.illnesses.had.been.growing.relative.to.deaths.from.other.diseases.84

In.agriculture,.PIPRA.constitutes.a.response.to.the.“orphan.drug”.problem.related.to.the.development.of.GM.crop.traits.for.horticultural.crops..Researchers.in.both.the.medical.and.agricultural.fields.also.endeavor.to.develop.international.partnerships.to.


mimic.the.successes.for.orphan.drugs.and.orphan.crops.for.the.purpose.of.delivering.valuable.technologies.to.poor.parts.of.the.world.

PREDICTIONS AND POLICY RECOMMENDATIONS

Since.1998,.there.has.been.a.downward.trend.in.R&D.intensity.for.crops.with.low.market.potential..Technology.has.marched.forward,.but.it.seems.to.have.left.horti-cultural.crops.behind..The.pace.of.innovation.has.been.slowed.by.market.resistance.and.regulatory.hurdles,.but.it.seems.to.have.slowed.disproportionately.for.specialty.crops.2.Despite.comparable.market.size.and.time.to.market,.pharmaceutical.R&D.investment.has.far.outpaced.crop.biotechnology.investment.22

Despite.the.decline.in.R&D.effort,.there.are.reasons.to.be.optimistic.that.rising.demand.for.horticultural.biotechnology.could.provide.a.jolt.to.lagging.R&D..Though.no.plant.breeding.technology.had.ever.elicited.consumer.opposition.before.agricul-tural.biotechnology,.other.revolutionary.technology.in.the.food.industry.has.caused.concerns.that.were.eventually.diminished..Huffman.and.Rousu.reported.that.it.took.40.years.for.consumer.opposition.to.pasteurization.of.milk.to.diminish.24.At.first,.the.opposition.was.widespread,.but.after.considerable.experience.with.pasteurized.milk.and.long.after.the.emergence.of.a.scientific.consensus.on.its.benefits,.full.acceptance.was.attained..The.slow.path.to.acceptance,.however,.was.not.without.costs.as.Pirtle.estimated.thousands.of.avoidable.deaths.occurred.because.acceptance.of.pasteurized.milk.was.not.more.immediate.85

General. acceptance. of. agricultural. and. horticultural. biotechnology. can. be.expected.to.grow.with.accumulating.experiences..Regardless,.clear. trends.in.food.demand.suggest. that.an.increasingly.wealthy,.educated,.and.discriminating.public.will. demand. more. and. more. differentiated. products. so. that. they. can. accomplish.heterogeneous.health,.environmental,.and.ethical.objectives..As.they.learn.to.dis-tinguish.organic,.low-fat,.low-carbohydrate,.and.farm.fresh.foods,.for.instance,.they.can.also.be.expected.to.identify.quality-enhancing.GM.traits.and.separate.irratio-nal.concerns.from.legitimate.risks..In.other.words,.niche.markets.for.GM.products.should.continue.to.grow..To.the.extent.there.is.a.consensus.view.on.consumer.accep-tance,.it.holds.that.the.right.quality-enhancing.attributes.will.be.warmly.received..As.consumers.become.more.accepting.and.demanding.of.GM.foods,.retailers.should.become.less.averse.to.stocking.them,.sending.appropriate.signals.up.the.supply.chain.to.seed.companies.

The. regulatory. environment. for. horticultural. biotechnology. is. also. changing.and. possibly. improving. with. growing. evidence. of. its. deficiencies.. Perhaps,. no.policy.change.has.broader.implications.than.Europe’s.decision.in.2008.to.lift.its.moratorium.on.GM.foods..Though.the.market.in.Europe.remains.constrained,.the.liberalizing.of.biotechnology.should.have.implications.for.policymakers.in.politi-cal.capitals.around.the.world,.especially.in.Africa,.and.should.provide.an.induce-ment. to. innovating. firms.. Costs. associated. with. regulatory. compliance. should.decline. as. policymakers. work. to. remove. socially. inefficient. barriers. to. entry..Governments.have.also.undertaken.to.improve.identity.preservation.infrastructure.throughout. their. food. systems. in. response. to. lapses. in. food. safety. and. risks. of.terrorist.attacks.on.the.food.supply..As.this.infrastructure.improves,.it.should.be.


easier. for. food.processors. to. serve.niche.markets. for.GM.foods.without. risking.liability.and.reputational.effects.associated.with.comingling..And.while.registra-tions. for. horticultural. crops. in. the. United. States. and. other. developed. countries.have.stagnated,.China.is.aggressively.moving.ahead.with.horticultural.biotechnology.applications..Perhaps,.competition.will.induce.other.countries.to.follow..The.rise.of.public–private.partnerships.like.PIPRA.that.intended.to.reduce.barriers.to.entry.is.also.likely.to.provide.increasingly.valuable.services.to.start-ups.and.horticultural.crop.operations.as.they.develop.

A.number.of.changes.to.IPR.and.regulatory.policy.could.also.improve.the.outlook.for.horticultural.biotechnology..First,.governments.should.move.from.a.system.of.event.and.variety-based.testing.to.one.that.is.trait-based..That.is,.innovators.should.not.be.required.to.undertake.the.same.testing.and.compliance.effort.to.introduce.an.already-approved.trait.into.a.new.variety.or.species..To.require.such.redundancy.in.testing. regimes. ignores. the.reduced. risks.associated.with.moving.a.probably.safe.trait.into.a.new.plant..Second,.governments.should.adopt.tolerances.for.adventitious.GM.content.in.foods.that.respect.consumer.choice.but.do.not.cause.the.market.for.GM.foods.to.collapse..The.tolerances.adopted.in.the.E.U..are.too.strict.to.permit.the.development.of.GM.food.markets..They.should.be.increased..Governments.should.also.pursue.standardized.policies.that.reduce.the.compliance.costs.to.firms.that.rely.on.exports.to.many.jurisdictions..Finally,.in.recognition.of.the.private.sector’s.insuf-ficient.incentives.to.invest.in.horticultural.crops.at.the.socially.optimal.level,.the.pub-lic.sector.should.step.up.its.effort.to.reduce.the.orphan-crop.problem..Horticulture.has.historically.been.publicly.supported..Even.though.policy.changes.have.provided.private.sector. inducements.for.agricultural.biotechnology,. it. is.clear.by.revealed.preference.that.firms.are.not.sufficiently.motivated.to.provide.biotechnology.appli-cations. for. small.markets.. An. orphan. crop. act,. like. the.Orphan.Drug.Act,. could.increase.the.incentive.for.research.on.GM.specialty.crops.

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193

9 Consumer Acceptance of Genetically Modified Foods: Traits, Labels, and Diverse Information

Wallace E. Huffman

The.United.States.has.more. than.a.decade.of.experience.with.commercially.mar-keted,.genetically.modified.(GM).horticultural.and.field.crops.and.is.the.dominant.player.in.GM.crops..The.first.GM.commercial.crops.were.the.Flavr-Savr.tomato.and.the.Russet.Burbank.New.Leaf.potato,.both.deregulated.in.1994.and.marketed.com-mercially.shortly.thereafter..At.about.the.same.time,.field.crop.varieties.possessing.“input.traits”.were.also.developed.and.first.marketed.commercially..GM.papaya.was.developed.by.the.public.sector.and.successfully.marketed.a.little.later..Some.of.the.economic.issues.facing.biotech.horticultural.crops.have.been.summarized.1,2

In.this.chapter,.new.experimental.economic.methods.are.described.and.used.to.assess.consumer.willingness.to.pay.for.food.products.that.might.be.made.with.new.transgenic.and.intragenic.GM.traits..Participants.in.my.lab.auctions.are.randomly.

CONTENTS

Early.Development.of.GM.Crops........................................................................... 194New.Development.of.GM.Vegetable.Crops............................................................ 197Food.Labels.and.Information.................................................................................. 198Experiments.Designed.to.Assess.Consumer.Willingness.to.Pay.for.GM.Foods....... 200Experimental.Evidence.on.Key.Bid-Price.Differences...........................................204

Simple.Differences.in.Mean.Bid.Prices.............................................................204Individual.Bid-Price.Differences.and.Diverse.Information...............................204Individual.Differences.in.Bid.Prices.and.Prior.Information..............................206Other.Related.Results.........................................................................................207

Discussion.of.GM.Technology.and.Mixed.Messages.............................................207Conclusions.and.Predictions.for.the.Future............................................................208Appendix.A:. Information.Injected.into.the.2001.Des.Moines.and.St..Paul.Experiments........................................................................................209Appendix.B:. Information.Injected.into.the.2007.Des.Moines.and.Harrisburg.Experiments................................................................................... 212References............................................................................................................... 215


chosen. adult. consumers. in. major. U.S.. metropolitan. areas.. Food. labels. are. kept.simple.and.focus.on.key.attributes.of.experimental.goods..Diverse.private.informa-tion.from.the.agricultural.biotech.industry.(largely.Monsanto.and.Syngenta),.envi-ronmental.groups.(largely.Greenpeace.and.Friends.of.the.Earth),.and.independent.third-party.information.(scientifically.objective.at.the.time.of.the.experiments).is.used.to.construct.the.information.treatments..Willingness.to.pay.is.determined.by.experimental. lab. auctions. under. random. food. label. and. information. treatments..Auctions.are.best.described.as.sealed.bid.random.nth.price.and.not.a.first.price.of.Vickery.2nd.price.auction..I.show.that.participants.in.these.experiments.respond.both.to.food.labels.and.information.treatments,.but.no.single.type.of.information.is.dominant.

The.first.section.of.the.chapter.reviews.early.development.of.GM.crops.that.were. largely. input. traits. of. herbicide. tolerance. and. insect. resistance. that. have.now.spread.unevenly.across. the.world,.and. the. second.section.describes.some.new.methods.for.developing.GM.vegetable.crops.with.enhanced.consumer.attri-butes..The.third.section.summarizes.the.development.of.U.S..food-label.policy.and.requirements..The.fourth.section.briefly.describes.experimental.evidence.on.consumer.willingness. to.pay. for.GM.foods.using. lab.auctions.of.experimental.commodities..In.the.final.section,.some.conclusions.and.predictions.about.likely.future.developments.in.commercial.horticultural.crops.are.presented.

EARLY DEVELOPMENT OF GM CROPS

The.first.commercial.GM.crops.were.the.Flavr-Savr.tomato,.developed.for.sale.in.the.United. States. by. Calgene,. and. the. Russet. Burbank. New. Leaf. potato. developed. by.Monsanto..These.two.products.were.the.first.whole.foods.produced.with.biotechnology.that.were.approved.by.the.U.S..Food.and.Drug.Administration.(FDA).for.retail.sale.*.The.Flavr-Savr.tomato,.“a.delayed-ripening.tomato,”.was.the.product.of.more.than.a.decade.of.research.to.develop.a.tomato.that.could.be.picked.when.ripe.and.transported.without.bruising..(pp..256–257).3.The.claim.was.that.it.would.have.a.longer.shelf-life.than.conventional.tomatoes.and.would.provide.consumers.and.processors.with.tastier.tomatoes.because.the.fruit.had.been.left.to.mature.on.the.vine.†.This.was.accomplished.

*.A.potential.food.safety.concern.was.raised.by.the.fact.that.in.creating.the.GM.tomato,.a.marker.gene.for. the.antibiotic.kanamycin.was.inserted..The.marker.gene.is.helpful. for.identifying.which.plants.have.been.affected.by.a.target.gene.3.When.the.marker.is.an.antibiotic,.technicians.test.for.presence.of.successfully.transferred.genes.of.interest.by.applying.the.antibiotic..Only.those.cells.that.contain.the.antibiotic.resistance.will.survive.this.treatment,.and.thereby.indicate.that.an.accompanying.target.gene.is.present..The.FDA,.however,.found.no.food.safety.concerns.due.to.insertion.of.foreign.antibiot-ics.into.plant.cells.

†. Because.U.S..winter.fresh.market.tomato.production.is.concentrated.in.Florida,.which.means.shipping.the.fruit.long.distances.to.the.U.S..retail.markets.in.the.East,.Midwest,.and.South,.it.is.important.that.the.fruit.does.not.perish.on.its.journey.to.market.because.of.its.soft.skin..(Winter.tomatoes.for.the.West.are.generally.supplied.by.Mexico.).The.conventional.solution.has.been.for.tomato.farmers.to.pick.the.fruits.while.they.are.green,.i.e.,.“mature.greens,”.transport.them.to.the.location.of.the.retail.market.and.then.spray.them.with.ethylene,.a.natural.ripening.agent,.to.artificially.ripen.and.redden.the.fruit..However,.the.artificially.ripened.tomatoes.have.an.inferior.flavor.relative.to.vine-ripened.tomatoes.

195Consumer Acceptance of Genetically Modified Foods

by. gene-silencing,. through. an. antisense. RNA. that. interfered. with. translation. and.reduced.the.production.of.specific.proteins.that.cause.ripening.(pp..256–257).3

The.first.Flavr-Savr.tomatoes.were.sold.in.U.S..grocery.stores.in.the.summer.of.1994.and.were.marketed.as.GM..They.sold.relatively.well.at.first.and.were.in.about.2500.stores.by.June.1995,.but. it.became.apparent. that. their.performance.did.not.match. expectations..First,. the.genes. for.delayed. ripening.were. inserted.into.a. tomato.variety.that.was.best.suited.for.processing,.not.direct.eating,.and.that.bruised.relatively.easily,.contrary.to.its.development.objective..Second,.con-trary.to.expectations,.it.had.a.bland.taste.relative.to.conventional.winter.tomatoes..Third,. the. new. tomato. variety. was. suited. to. California’s. dry. summer. growing.conditions,.but.not. to.the.humid.winter. tomato.growing.regions.of.Florida.and,.as.a.result,.was.susceptible.to.Florida’s.tomato.fungal.diseases..Fourth,.the.retail.price.was.more.than.two.times.higher.than.conventional.fresh.market.tomatoes..Hence,.a.number.of.factors.contributed.to.the.failure.of.the.Flavr-Savr.tomato.in.the.U.S..market.(pp..256–257).3,4

At. the.same.time,.Zeneca.produced.a.related.high-solid.GM.tomato.for.use. in.purees. and. soups,. obtained. approval. for. sale. in. the. United. Kingdom,. and. began.marketing. in.1996.under. the.brand.names.Safeway.Double.Concentrated.Tomato.Puree.and.Sainsbury’s.California.Tomato.Puree..These.products.were.sold.at.a.lower.per.unit.price.than.purees.from.conventional.tomatoes.and.were.marketed.in.larger.containers.to.make.the.product.appear.to.consumers.as.a.“better.value.”.By.1999,.the. GM. puree. had. captured. up. to. 60%. of. the. processed. tomato. market. share. in.the.United.Kingdom..However,.when.unrelated.food.scares.(e.g.,.BSE.in.sheep.and.cattle,.dioxin.in.livestock.feed).started.to.unfold.in.the.United.Kingdom.in.the.late.1990s,.Zeneca’s.GM.high-solid.tomato.varieties.were.a.casualty,.and.they.were.with-drawn.from.the.market.4

Monsanto.engineered.the.Russet.Burbank.New.Leaf.potato.to.be.resistant.to.the.Colorado.potato.beetle,.a.major.potato.pest,.and.this.potato.was.deregulated.in.1994..This.new.variety.offered.growers.the.advantage.of.significantly.reducing.the.need.for. chemical. pesticide. applications,. and. initially. gained. favor. in. the. fresh. potato.market..However,.under.pressure.from.consumer.groups,.the.fast-food.industry.(e.g.,.McDonalds). and. grocery. store. chains. would. not. purchase. it. or. halted. early. pur-chases,.and.this.prompted.Monsanto.to.withdraw.the.GM.potato.variety.from.the.retail.market.in.1999.5

To.date,.the.most.commercially.successful.GM.crops.in.the.United.States.have.been.those.engineered.with.so-called.input.traits,.namely,.insect.resistance.(Bt).and.herbicide. tolerance. (HT.or.RR/RoundUp.Ready)..These.GM.traits.were.obtained.by.transferring.genes.largely.from.soil.bacteria.into.selective.species.of.commercial.field.crops.to.induce.resistance.or.tolerance.to.target.organisms,.thereby.creating.a.so-called.transgenic.crop.variety.(pp..250–256).3.The.target.of.these.traits.has.been.canola,.soybean,.cotton,.and.corn,.with.commercial.GM.varieties.of.these.four.crops.first.introduced.in.the.mid-1990s..These.GM.field.crops,.whose.products.are.largely.destined.for.oil,.feed,.and.fiber,.have.had.very.rapid.grower.adoption.(Figure.9.1)..For.instance,.HT.soybeans.were.first.marketed.to.farmers.in.1996.and.now.account.for.more.than.90%.of.U.S..soybean.acreage..Canola.with.HT.has.also.been.successful,.although.it.is.a.relatively.small.acreage.crop.in.the.United.States..Bt.and.HT.cotton.


also.got.off.to.a.fast.start.in.1996..By.2005,.about.60%.of.U.S..cotton.carried.HT.or.Bt.genes,.with.the.most.recent.varieties.carrying.both.traits.as.a.“stacked.gene”.variety..GM.field.corn.got.off.to.a.slow.start,.and.in.2005.about.30%.of.the.corn.acreage.was.planted.to.Bt.and.15%.to.HT.*.In.corn,.recent.stacking.of.multiple.Bt.genes,.impart-ing.resistance.against.European.corn.borer.and.rootworm,.often.together.with.HT,.has.pushed.GM.corn.acreage.up.in.the.last.2.years.and.is.anticipated.to.become.the.“gold.standard”.in.the.future.†

Genetic. improvement. of. papaya. remains. the. one. bright. example. of. successful.public.sector.bioengineering.of.a.horticultural.food.crop..Starting.in.the.1940s,.the.Hawaiian.papaya.fruit. industry.was.ravaged.by.papaya.ringspot.virus.and,.by.the.1980s,.Hawaii’s.papaya.production.had.fallen.significantly.and.was.concentrated.in.the.Puna.district.of.the.Big.Island..However,.by.the.early.1990s,.ringspot.virus.was.invading.that.area,.too..The.University.of.Hawaii.at.Mãnoa,.Cornell.University.and.the.U.S..Department.of.Agriculture-Agricultural.Research.Service.(USDA-ARS).then. initiated.new. research. and.developed.a. transgenic. papaya.variety. that. was.resistant. to. ringspot,. labeled. Rainbow,. and. it. was. released. freely. to. farmers. in.

*.Zilberman.has.argued.that.rational.regulation.of.transgenic.products.should.compare.their.risks.and.benefits.with.the.risks.and.benefits.of.alternative.technologies..Current.regulations.ignore.the.alterna-tives,.and.this.is.costly.to.society.6

†. Although.herbicide-tolerant.wheat.varieties.have.been.developed.for.the.United.States.by.Monsanto,.they.have.not.been.marketed.widely,.nor.are.they.currently.available.to.farmers.because.of.consumer.resistance.due.to.the.use.of.wheat.largely.for.food.such.as.breads,.pastas,.etc.

Percent of acres100

80

60

40

20

01996 1997 1998 1999 2001 2002 2003 2004 2005 2006 20072000

HT

HT cotton

Bt cottonHT cornBt corn

soybeans

FIGURE 9.1 (See color insert.).Adoption.of.genetically.engineered.crops:.United.States,.1996–2007..Note:.Data.for.each.crop.category.include.varieties.with.both.HT.and.Bt.(stacked).traits.. (Data. for. 1996–1999. are. from. Femandez-Cornejo. and. McBride. (2002);. data. for.2002–2007.are.available.in.the.ERS.Data.Product,.adoption.of.genetically.engineered.crops.in.the.United.States,.Tables.1–3.)


1998.7,8.Since. its. introduction,. the.Rainbow.variety.of.papaya.has.been.used.stra-tegically.to.create.a.virus-free.ring.as.a.buffer.to.slow.down.the.spread.of.papaya.ringspot.virus.in.the.Puna.area.and.to.slow.down.or.reduce.resistance.of.consumers.and/or.some.growers.to.the.new.GE.technology.7,9.Papaya.is.the.only.U.S..crop.in.which.public. sector. scientists.have.pioneered. the. development.of. a. commercially.successful.GM.crop.variety.

Overall,.the.early.commercial.successes.with.GM.crops.were.not.with.horticul-tural.crops,.but.rather.with.a.small.set.of.field.crops..The.most.likely.reason.is.that.consumers.failed.to.personally.see.enhanced.value.from.many.of.the.new.GM.traits.that.were.scientifically.possible..In.fiber.crops.and.crops.that.are.used.heavily,.but.not.exclusively,.for.livestock.feed,.consumer.acceptance.was.less.important..However,.the.image.of.GM.crops.with.consumers.has.been.damaged.by.the.fact.that.new.GM.products.with.unique.enhanced.consumer.attributes.have.been.slow.to.develop.

NEW DEVELOPMENT OF GM VEGETABLE CROPS

As.transgenic.GM.technology.has.been.developed.and.marketed.for.a.small.set.of.field.crops,.a.new.line.of.research.has.recently.emerged.around.intragenic.GM.hor-ticultural.crops.10,11.Prompted.by.continued.consumer.resistance.to.transgenic.food.crops,.these.new.methods.introduce.new.traits.into.a.crop.variety.by.using.only.DNA.from.the.same.species,.thereby.yielding.an.“intragenic”.genetic.modification..This.research.was.made.possible.by.a.small.set.of.scientists.recognizing.that.a.huge.range.of.genetic.diversity.exists.within.horticultural.crops.that.have.been.grown.for.a.long.period.of. time. under. diverse. environments. and. human. needs,. perhaps. in. relative.isolation..For.example,.major.genetic.diversity.exists.in.potato.and.tomato,.stretching.from.very.old.primitive.or.landrace.varieties.to.the.modern.commercial.varieties.of.today..However,.in.the.case.of.the.potato,.inbreeding.depression,.tetraploid.genetics,.and.clonal.propagation.conspire.to.make.traditional.breeding.difficult.and.slow..By.taking.a.new.intragenic.bioengineering.approach,.genomic.and.metabolic.pathway.discoveries.can.be.quickly.introduced.into.established.commercial.varieties.to.fast-track.the.breeding.process.without.introducing.foreign.DNA.or.antibiotic.markers.

Economists.have.shown.recently.that.consumer.acceptance.of.GM.food.crops.is. intimately. linked. to. the. type.of. traits. engineered. into. the. crops,. the. types.of.food.labels.on.retail.food.products,.and.the.information.environment..For.example,.the.GM.food.market.has.been. subjected. to.diverse.and.conflicting. information,.and.this.makes.informed.decision.making.by.consumers.and.producers.difficult.12.Although.the.first.commercial.GM.crop.was.a.horticultural.crop.(the.Flavr-Savr.tomato).with.“enhanced.consumer.attributes,”.all.of.the.other.commercially.suc-cessful. GM. crops. in. the. United. States. have. possessed. input. traits—traits. that.reduce.either. the.cost.of.production.or. the.variance. in. the.cost.of.production. to.farmers. and,.hence,. have.only. benefited. consumers. to. the. extent. that. they.have.lowered.food.prices.or.increased.food.availability..Although.Falck-Zepeda.et.al.13.and.Moschini.et.al.14.show.that.consumer.surplus.benefits.from.these.technologies.have.been.sizeable,.these.benefits.have.not.registered.effectively.with.consumers..With.new.intragenic.potatoes.and.tomatoes.that.are.engineered.for.dramatically.


enhanced.antioxidants. and.vitamins. (e.g.,. vitamin.C,.A,.or.E),. improved. starch.content,.and/or.reduced.bruising.becoming.scientifically.possible,.U.S..consumers.may,.for.the.first.time,.see.GM.crops.as.having.direct.positive.value.to.them.over.conventionally.bred.crops.*

FOOD LABELS AND INFORMATION

Economists. have. shown. that. food. labels. and. information. are. important. factors.conditioning. consumer. response. to. GM. foods. (e.g.,. see. Rousu. et. al.,12. Huffman.and.Rousu,15. and.Moschini.and.Lapan16)..Food.products. in. the.United.States.can.be. labeled. for.nutritional.claims.and. for. safety..The.1990.Nutrition.Labeling. and.Education.Act.dramatically.changed.nutrition.labels.on.packaged.foods.sold.in.U.S..supermarkets.17,†. This. law. requires. packaged. foods. to. display. nutrition. informa-tion.prominently. in.a.new.label. format,.namely,. the.Nutrition.Facts.panel.. It.also.regulates.serving.size,.health.claims.(that.link.a.nutrient.to.a.specific.disease),.and.descriptor.terms,.for.example,.“low.fat,”.on.food.packages..The.goal.of.this.legisla-tion.was.to.improve.consumer.welfare.by.providing.nutrition.information.that.would.assist.consumers.in.making.healthy.food.choices.

As.an.indication.of.the.costliness.of.effective.food.nutrient.labeling,.it.is.esti-mated.that.the.U.S..food.industry.spent.$2.billion.to.comply.with.the.1990.Nutrition.Labeling.and.Education.Act.(NLEA).(Public.Law.101-535).18.However,.some.attri-butes,.such.as.enhanced.calcium.and.vitamins.A.and.C,.are.viewed.positively.by.consumers,.that.is,.more.is.better.in.the.case.of.positive.consumer.attributes..But.other.food.attributes,.such.as.salt,.fat,.and.pesticide.residue,.are.negative,.and.then.the.consumer.views.less.to.be.better..Food.labels.before.the.Nutrition.Labeling.and.Education.Act.had.a.seeming.emphasis.on.negative.labeling..Balasubramanian.and.Cole17.suggest.that.this.tendency.can.be.explained.by.consumers.having.an.asym-metric.value.function,.weighing.a.dollar.of.loss.more.heavily.than.a.dollar.of.gain,.which.is.Tversky.and.Kahneman’s19.prospect.theory.

The.policies.under.the.Nutrition.Labeling.and.Education.Act.also.tend.to.emphasize.negative.rather.than.positive.labeling..First,.permissible.health.claims.are.ones.that.asso-ciate.specific.nutrients.with.reduced.risk.of.specific.diseases..Of.the.seven.health.claims.approved.by.the.FDA.at.the.onset.of.the.new.nutrient.labeling.act,.three.linked.negative.attributes.exclusively.with.deadly.diseases,.that.is,.dietary.fat.with.cancer,.sodium.with.hypertension,.and.dietary.saturated.fat.with.high.cholesterol.and.heart.disease,.and.only.one.claim.featured.a.positive.attribute,.that.is,.calcium.and.osteoporosis..Later.claims.have,.however,.been.more.balanced..Second,.regulations.on.nutrient-content.claims.tend.to.focus.more.heavily.on.negative.attributes.(calories,.sugar,.sodium,.fat,.fatty.acids,.and.cholesterol).than.on.positive.attributes.such.as.fiber.and.vitamins.

*.Antioxidants.are.substances.that.may.protect.human.cells.from.the.damage.otherwise.caused.by.unsta-ble.molecules.known.as.free.radicals..Free.radical.damage.over.time.is.believed.to.cause.some.types.of.cancer..Antioxidants.interact.with.and.stabilize.free.radicals.and.may.prevent.some.of.the.damage.free.radicals.might.otherwise.cause..Antioxidants.include.beta-carotene,.lycopene,.vitamins.C,.E,.and.A,. and. other. substances.. These. compounds. are. sometimes. called. phytonutrients. and. are. naturally.occurring.in.at.least.low.levels.in.most.fruits.and.vegetables.

†. Unpackaged.foods—for.example,.fresh.fruits.and.vegetables,.are.not.affected.


GM.content.has.not.been.shown.to.have.negative.human.health.consequences..Nevertheless,.with.foods.made.currently.from.crop.varieties.that.contain.GM.input.traits,.adding.a.label.for.GM.content.would.be.an.example.of.labeling.a.negative.food.attribute.

Genetically.engineered.products.used.for.food,.however,.do.have.to.pass.a.food.safety.test..In.1992,.the.FDA.announced.its. landmark.decision.that.GM.food.and.food.products.will.be.regulated.the.same.as.those.created.by.conventional.means..This.policy.allows.new.GM.foods.to.be.treated.as.conventional.foods.as.long.as.they.meet.three.conditions:.their.nutritional.value.has.not.been.lowered;.they.incorporate.new.substances.that.are.already.a.part.of.the.human.diet;.and.they.contain.no.new.allergenic.substances..In.January.2001,.the.FDA.issued.a.“Guidance.for.Industry”.statement.reaffirming.this.policy..In.this.statement,. the.FDA.stated.to.the.biotech.industry.that.the.only.GM.foods.that.need.to.be.labeled.are.foods.that.have.different.characteristics.from.the.non-GM.version,.for.example,.elevated.vitamin.A.levels..In.the.United.States,. labeling.food.for.GM.content. is.not.otherwise.required..Firms,.however,. are. to.notify. the.FDA.at. least.4.months.before.putting. a.new.GM.food.product.on.the.market,.and.the.scientific.description.of.the.product.is.posted.on.the.Internet.for.review.during.this.time.20.Only.minor.changes.have.been.made.in.these.guidelines.since.2001.

Hence,.the.GM-labeling.policy.in.the.United.States.can.be.classified.as.being.volun-tary..If.a.voluntary.label.is.affixed,.the.FDA.has.mandated.that.it.cannot.use.the.phrase.“genetically.modified.”.The.FDA.prefers.the.phrase.“genetically.engineered”.or.“made.through.biotechnology.”*.Effective.GM.labeling,.however,.involves.real.costs,.espe-cially.the.costs.of.testing.for.the.presence.of.GM.content,.segregating.GM.and.non-GM.products,.variable.costs.of.monitoring.for.truthfulness.of.labeling.and.enforcement.of.the.regulations.that.exist,.and.risk.premiums.for.being.out.of.contract.21,22

An.effective.GM-labeling.policy.includes.effective.segregation.of.GM.from.non-GM.commodities..If.one.or. the.other.of. these.products.could.be. inexpen-sively.color.coded,.segregation.might.not.be.very.expensive..If,.however,. iden-tity.preservation. through. the.production,.marketing,.and.processing.chain.was.required,. this. system. would. be. substantially. more. costly.21. To. the. extent. that.there.is.a.market.for.non-GM.products,.buyers.would.be.expected.to.specify.in.their.purchase.contracts.some.limit.on.GM.content.and/or.precise.prescriptions.regarding.production/marketing/handling.processes..One.can.envision.a.market-place.of.buyers.with.differentiated.demand.according. to. their. aversion. to.GM.content..To.make.this.differentiation.effective,.new.costs.and.risks.are.incurred..Additional. testing. involves. costs. of. conducting. the. tests,. for. which. there. are.several.technologies.of.varying.accuracy..The.risk.is.that.GM.products.will.be.commingled.with.non-GM.products,.so.the.detection.system.must.test.to.see.that.customers’.shipments.are.within.contract.limits.for.GM.content..This.is.a.serious.

*.In.contrast,. the.European.Commission.adopted.GM.food.labels. in.1997..The.Commission.requires.each.member. country. to. enact. a. law. requiring. labeling.of. all. new.products. containing. substances.derived.from.GM.organisms..Japan,.Australia,.and.many.other.countries.have.also.passed.laws.requir-ing. GM. labels. for. major. foods.. The. international. environmental. lobby. has. frequently. argued. that.“consumers.have.the.right.to.know.whether.their.food.is.GM.or.not.”23


economic.problem,.as.agents.seek.to.determine.the.optimal.strategy.for.testing.and.other.risk.mitigation.strategies.*

While.private.sector.handlers.routinely.segregate.and.blend.grains.and.beans.as.a.primary.function.of.their.business,.new.risks.arise.when.handling.GM.and.non-GM.products,.due.to.the.added.risk.of.adventitious.commingling..When.GM.is.the.inferior.product,.growers.and.handlers.of.GM.products.have.an.incentive.to.mix.GM.with.non-GM.products..For.U.S..grains,.Wilson.and.Dahl.suggest.that.this.risk.may.be.about.4%.at.the.grain.elevator.level.21.Farmer-processor.contracting.in.horticul-tural.or.specialty.crops,.however,.could.reduce. this.margin.by.specializing. in. the.product.being.delivered,.such.as.non-GM.or.a.positive.GM.trait..Another.source.of.risk.is.testing,.because.no.test.is.100%.accurate..Testing.risk,.however,.varies.with.the.technology,.tolerance,.and.variety.of.products.handled,.and.seems.likely.to.fall.over.time,.as.the.technology.of.testing.advances.

In.markets.where.there.is.imperfect.information.due.to.one.or.more.parties.hav-ing.private. information,.private.parties.have.an. incentive. to.use. their. information.to.enhance.their.private.goals.24,25.Highly.conflicting.information.has.been.injected.into.the.GM.food.market.by.interested.parties..These.vested.parties.are.the.agricul-tural.biotech.industry.(pp..153–183),26.including.Monsanto,.DuPont/Pioneer.Hi-Bred,.Dow,.Syngenta,.and.BASF,.that.have.disseminated.information.that.is.very.favorable.to.GM. technologies,. crops. and. food.products,. and.environmental. groups,. includ-ing. Greenpeace,. Friends. of. the. Earth,. Action. Aid,. and. Earth. Watch,. that. have.disseminated.information.that. is.very.negative.about.GM.crops,.such.as.calling.it.“Frankenfood.”27.This.diverse.information.has.undoubtedly.contributed.to.the.GM.food.controversy.and.may.be.one.factor.explaining.differences.across.Western.coun-tries. in. their. acceptance.of.GM.crops..Also,.consistent.with.consumer.education,.independent. third-party.or.verifiable.information.about.agricultural. .biotechnology.may. have. considerable. value. if. available. and. disseminated. to. consumers.12,28,29.Verifiable. information.provides.an.objective.assessment.of. the.benefits.and.costs,.including.environmental.risks,.of.GM.crop.varieties.and.the.foods.made.from.these.raw.materials..Hence,.society.can.avoid.losses.due.to.the.strategic.behavior.of.inter-ested.parties.toward.new.technologies.and.products.if.decision.makers.have.access.to.and.use.independent.third-party.or.verifiable.information.

EXPERIMENTS DESIGNED TO ASSESS CONSUMER WILLINGNESS TO PAY FOR GM FOODS

Because.GM.foods.are.relatively.new,.my.research.team.chose.to.use.an.auction.market.setting30.to.collect.information.about.consumer.willingness.to.pay.for.(or.demand).GM.foods.12,31.This. reflects. the. reality. that.GM.food.products.are.not.

*. “Tolerances”.are.an.important.issue.in.segregation.and.identity.preservation..GM.tolerance.refers.to.the.maximum.impurity.level.for.GM.content.that.is.tolerated.in.a.product.that.still.carries.the.non-GM.label..There.are.two.levels.where.tolerances.apply:.one.is.defined.by.regulatory.agencies.such.as.the.FDA,.and.the.other.is.commercial.tolerance..Individual.firms.can.and.seem.likely.to.adopt.different.tolerance.lev-els,.subject.to.any.regulation..Moreover,.different.countries.are.likely.to.have.different.tolerance.levels,.and.this.increases.the.risks.and.costs.of.segregation.or.identity.preservation.


generally.labeled.in.the.United.States,.so.grocery.store.purchases.are.not.informa-tive. on. this. issue.. Some. scientists. have. used. contingent. value. or. stated. prefer-ence.surveys.of.consumer.willingness.to.pay.for.new.products..These.surveys.are.known.to.contain.hypothetical.bias;.participants.in.these.surveys.are.not.required.to.execute. their.stated.preferences,. that. is,.“participants.don’t.have.to.pay.what.they.say.”32–34

In.contrast,.in.our.auction.market.settings,.consumers.were.expected.to.execute.their.winning.bids.by.purchasing.one.unit.of. the.auctioned.commodity..Also,.in.contrast.to.most.economics.experiments.that.use.university.undergraduate.students.from.the.investigator’s.class.as.auction.participants,.we.used.randomly chosen adult consumers in major metropolitan areas.that.were.identified.by.an.independent..survey.agency.and.told.that.a.university.project.was.being.undertaken.to.obtain.consumers’.assessments.of.food.and.new.household.products..In.particular,.screened.individuals.were.not.told.that.they.would.be.assessing.genetically.modified.organisms.(GMOs).or.even.would.be.participating.in.an.experimental.auction.

Economists.frequently.choose.a.Vickery.2nd.price.auction.(http://en.wikipedia.org/wiki/Vickrey_auction).for.valuing.goods..However,.it.is.well.known.that.individ-uals.who.anticipate.that.they.are.far.from.placing.the.margin.bid.will.bid.randomly.and.insincerely..These.participants.have.a.real.sense.that.their.bid.is.not.pivotal.in.determining.the.market.price..We.chose.the.random.nth.price.auction.35.In.this.auc-tion,.the.winning.bidders.are.chosen.from.a.uniform.distribution.over.1.to.n,.the.total.number.of.bidders.in.a.session..For.example,.if.there.are.15.participants.in.a.session,.the.bids.are.first.ranked.from.1.to.15,.and.the.randomly.drawn.n.is.5;.then.the.four.highest. bidders.pay. the.fifth.highest. price..With. this. type.of. auction. mechanism,.all. bidders. are. engaged.because. they. sense. that. their. bid.will. help.determine. the..market.price,.or.bidding.their.true.willingness.to.pay.is.a.weakly.dominate.strategy.36.Moreover,.our.auction.is.best.described.as.being.a sealed-bid random nth price auc-tion,.because no information about willingness to pay for experimental products is released before all bids are placed.

Individuals.who.agreed.to.participate.came.to.a.central.location,.signed.a.personal.consent.form,.were.paid.$40.for.their.participation,.and.completed.a.short.question-naire.on.their.social-demographic-economic.characteristics.and.beliefs.about.a.few.technologies,.including.GMOs..They.received.instruction.in.the.mechanics.of.a.ran-dom.nth.price.auction,.and.participated.in.an.auction.practice.session..Next,.they.took.a.short. test.on. their.understanding.of. the.auction.mechanism.and.any.ques-tions.were.answered..The.auctioning.of.experimental.commodities.followed..After.winning. bids. were. determined,. the. participants. completed. another. short. survey,.and.then.were.told.to.execute.winning.binds.by.completing.purchases.of.auctioned.commodities.in.an.adjacent.stock.room..Otherwise,.they.were.told.that.they.were.free.to.leave.

In. all. of. our. experiments,. we. used. three. sets. of. diverse. information. about.genetic.modification.and.GM.foods.to.construct. information.treatments:. (1). the.industry (pro-biotech) perspective—a.collection.of. statements. and. information.on.genetic.modification.provided.by.a.group.of.leading.biotechnology.companies,.including. Monsanto. and. Syngenta;. (2). the. environmental group (anti-biotech)


perspective—a.collection.of.statements.and.information.on.genetic.modification.from.Greenpeace,.a.leading.environmental.group;.and.(3).the.independent, third-party (verifiable information) perspective—a.statement.on.genetic.modification.approved. by. a. third-party. group,. consisting. of. a. variety. of. people. knowledge-able.about.GM.goods,. including.scientists,. professionals,. religious. leaders,. and.academics,.who.do.not.have.a.financial.stake.in.GM.foods..We.limited.the.infor-mation.statements. to.one.page.and.organized.the.information.under.five.differ-ent.headings,.General.Information,.Scientific.Impact,.Human.Impact,.Financial.Impact,.and.Environmental.Impact,.to.reduce.the.information.load.on.participants.(Appendices.A.and.B)..Information.treatments,.consisting.of.one-to-three.of.the.above.information.types,.were.injected.into.each.of.the.sessions.or.experimental.trials.. For. example,. in. the. first. set. of. experiments,. the. information. treatments.were:.(1).only.the.industry.perspective;.(2).only.the.environmental.group.perspec-tive;. (3). industry. and.environmental. perspectives;. (4). environmental. and. third-party. perspectives;. (5). industry,. environmental,. and. third-party. perspectives..Information. treatments. were. randomly. assigned. sessions. without. replacement..When.a.session.received.industry.and.environmental.perspectives,.the.order.was.randomized..When.a.session.received.the.third-party.perspective,.it.was.always.displayed.last.

In. the. 2001. Des. Moines. and. St.. Paul. experiments,. each. experimental. unit.(or.session).of.13–16.individuals/consumers.participated.in.only.two.rounds.of.bidding.on.experimental.food.items..The.rounds.were.differentiated.by.the.food.label..In.one.round,.which.could.be.round.1.or.2.depending.on.the.experimental.unit,. participants/consumers. bid. on. three. food. products,. each. with. a. conven-tional.food.label.that.stated.only.the.type of food.and.weight..In.the.other.round,.participants.bid.on.the.same.three.food.products.with.a.GM.label,.which.differed.from. the. conventional. food. label. by. the. inclusion. of. only. one. extra. sentence:.“This.product.is.made.using.genetic.modification.(GM)”.(Figure.9.2)..Each.ses-sion. or. experimental. unit. received. an. information. treatment. chosen. randomly.from.the.six.available.treatments..A.total.of.172.individuals.participated.in.these.experiments.

In.the.2007.Des.Moines.and.Harrisburg.experiments,.each.experimental.unit.(or.session).of.13–16.individuals/consumers.participated.in.four.rounds.of.bidding.on.experimental.food.items..The.rounds.were.differentiated.by.the.composition.of.the.food.label,.which.had.seven.variants.including.a.baseline.with.no.information.(with.a.maximum.of.four.used.in.any.session).(Figure.9.3).

Although. all. bidders. in. a. session. or. round. of. bidding. saw. the. same. food.labels,. they. received. different. information. treatments. in. the. Des. Moines. and.Harrisburg. experiments.. The. Des. Moines. and. Harrisburg. experiments. were.unique.in.not.only.distinguishing.the.type.of.GMO,.but.also.for.injecting.a.treat-ment.with.“No Information.”.Also,.the.exact.wording.of.the.three.types.of.infor-mation.was.modified.to.be.appropriate.to.the.emphasis.of.these.experiments;.for.example,.the.industry.perspective.and.third-party.perspective.described.the.key.differences.between.“Transgenic”.and.“Intragenic.GM.Products”.(Appendices.A.and.B).


Russet potatoes

Net weight 5 lb.

Tortilla chips

Net weight 16 oz.fresh made Thursday April 5th

Russet potatoes

Net weight 5 lb.

This product is made usinggenetic modification (GM).

Tortilla chips

Net weight 16 oz.fresh made Thursday April 5th


Vegetable oil

Net weight 32 fl. oz.


Vegetable oil

Net weight 32 fl. oz.

FIGURE 9.2 GM.and.Plain.Food.Labels.for.2001.Des.Moines.and.St..Paul.Experiments.

Potato (5 lbs.)Potato (5 lbs.)

GM free product

Potato (5 lbs.)Intragenic GM product

Potato (5 lbs.)Transgenic GM product

Potato (5 lbs.)Enhanced levels of

antioxidants and vitamin CGM product


antioxidants and vitamin CIntragenic GM product


antioxidants and vitamin CTransgenic GM product

FIGURE 9.3 Food.Labels.for.Potato.in.the.2007.Des.Moines.and.Harrisburg.Experiments.


EXPERIMENTAL EVIDENCE ON KEY BID-PRICE DIFFERENCES37

SImple dIfferenceS In mean bId prIceS

We.first.examine.simple.bid-price.differences.for.consumer.willingness.to.pay.for.food.items.labeled.as.being.genetically.modified.versus.having.a.plain/conventional.food.label,.specifying.only.the.type.and.weight.of.the.food..The.2001.Des.Moines.and.St..Paul.data.showed.that.bidders.discounted.the.GM-labeled.product.by.about.15%.relative.to.its.plain-labeled.counterpart.*

Turning. to. the. 2007. Des. Moines. and. Harrisburg. samples,. bidders. were,. on.average,.willing.to.pay.a.sizeable.premium.for.food.products.containing.the.label.“Enhanced.Levels.of.Antioxidants.and.Vitamin.C—Intragenic.GM.Product”.relative.to.a.plain/conventionally.labeled.food.product..The.premium.ranged.from.39.to.45.cents.per.unit.on.the.three.food.products,.or.19%–26%.higher..The.mean.price.for.each.of.the.three.commodities—broccoli,.tomatoes,.and.potatoes—with.enhanced.attributes. relative. to. products. with. a. plain/conventional. label,. was. different. from.zero.at.the.5%.significance.level.†.Hence,.when.consumers.bid.on.GM.products.con-taining.input.traits,.the.GM.product.was.weakly inferior.to.the.plain/conventionally.labeled.product..However,.when.consumers.bid.on.fresh.intragenic.GM.horticultural.products.containing.enhanced.levels.of.antioxidants.and.vitamin.C,.they.were.will-ing. to.pay.a.premium.and,.hence,. the. intragenic.products.were.economically.and.statistically.superior.to.a.plain/conventionally.labeled.product.‡.These.results.imply.a.dramatic.difference.in.the.incentives.for.private.industry.to.label.new.GM.products.with.enhanced.consumer.attributes.relative.to.those.derived.from.raw.materials.that.contain.input.traits.22

IndIvIdual bId-prIce dIfferenceS and dIverSe InformatIon37

When.bidders.received.both.pro-biotech.and.anti-biotech.information,.the.bid.price.difference. was. reduced,. reflecting. the. opposing. forces. of. the. two. types. of. infor-mation.in.this.information.treatment..Moreover,.this.information.treatment.did.not.have.a.statistically.significant.effect.on.bid.price.differences..From.these.results,.we.conclude.that.in.those.sessions.where.bidders.received.only.anti-biotech.or.both.pro-biotech.and.anti-biotech.information,.they.bid.differently.than.when.they.received.only.pro-biotech.information.

When.bidders.were.in.sessions.that.received.the.pro-biotech.and.verifiable.infor-mation. treatment,. the. impact.of. this.combination.was.not. statistically. significant..

*.All.three.differences.are.significantly.negative.at.better.than.the.0.07.significance.level.†. The.mean.bid.prices.where.the.biotech.method.was.switched.from.intragenic.to.transgenic,.but.other-

wise.containing.enhanced.consumer.attributes,.were.larger.than.for.the.plain.label.but.14%–22%.lower.than.for.the.intragenic.label.

‡. We.have.not.undertaken.sensory.tests.to.determine.whether.consumers.can.perceive.any.differences.in.the.conventional.product.relative.to.one.with.intragenic.enhanced.consumer.attributes..Given.that.no.“foreign”.DNA.nor.antibiotic.marker.is.present.in.the.intragenic.product,.my.hypothesis.is.that.no.perceived.sensory.differences.exist..Moreover,.Zhao.et.al..found.no.difference.in.a.sensory.analysis.of.conventional.and.organically.grown.fruits.and.vegetables,.except.that.conventionally.grown.tomatoes.scored.slightly.higher.for.ripeness,.which.is.positively.correlated.with.flavor.intensity.38


When. bidders. were. in. sessions. that. received. anti-biotech. and. verifiable. informa-tion,.bid-price.differences.are.reduced.(less.negative).and.the.difference.is.signifi-cantly.different. from.zero..Hence,. those.who. received.a. treatment.of.anti-biotech.information. and.verifiable. information.discounted.GM.foods. less. than. those.who.received.only.a.treatment.of.anti-biotech.information..When.bidders.were.in.sessions.that.received.a.treatment.that.contained.all.three.types.of.information.(pro-biotech,.anti-biotech,. and.verifiable),. the. impact.of. this. treatment.on.bid-price.differences.was.small.and.not.statistically.significant..Hence,.in.this.complex.setting,.verifiable.information.did.not.have.a.distinguishable.effect.

Bidders.who.had.larger.household.incomes.discounted.GM.by.a.larger.amount.than.those.with.less.household.income..This.result.is.statistically.significant.at.the.5%.level.and.is.consistent.with.non-GM.products.being.viewed,.on.average,.by.bid-ders. as. a. superior. product.. Participants. coming. into. our. experiments. were. asked.about. how. well-informed. they. were. about. genetic. modification.. This. subjective.information.was.then.coded.into.a.dichotomous.variable..Those.bidders.who.con-sidered.themselves.to.be.at.least.“somewhat.informed.about.GM.foods”.discounted.GM-labeled.foods.more.than.did.other.bidders..This.effect.is.statistically.significant.(at. the.10%. level).. Moreover,. this. result. suggests. that. bidders. in.our. experiments.who.were.“GM-informed”.had,.on.average,.acquired/received.negative.information.about.GM.foods.prior.to.the.experiment.*.Bids.also.were.affected.by.the.labeling.sequence..Bidders.in.sessions.that.bid.on.the.GM-labeled.food.products.in.round.one.(and.the.plain-labeled.food.products.in.round.two).discounted.GM-labeled.foods.by.less.than.those.who.were.in.sessions.that.bid.on.the.products.in.the.opposite.order..This result reinforces the importance of randomized assignments of treatments to sessions in experimental auctions,.which.is.an.innovation.in.our.methodology.

Next,.we.turn.to.the.2007.sample.of.individuals.from.Des.Moines.and.Harrisburg..In. these. results,. the. base. case. with. no-information. treatment. gives. a. bid-price.difference.of.intragenic.GM-.over.plain-labeled.food.products.by.a.statistically.sig-nificant.52.cents.per.unit.of.product..When.pro-biotech.information.treatment.was.injected.into.the.experiments,.the.bid-price.difference.was.a.statistically.significant.73.cents.per.unit.or.21.cents.more.than.for.the.no-information.treatment,.suggesting.a.net.positive.influence.of.industry-provided.biotech.information.on.willingness.to.pay.for.intragenic.GM.products..The.injection.of.the.anti-biotech.information.treatment.gives.a.bid-price.difference.of.only.25.cents.per.unit,.which.is.27.cents.per.unit.lower.than.for.the.no-information.treatment..However,.this.coefficient.is.not.different.from.zero.at.the.5%.or.10%.significance.levels,.suggesting.that.environmental.groups.do.not.distinguish.between.intragenic.and.transgenic.biotech.methods.for.engineering.new.crops..The.injection.of.a.pro-biotech.and.anti-biotech.treatment.(where.the.order.is. random.across.participants. in.a.session). increases. the.bid.price.difference.by.a.statistically.significant.56.cents.per.unit..The.impact.of.this.information.treatment.on.bid-price.differences.is.slightly.higher.(4.cents.per.unit).than.for.the.no-information.treatment,.which.is.a.reflection.of.the.opposing.forces.of.pro-biotech.and.anti-biotech.information,. but. with. the. edge. going. to. the. pro-biotech. information.. When. the.

*.See.Huffman.et.al.39.for.an.analysis.of.the.impact.of.bidders’.prior.beliefs.about.GM.technology.and.food.products.on.their.willingness.to.pay.for.food.items.that.are.potentially.GM.


injected.information.treatment.includes.all.three.types.of.information.(with veri-fiable. information. always. last),. the.bid-price.difference. is. larger.by. a. statistically.significant.44.cents.per.unit,.which.is.8.cents.less.than.for.the.no-information.treat-ment..Comparing.this.result.to.the.previous.one,.verifiable.information.seems.to.be.a.moderating.force.relative.to.the.pro-biotech.industry.perspective.

Consumers.have.expressed.some.skepticism.of.transgenic.GM.food.products,.and.our.2007.Des.Moines.and.Harrisburg.data.set.shows.that.consumers.bid.significantly.higher. prices. for. intragenic. than. transgenic. food. products. that. contain. enhanced.levels.of.antioxidants.and.vitamin.C..However,. the.differences.in.these.bid.prices.may.be.affected.by.the.information.environment.in.which.consumers.are.bidding..To.test.this.hypothesis,.we.examined.the.differences.in.bid.prices.for.products.with.an.intragenic.GM.label.versus.one.with.a.transgenic.GM.label.

Bid-price.differences. for. the.baseline.case.of. the.no-information. treatment.are.18 cents.per.unit.higher.for.intragenic.than.transgenic,.but.this.number.is.not.signifi-cantly.different.from.zero.at.the.10%.or.5%.levels,.suggesting.that.this.information.is.truly.uninformative.about.these.biotech.methods..In.contrast,.an.injection.of.the.pro-biotech.information.treatment.gives.a.bid-price.difference.by.a.statistically.significant.67.cents.per.unit,.suggesting.that,.on.net,.the.industry.perspective.favors.intragenic. over. transgenic.. In. contrast,. an. injection. of. anti-biotech. information.treatment.gives.a.bid-price.difference.of.only.17.cents.per.unit,.which.is.slightly.lower.than. the. point. estimate. for. the. no-information. treatment. and. 50. cents. per. unit.lower. than.for. the.pro-biotech. information. treatment.. Injection.of. the.pro-biotech.and.anti-biotech.information.treatment.gives.a.bid-price.difference.of.a.statistically.significant.42.cents.per.unit,.which. is. in. the.middle.of. the.estimates. for. the.pro-biotech.and.anti-biotech.treatment.estimates..The.injection.of.the.information.treat-ment.containing.all.three.information.types.gives.a.bid.price.difference.of.38.cents,.which. suggests. that. objective. information. moderates. the. positive. impact. of. the.pro-biotech.information.on.bid-price.differences.

The.estimated.coefficient.for.the.dummy.variable.denoting.that.the.food.product.has.enhanced.antioxidants.and.vitamin.C.is.a.positive.12.cents.per.unit.and.signifi-cant.at.the.10%.level..This.result.suggests.that.the.presence.of.enhanced.consumer.attributes.increases.the.value.to.bidders.of.the.intragenic.over.transgenic.methods..Our.results.also.showed.a.significant.“bidding.round.or.order.effects”.in.the.data,.which.supports.methodological.advances.using.randomization..Household.income.has.no.impact.on.bid-price.differences.in.these.results..In.conclusion,.I.can.say.that.the.information.setting.affects.consumer.discounting.of.transgenic.relative.to.intra-genic.horticultural.products.

IndIvIdual dIfferenceS In bId prIceS and prIor InformatIon

New. food. products. using. GM. crops. appeared. in. U.S.. supermarkets. starting. in.1996,. and. consumers. perceived. some. risks.. Because. consumers. are. exposed. to.diverse.and.sometimes.conflicting.perspectives.about.GM.technologies.and.foods,.they.form.subjective.beliefs..Huffman.et.al..examined.the.role.of.these.prior.beliefs.on.consumer.willingness.to.pay.for.foods.that.might.be.genetically.modified.39.The.data.are.from.the.2001.Des.Moines.and.St..Paul.experiments,.where.participants.


were.asked.before.the.experimental.auction.how.well-informed.they.were.about.genetic.modification—extremely,.well,.somewhat,.not.very.well,.or.not.informed.at.all..Huffman.et.al..show.that.participants.who.had.informed.prior.beliefs.about.genetic. modification. discounted. GM-labeled. food. products. by. a. larger. amount.than.those.who.had.uninformed.prior.beliefs..Also,.uninformed participants.were.especially.susceptible.to.information.from.interested.and.third.parties..In.contrast,.informed participants.were.generally.not.affected.significantly.by.new.informa-tion..These.results.contradict.some.earlier.psychological.studies.that.claimed.that.individuals. tend. to.base.rates.19.The.results.show.how.both.skeptics.and.propo-nents.of.new.technologies.might.try.to.manage.information.to.achieve.private.or.group-wide,.but.not.social,.objectives.

other related reSultS

Rousu.et.al..examine.the.impact.of.tolerance.levels,.or.the.impact.of.the.minimum.level.of.GM.contamination.that.will.pass.as.GM-free.40.Using.the.2001.Des.Moines.and.St..Paul.data,.they.examine.bids.on.three.food.products.that.have.different.toler-ance.labels..In.one.trial,.all.consumers.bid.on.foods.with.a.non-GM.label,.certified.to.be.completely.free.of.genetically.engineered.material,.and.in.the.other.trial,.con-sumers.bid.on.foods.with.a.non-GM.label.indicating.that.a.certain.percentage.of.GM.material,.either.1%.or.5%.percent,.was.tolerated..Consumers.in.these.treatments.did.not.receive.any.information.on.GM.food.products..This.experiment.contained.three.experimental.units/sessions.with.a.total.of.44.participants.

Rousu. et. al.. found. evidence. that. consumers. preferred. foods. that. were. 100%.non-GM,.relative.to.food.products.with.small.amounts.of.GM.material.(1%.or.5%)..Consumers.bid.approximately.10%.less.for.the.GM-tolerant.food.products.than.they.did.for.the.certified.GM-free.products..However,.they.found.that.once.GM.content.was.present,.no.difference.existed.in.bids.between.foods.that.contained.1%.versus.5%.GM.content.40.Thus,.while.these.findings.indicate.that.a.significant.percentage.of.consumers.will.pay.more.for.GM-free.labeled.food.products.relative.to.convention-ally.labeled.food.products,.it.does.not.appear.that.1%.or.higher.tolerance.levels.for.GM.material.matters.

DISCUSSION OF GM TECHNOLOGY AND MIXED MESSAGES

Although.consumers.in.the.United.States.are.relatively.tolerant.of.alternative.produc-tion.methods.for.their.food,.they.do.respond.adversely.to.some.risks..For.example,.when.genetic.modification.refers.to.input.traits,.consumers.in.our.experiments.dis-counted.GM.food.products.by.15%,. relative. to. a.plain-.or. conventionally. labeled.alternative..This.seems.to.arise.from.environmental,.biodiversity,.or.health.concerns.from.introducing.foreign.DNA.into.food.crops..To.circumvent.these.concerns,.new.methods.have.been.developed.for.intragenic.genetic.modification,.where.no.foreign.DNA.is.introduced.in.the.GM.varietal.development.process..Our.results.from.the.2007.data.support.the.hypothesis.that.consumers.have.a.more.favorable.perspective.about. these.genetic.modifications,.and. that. they.are,. in. fact,.willing.to.pay.a.pre-mium.for.enhanced.levels.of.antioxidants.and.vitamin.C.by.intragenic.GM.methods..


However,. when. we. were. experimenting. with. food. traits. to. consider. including.enhanced.food.products,.we.experimented.with.“low.pesticide.residual”.as.a.food.quality. attribute.. Consumer. reaction. to. this. trait. was. complex,. because. the. label.raised.a.dormant.issue.that,.yes,.there.is.pesticide.residue.in.our.food..Mentioning.that.insect-resistant.(Bt).traits.could.be.introduced.so.as.to.reduce.the.need.for.farmers.to.apply.commercial.pesticides,.and.thereby.reduce.chemical.pesticide.residual,.was.a.hard.sell.

Along. a. similar. line. of. mixed. messages,. Markosyan. et. al.. conducted. surveys.of. consumers. in. grocery. stores. in. October. 2006. in. the. Pacific. Northwest. to. test.their.willingness.to.pay.for.“naturally.enriched.antioxidant.coatings”.embedded.in.the.wax.on.retail.fresh.apples.41.In.general,.consumers.were.willing.to.pay.a.little,.4%–8%.more,.for.the.antioxidant.enhanced.apples,.but.a.number.of.consumers.were.quite.negative.about. the.technology,.for.example,.“I.don’t.want. to.eat.wax,”.“it. is.unnatural,”.“additives.to.fruit.are.not.necessary,”.“washing.apples.removes.the.wax,”.“prefer.foods.without.additives,”.and.“it.is.better.to.get.nutrients.naturally.”.Hence,.adding.antioxidants. to. the.wax.of. apples.also. raised. the.dormant. issue. that. fresh.commercial.apples.are.waxed.

CONCLUSIONS AND PREDICTIONS FOR THE FUTURE

More.than.a.decade.has.passed.since.the.first.GM.foods.appeared.in.U.S..grocery.stores..Early.attempts.to.market.fresh.horticultural.products,.in.particular,.the.Flavr-Savr.tomato.and.the.Russet.Burbank.Newleaf.potato.failed.after.very.brief.appear-ances.in.the.market..Input.traits.developed.by.transgenetic.methods.applied.to.field.crops. have. been. much. more. successful,. but. consumers. continue. to. express. some.resistance.to.them.because.of.environmental,.biodiversity,.or.human.health.concerns..Recent.developments.of.new.intragenic.GM.methods.are.exciting.because.they.per-mit.scientists.to.use.diverse.genes.and.attributes.identified.in.the.genomes.of.particu-lar.horticultural.plants.to.quickly.enhance.quality.attributes.of.commercial.varieties.of.crops.like.potato.and.tomato..This.is.very.important.in.potato.breeding,.where.it.is.impossible.for.scientists.to.dramatically.enhance.conventional.levels.of.vitamin.C.and.antioxidants.using.a.range.of.conventional.non-GM.breeding.methods..The.new.intragenic.GM.potato.varieties.enhanced.with.antioxidants.and.vitamin.C.promise.to.be.the.first.successful.commercial.GM.product.with.consumer.traits.on.the.market.

Economists’.research.has.shown.that.U.S..consumer.acceptance.and.willingness.to.pay.for.GM.food.crops.is.not.only.conditioned.by.the.nature.of.the.new.trait.and.the.method. of.DNA. transfer,. but. also.by. the. content. of. food. labels,. prior.beliefs.of. consumers,. and. content. of. diverse. information. injected. into. the. food. market.about.GM.technologies.and.food.products..During.the.era.of.input-trait.dominated.GM.foods,.consumers.have.revealed.that.GM.food.products.are.weakly.inferior.to.conventional.products,.which.means.that.marketers.of.GM.food.products.will.not.label.voluntarily..With.the.commercialization.of.new.intragenic.GM.products.with.enhanced.consumer.attributes,.these.new.GM.food.products.promise.to.command.a.premium.relative.to.conventional.food.products..Hence,.the.private.sector’s.incentive.to.voluntarily. label.GM.products.will.change.dramatically..This.promises.to.be.a.positive.development.in.the.commercialization.of.GM.food.products.


During.the.era.of.input.traits,.consumers’.informed.prior.beliefs.were.somewhat.negative.about.agricultural.biotechnology.and.GM.foods..These.prior.beliefs,.how-ever,.have.the.potential.to.become.more.favorable.toward.GM.horticultural.crops.as.new.products.with.intragenic.GM-enhanced.consumer.attributes.become.generally.available.in.the.food.market..Also,.strong.evidence.exists.that.consumers.are.posi-tively. influenced. by. biotech. industry. or. pro-biotech. information,. and. negatively.affected.by.environmental.group.or.anti-biotech.information..Moreover,.third-party.verifiable.information.has.been.shown.to.be.a.moderating.influence.on.consumer.interpretation. of. anti-biotech. and. pro-biotech. information. and. on. willingness. to.pay.for.GM.foods..Hence,.a.future.role.exists.for.public.sector.provision.of.third-party.verifiable.information.about.GM.technologies.and.GM.food.products..This.new.information.will.affect.prior.beliefs.of.consumers.about.GM.food.products.and.be.a.useful.input.to.objective.assessments.of.new.GM.food.products.that.enter.the.market,.which.could.be.important.to.the.commercial.successes.of.new.GM.horti-cultural.crops.

APPENDIX A: INFORMATION INJECTED INTO THE 2001 DES MOINES AND ST. PAUL EXPERIMENTS42

A..The.following.is.a.collection.of.statements.and.information.on.genetic.modification.from.Greenpeace,.a.leading.environmental.group.

General InformationGenetic. modification. is. one. of. the. most. dangerous. things. being. done. to. your.food. sources. today.. There. are. many. reasons. that. genetically. modified. foods.should. be. banned,. mainly. because. unknown. adverse. effects. could. be. cata-strophic!. Inadequate. safety. testing.of.GM.plants,. animals,. and. food.products.has.occurred,.so.humans.are.the.ones.testing.whether.or.not.GM.foods.are.safe..Consumers.should.not.have.to.test.new.food.products.to.ensure.that.they.are.safe.

Scientific ImpactThe.process.of.genetic.modification. takes.genes.from.one.organism.and.puts.them. into. another.. This. process. is. very. risky.. The. biggest. potential. hazard.of.GM.foods.is.unknown..This. is.a.relatively.new.technique,.and.no.one.can.guarantee.that.consumers.will.not.be.harmed..Recently,.many.governments.in.Europe.assured.consumers.that.there.would.be.no.harm.to.consumers.over.mad.cow.disease,.but.unfortunately,.their.claims.were.wrong..We.do.not.want.con-sumers.to.be.harmed.by.GM.food.

Human ImpactGM.foods.could.pose.major.health.problems..The.potential.exists.for.allergens.to.be.transferred.to.a.GM.food.product.that.no.one.would.suspect..For.example,.if.genes.from.a.peanut.were.transferred.into.a.tomato,.and.someone.who.is.allergic.to.peanuts.eats.this.new.tomato,.they.could.display.a.peanut.allergy.

Another.problem.with.GM.foods.is.a.moral.issue..These.foods.are.taking.genes.from.one.living.organism.and.transplanting.them.into.another..Many.people.think.it.is.morally.wrong.to.mess.around.with.life.forms.on.such.a.fundamental.level.


Financial ImpactGM.foods.are.being.pushed.onto.consumers.by.big.businesses,.which.care.only.about. their.own.profits.and. ignore.possible.negative.side.effects..These.groups.are.actually.patenting.different.life.forms.that.they.genetically.modify,.with.plans.to.sell.them.in.the.future..Studies.have.also.shown.that.GM.crops.may.get.lower.yields.than.conventional.crops.

Environmental Impact GM.foods.could.pose.major.environmental.hazards..Sparse.testing.of.GM.plants.for. environmental. impacts. has. occurred.. One. potential. hazard. could. be. the.impact.of.GM.crops.on.wildlife..One.study.showed.that.one.type.of.GM.plant.killed.Monarch.butterflies.

Another.potential.environmental.hazard.could.come. from.pests. that.begin. to.resist.GM.plants.that.were.engineered.to.reduce.chemical.pesticide.application..The.harmful.insects.and.other.pests.that.get.exposed.to.these.crops.could.quickly.develop.tolerance.and.wipe.out.many.of.the.potential.advantages.of.GM.pest.resistance.

B..The.following.is.a.collection.of.statements.and.information.on.genetic.modifica-tion.provided.by.a.group.of.leading.biotechnology.companies,.including.Monsanto.and.Syngenta.

General InformationGM.plants.and.animals.have.the.potential.to.be.one.of.the.greatest.discoveries.in.the.history.of.farming..Improvements.in.crops.so.far.relate.to.improved.insect.and.disease.resistance.and.weed.control..These.improvements.using.bioengineering/GM.technology.lead.to.reduced.cost.of.food.production..Future.GM.food.prod-ucts.may.have.health.benefits.

Scientific ImpactGenetic.modification.is.a.technique.that.has.been.used.to.produce.food.products.that.are.approved.by.the.FDA..Genetic.engineering.has.brought.new.opportuni-ties. to. farmers. for. pest. control. and. in. the. future. will. provide. consumers. with.nutrient-enhanced. foods.. GM. plants. and. animals. have. the. potential. to. be. the.single.greatest.discovery.in.the.history.of.agriculture..We.have.just.seen.the.tip.of.the.iceberg.of.future.potential.

Human ImpactThe.health.benefits.from.genetic.modification.can.be.enormous..A.special.type.of.rice.called.“golden.rice”.has.already.been.created.which.has.higher.levels.of.vitamin.A..This.could.be.very.helpful.because.the.disease.vitamin.A.deficiency.(VAD).is.dev-astating.in.third-world.countries..VAD.causes.irreversible.blindness.in.over.500,000.children,.and.is.also.responsible.for.over.1.million.deaths.annually..Since.rice.is.the.staple.food.in.the.diets.of.millions.of.people.in.the.third.world,.golden.rice.has.the.potential.of.improving.millions.of.lives.a.year.by.reducing.the.cases.of.VAD.

The.FDA.has.approved.GM.food.for.human.consumption,.and.Americans.have.been.consuming.GM.foods.for.years..While.every.food.product.may.pose.risks,.there.has.never.been.a.documented.case.of.a.person.getting.sick.from.GM.food.


Financial ImpactGM.plants.have.reduced.the.cost.of.food.production,.which.means.lower.food.prices,.and.that.can.help.feed.the.world..In.the.United.States,.lower.food.prices.help.decrease.the.number.of.hungry.people.and.also.let.consumers.save.a.little.more.money.on.food..Worldwide. the.number.of.hungry.people.has.been.declining,.but. increased.crop.production.using.GM.technology.can.also.help.further.reduce.world.hunger.

Environmental ImpactGM.technology.has.produced.new.methods.of.insect.control.that.reduce.chemical.insecticide.application.by.50%.or.more..This.means.less.environmental.damage..GM.weed.control.is.providing.new.methods.to.control.weeds,.which.are.a.special.problem.in.no-till.farming..Genetic.modification.of.plants.has.the.potential.to.be.one.of.the.most.environmentally.helpful.discoveries.ever.

C..The.following.is.a.statement.on.genetic.modification.approved.by.a.third-party.group,.consisting.of.a.variety.of.individuals.knowledgeable.about.GM.foods,.includ-ing.scientists,.professionals,.religious.leaders,.and.academics..These.parties.have.no.financial.stake.in.GM.foods.

General InformationBioengineering. is. a. type. of. genetic. modification. where. genes. are. transferred.across.plants.or.animals,.a.process.that.would.not.otherwise.occur.(in.common.usage,. genetic. modification. means. bioengineering).. With. bioengineered. pest.resistance.in.plants,.the.process.is.somewhat.similar.to.the.process.of.how.a.flu.shot.works.in.the.human.body..Flu.shots.work.by.injecting.a.virus.into.the.body.to.help.make.a.human.body.more.resistant. to.the.flu..Bioengineered.plant-pest.resistance.causes.a.plant.to.enhance.its.own.pest.resistance.

Scientific ImpactThe.FDA.standards.for.GM.food.products.(chips,.cereals,.potatoes,.etc.).are.based.on. the.principle. that. they.have.essentially. the. same. ingredients,. although. they.have.been.modified.slightly.from.the.original.plant.materials.

Oils.made.from.bioengineered.oil.crops.have.been.refined,.and.this.process.removes.essentially.all.the.GM.proteins,.making.them.like.non-GM.oils..So.even.if.GM.crops.were.deemed.to.be.harmful.for.human.consumption,.it.is.doubtful.that.vegetable.oils.would.cause.harm.

Human ImpactWhile.many.GM.foods.are.in.the.process.of.being.put.on.your.grocers’.shelves,.there.are.currently.no.foods.available.in.the.United.States.where.genetic.modifi-cation.has.increased.nutrient.content.

All.foods.present.a.small.risk.of.an.allergic.reaction.to.some.people..No.FDA-approved.GM.food.poses.any.known.unique.human.health.risks.

Financial ImpactGM.seeds.and.other.organisms.are.produced.by.businesses.that.seek.profits..For.farmers.to.switch.to.GM.crops,.they.must.see.benefits.from.the.switch..However,.genetic.modification.technology.may.lead.to.changes.in.the.organization.of.the.


agri-business.industry.and.farming..The.introduction.of.GM.foods.has.the.poten-tial.to.decrease.the.prices.to.consumers.for.groceries.

Environmental ImpactThe. effects. of. genetic. modification. on. the. environment. are. largely. unknown..Bioengineered. insect. resistance. has. reduced. farmers’. applications. of. environ-mentally.hazardous. insecticides..More.studies.are.occurring. to.help.assess. the.impact.of.bioengineered.plants.and.organisms.on.the.environment..A.couple.of.studies.reported.harm.to.Monarch.butterflies.from.GM.crops,.but.other.scientists.were.not.able.to.recreate.the.results..The.possibility.of.insects.growing.resistant.to.GM.crops.is.a.legitimate.concern.

APPENDIX B: INFORMATION INJECTED INTO THE 2007 DES MOINES AND HARRISBURG EXPERIMENTS

A..The.following.is.a.collection.of.statements.and.information.on.genetic.modifica-tion.from.Greenpeace,.a.leading.environmental.group.

General InformationGenetic. modification. takes. genes. from. one. organism. and. places. them. into.another.. The. process. lets. scientists. manipulate. genes. in. an. unnatural. way..Inadequate.safety.testing.of.GM.plants.and.food.products.has.occurred..Humans.and.the.Earth.are.being.used.as.guinea.pigs.for.testing.whether.“Frankenfoods”.are.safe..GM.foods.should.be.banned.because.their.effect.on.consumers.and.the.environment.is.unknown.and.potentially.catastrophic!.Genetic.modification.is.one.of.the.most.risky.things.being.done.to.your.food.sources.today.and.should.be.stopped.before.more.damage.is.done.

Scientific ImpactAll.genetic.modifications.of.plants.are. risky..All.GM.techniques.are. relatively.new.and.no.one.can.guarantee. that.consumers.or. the. environment.will.not.be.harmed..The.biggest.potential.hazard.of.GM.foods.is.unknown.

Human ImpactGM.foods.could.pose.serious.risks.to.human.health..Some.foods.contain.aller-gens,.and.the.potential.exists.for.allergens.to.be.transferred.into.a.GM.food.prod-uct. that. no. one. would. suspect.. For. example,. if. the. genes. from. a. peanut. were.transferred.into.a.tomato,.and.someone.who.is.allergic.to.peanuts.eats.this.GM.tomato,.he.could.display.a.peanut.allergy.

Another.problem.with.transgenic.foods.is.a.moral.issue..Many.GM.techniques.transfer.genes.across.species..We.believe.it.is.morally.wrong.to.alter.life.forms.on.such.a.fundamental.level.

Financial ImpactGM.foods.are.being.pushed.onto.consumers.by.big.businesses.which.only.care.about.their.own.profits.and.ignore.possible.negative.side.effects..These.groups.are.actually.patenting.new.life.forms.they.create.with.plans.to.sell.for.profits..Studies.have.shown.that.GM.crops.may.even.get.lower.yields.than.conventional.crops.


Environmental ImpactGM.foods.could.pose.major.environmental.hazards..Little.testing.of.GM.plants.for.environmental.impacts.has.occurred..One.potential.risk.of.GM.crops.is.their.impact.on.wildlife,.including.wild.species.of.plants.and.insects..A.study.showed.that.one.type.of.GM.plant.killed.Monarch.butterflies.

Another.potential.environmental.hazard.could.come.from.pests.that.become.resis-tant.to.new.naturally.occurring.toxic.substances.engineered.into.plants.to.kill.pests—insects.and.worms—or.to.make.a.plant.resistant.to.a.particular.herbicide.application..The.target.pests.that.get.exposed.to.these.new.GM.crops.could.quickly.develop.toler-ances.and.wipe.out.many.of.the.potential.advantages.of.GM.pest.resistance.

B..The.following.is.a.collection.of.statements.and.information.on.genetic.modifica-tion.provided.by.a.group.of.leading.biotechnology.companies,.including.Monsanto,.Pioneer,.and.Syngenta.

General InformationGM.plants.have.the.potential.to.be.one.of.the.greatest.discoveries.in.the.history.of.farming..GM.crops.have.lowered.food.production.costs.by.improving.insect.and.disease.resistance.and.weed.control.in.plants..New.genetic.engineering.tech-niques.could.dramatically.enhance.consumer.benefiting.attributes.of.food.such.as.vitamins,.antioxidants,.flavor,.and.shelf.life..These.improvements.to.plant.quality.can.only.be.attained.through.GM,.not.conventional.breeding.

The.process.of.genetic.modification.takes.genes.from.one.organism.and.places.them.into.another..There.are.two.distinct.types.of.GM.used.by.biotechnology.compa-nies..Transgenic.GM.transfers.genes.between.two.unrelated.organisms,.for.example,.from.soil.bacteria.to.corn..Intragenic.GM.involves.transferring.genes.between.two.breeds.of.the.same.organism,.for.example,.from.wild.species.of.corn.to.a.commercial.variety.of.corn.

Scientific ImpactBoth. transgenic. and. intragenic. techniques. are. used. to. produce. food. products.that.are.approved.by.the.FDA..Intragenic.modification.is.a.genetic.technique.for.significantly.speeding.up.the.conventional.process.of.plant.cross-breeding,.which.has.been.undertaken.by.farmers.and.plant.breeders.for.thousands.of.years..Many.industry.groups.believe.intragenics.should.require.minimal.FDA.testing.because.no.foreign.genes.or.proteins.are.added.to.the.GM.plant..We.have.only.seen.the.tip.of.the.iceberg.of.the.future.potential.of.GM.for.improving.worldwide.health.and.nutrition.through.enhanced.plants.

Human ImpactThe.potential.exists.for.GM.to.dramatically.enhance.traits.that.have.direct.value.to.consumers,.such.as.increased.vitamins.and.antioxidants,.more.flavor,.longer.shelf.life,.lower.pesticide.use,.and.reduced.cost.of.production..Superior.GM.plants.will.help.reduce.worldwide.malnutrition.and.improve.the.healthiness.of.foods..The.FDA.has.approved.GM.food.for.human.consumption,.and.Americans.have.been.consum-ing.GM.foods.for.a.decade..While.every.food.(modified.or.not).poses.some.risks,.there.has.never.been.a.documented.case.of.a.person.getting.sick.from.GM.food.


Financial ImpactWith.the.introduction.of.enhanced.nutrition,.antioxidants,.shelf.life,.flavors,.and.other.consumer-desired.attributes.using.GM.technology,.consumers.will.for.the.first.time.enjoy.the.direct.benefits.of.genetic.engineering..GM.plants.have.reduced.farmers’.costs,.which.mean.lower.food.prices..Worldwide.the.number.of.hungry.people. is. declining.. GM. technology. is. helping. to. feed. the. world. and. improve.worldwide.nutrition.

Environmental ImpactGenetic.modification.of.plants.has.the.potential.to.be.one.of.the.most.environ-mentally.helpful.discoveries.ever..GM.technology.has.produced.new.methods.of.insect.control.that.reduce.chemical.insecticide.application.by.50%.or.more..GM.weed.control.is.providing.new.methods.to.control.weeds,.which.are.a.problem.in.no-till.farming..This.means.greater.crop.yields.and.less.environmental.damage.

C..The.following.is.a.statement.on.genetic.modification.approved.by.a.third-party.group.consisting.of.a.variety.of.individuals.knowledgeable.about.GM.foods,.includ-ing.scientists,.professionals,.religious.leaders,.and.academics..These.parties.have.no.financial.stake.in.GM.foods.

General InformationThe.process.of.genetic.modification.takes.genes.from.one.organism.and.places.them. into. another..There.are. two.distinct. types.of.GM.used.by.biotechnology.companies..Transgenic.GM.transfers.genes.between.two.unrelated.organisms,.for.example,.from.soil.bacteria.to.corn..Intragenic.GM.involves.transferring.genes.between.two.breeds.of.the.same.organism,.for.example,.from.wild.species.of.corn.to.a.commercial.variety.of.the.crop..Hence,.intragenic.modification.has.much.in.common.with.conventional.plant.breeding.

Scientific ImpactThe. FDA. standard. for. GM. food. products. is. based. on. the. principle. that. they.have.essentially.the.same.ingredients,.although.modified.from.the.original.plant..Almost.all.GM.crops.meet.the.FDA’s.substantive.equivalent.requirement..Hence,.they.do.not.require.special.testing.before.commercial.marketing.can.occur.

Human ImpactMany.scientists.see.intragenics.as.having.real.potential.for.enhancing.consumer.attributes.of.plants.such.as.dramatically.increasing.vitamin.and.antioxidant.lev-els,.extending.shelf.life,.and.reduced.chemical.pesticide.application.without.con-cerns.about.gene.transfer.across.species..These.improvements.to.plants.are.only.possible.using.genetic.modification.and.not.conventional.breeding.

All.foods.present.a.risk.of.an.allergic.reaction.to.a.small.fraction.of.the.popula-tion..No.FDA-approved.GM.food.poses.any.known.unique.human.health.risks,.but.when.genes.are. transferred.across.species,.a.new.allergen. is.possible..This.is.more.likely.with.transgenics.than.intragenics..While.GM.crops.can.result.in.higher.yields.and.enhanced.nutrition,.there.is.no.consensus.whether.GM.foods.have.or.will.reduce.worldwide.hunger.


Many. people. have. moral. or. religious. objections. to. GM.. Some. groups. see.intragenics.as.being.more.acceptable.because.genes.are.transferred.between.two.breeds.of.the.same.species.

Financial ImpactGM.seeds.and.other.organisms.are.produced.by.businesses.that.seek.profits..For.farmers. to.switch. to.GM.crops,. they.must.see.benefits. from.making.a.change..Consumers. must. also. see. benefits. from. consuming. GM. foods—lower. price. or.enhanced.consumer.attributes..However,.GM.technology.may.lead.to.changes.in.the.organization.of.the.agri-business.industry.and.farming.

Environmental ImpactThe. long-term. effects. of. GM. on. the. environment. are. largely. unknown..Bioengineered. insect. resistance. has. reduced. farmers’. applications. of. envi-ronmentally.hazardous.insecticides,.but.resistance.to.this.bio-control.system.will.increase.over.time..More.studies.are.occurring.to.help.assess.the.impact.of.bioengineered.plants.on.the.environment..Some.studies.reported.harm.to.Monarch.butterflies.from.GM.crops,.but.other.scientists.were.not.able.to.rec-reate.the.results.

Enhanced. consumer. attributes,. such. as. vitamins,. antioxidants,. and. longer.shelf.life.due.to.intragenics.pose.no.known.environmental.hazards.

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219

10 Intellectual Property and Development of Transgenic Horticultural Crops

Cecilia L. Chi-Ham and Alan B. Bennett

INTELLECTUAL PROPERTY IN HORTICULTURAL BIOTECHNOLOGY

For. centuries,. horticulturists. have. applied. science. and. art. to. genetically. improve.edible.and.ornamental.plant.varieties..Horticultural. industries.have.produced.new.flowers,.fruit.and.perennial.trees,.landscape.plants,.and.vegetables..Also,.the.indus-try.has.provided.technological.advances.to. improve.plant.cultivation.and.posthar-vest. practices.. The. time. and. economic. investment. by. plant-breeding. programs. is.significant.because.it.may.take.decades.for.the.development.of.a.new.plant.variety.or. improved. seed.. Historically,. breeders’. inventions. have. been. freely. distributed,.diminishing.the.incentive.to.invest.and.the.ability.to.capture.value..In.order.to.rec-oncile. societal.needs. for.new.crops,. recognize.breeders’.creativity,. and.provide. a.means.that.ensures.financial.returns.for.the.economic.investment,.international.and.national.legal.intellectual.property.protections.systems.have.been.developed..As.a.consequence,.there.is.an.increasing.need.for.horticultural.scientists.to.complement.their.technical.knowledge.with.intellectual.property.information.and.awareness.

This.chapter.is.intended.to.provide.horticulturists.with.an.understanding.of.intel-lectual. property. issues. affecting. the. development. and. deployment. of. genetically.engineered.horticultural.crops..The.chapter.is.divided.into.three.sections..First,.we.describe. the. different. forms. of. intellectual. and. tangible. property. rights. and. their.application.in.the.development.of.horticultural.crops,.particularly.transgenic.crops..The.second.part.of.the.chapter.addresses.scientists’.increasing.needs.to.complement.their. technical.knowledge.with.intellectual.property. information.and.awareness..

CONTENTS

Intellectual.Property.in.Horticultural.Biotechnology............................................. 219Types.of.Intellectual.Property.to.Protect.Horticultural.Inventions.........................220Freedom.to.Operate.................................................................................................224Strategies.for.IP.Access..........................................................................................228References...............................................................................................................230


We. specifically. define. freedom-to-operate,. a. systematic. way. of. considering. legal.implications.and.navigating.through.IP.landscape.and.issues.in.research.and.com-mercial. development.. Finally,. the. third. section. describes. strategies. of. accessing.intellectual. property. rights. (IPRs). to. enhance. broad. access. and. dissemination. of.new.transgenic.horticultural.crops..We.present.PIPRA.as.a.model.of.a.public.sector.initiative.that.provides.expertise.to.understand.and.to.facilitate.access.to.IP.for.the.development.and.deployment.of.horticultural.crops.

TYPES OF INTELLECTUAL PROPERTY TO PROTECT HORTICULTURAL INVENTIONS

In.the.case.of.horticultural.crops,.the.legal.systems.protect.human-made.improve-ments,.such.as.a.new.plant.variety,.with.IPRs.and. the.physical.materials,.such.as.seeds,.with.tangible.property.rights.(TPRs)..These.forms.of.IPRs.and.TPRs.can.be.used.by.themselves.or.compounded.to.protect.and.manage.innovations.in.horticul-tural.biotechnology..The.main.types.of.intellectual.property.protection.applicable.to.horticultural.inventions.include.patents,.plant.variety.rights,.trade.secrets,.trade-marks,.and.copyrights.(Table.10.1).1,2.Tangible.property.rights.are.also.important.and.are.often.managed. through.material. transfer.agreements. (MTAs).or.bailments.and.can.be.used.to.limit.the.use.of.physical.materials..These.forms.of.IPRs.and.TPRs.are.the.cornerstone.of.the.innovation.process.in.the.United.States.and.Europe,.and.these.rights.are.increasingly.expanding.into.other.countries.in.the.world.

Below,. we. describe. the. different. forms. of. IPRs. and. TPRs. and. how. these. are.related.to.the.development.of.horticultural.crops..There.are.significant.differences.between.intellectual.and.tangible.property.rights,.particularly.in.terms.of.geographic.and.time.limitations..IPRs,.like.plant-variety.protection.(PVP).or.plant.patents,.are.granted.by.national.offices.for.a.specific.country-jurisdiction.and.for.a.specified.time.period.. In.contrast,. tangible.property.rights.are.not. limited.by.geography.or. time.and.the.terms.governing.their.use.are.left.to.the.discretion.of.the.tangible.property.owner..The. terms.governing. tangible.property.use.are.usually.specified. in.MTAs.or.bailments..Thus,.the.tangible.property.rights.may.extend.beyond.the.protection.offered.by.IPRs.

Of.the.many.forms.of.IPRs,.patents.provide.the.legal.rights.to.exclude.others.from.using.your.invention.that.must.be.novel,.useful,.and.nonobvious.to.one.skilled.in.the.art.of.horticultural.breeding..Patent.rights.are.conferred.by.national.governments.for.a.specified.period.of.time,.usually.20.years,.and.are.limited.to.the.country.in.which.the.patent.is.granted..In.exchange.for.exclusivity,.the.inventor.makes.a.public.disclo-sure.of.the.invention..In.the.United.States,.the.two.forms.of.patents.most.pertinent.to.the.horticultural.industry.are.plant.patents.and.utility.patents.

Plant patents.are.specific.to.the.U.S..legal.system.and.are.granted.only.for.asexu-ally. propagated. plants,. such. as. strawberries. and. fruit. trees,. and. exclude. tuber-propagated. and. uncultivated. species.. At. the. time. the. U.S.. Plant. Patent. Act. was.enacted.in.the.1930s,.sexually.produced.plants.were.believed.to.be.unstable.and.non-uniform.and,.as.a.result,.were.excluded.from.plant.patent.protection..Utility patents.offer.one.of.the.most.robust.forms.of.IP.protection..In.horticultural.biotechnology,.utility.patents.can.be.used.to.protect. the.genetically.modified.seeds.or.plants.and.

221Intellectual Property of Transgenic Horticultural Crops

TABLE 10.1Overview of IP Protection Systems

Means of Protection

What Can Be Protected

How to Protect Term of Protection Rights of the Owner

Utility.patent

Any.useful,.novel,.and.nonobvious.invention

USPTO If.filing.date.is.after.June.8,.1995,.20 years.from.filling.date;.if.filing.date.is.before.June.8,.1995,.17.years.from.issue

Right.to.exclude.others.from.making,.using,.manufacturing,.selling,.and.offering.to.sell

U.S..plant.patent

Asexually.produced.plants.(excludes.uncultivated.and.tuber.propagated.plants)

USPTO If.filing.date.is.after.June.8,.1995,.20 years.from.filling.date;.if.filing.date.is.before.June.8,.1995,.17.years.from.issue.date

Right.to.exclude.others.from.asexually.reproducing,.selling,.or.using.claimed.plant

U.S..PVP Sexually.produced.plants.(excludes.first.generation.hybrids,.uncultivated.plants)

USDA 20.years.most.crops;.25.years.trees,.vines

Right.to.exclude.others.from.importing.or.selling,.sexually.or.asexually.reproducing,.distributing.without.proper.notice,.producing.a.hybrid.or.new.variety,.and.using.the.claimed.patent

UPOV91 All.genera.and.species

UPOV 20.years.most.crops;.25.years.trees,.shrubs,.and.vines

Prevents.others.from.producing.or.reproducing,.conditioning.for.the.purpose.if.propagation,.offering.for.sale,.selling.or.other.marketing,.importing,.exporting,.and.stocking.for.any.purposes

Trademarks Words,.phrases,.and.logos.that.can.distinguish.the.goods.and.services.from.those.of.others

Use.or.have.a.bona.fide.intent.to.use.and.apply.for.a.federal.registration

Unlimited.duration.as.long.as.the.mark.is.in.use;.the.mark.has.to.be.renewed.every.10th.year

Right.to.exclude.others.from.using.the.mark.and.other.marks.so.similar.they.cause.confusion

(continued)


the.methods.embodied.in.the.invention.such.as.plant.transformation.and.selection.methods..Utility.patents.may.protect.different.aspects.of.the.invention..For.instance,.Calgene.Inc.,.now.Monsanto.Company,.used.a.series.of.claims.in.the.family.of.utility.patents.to.protect.the.first.horticultural.biotechnology.crop,.the.FlavrSavr™.tomato.that.was.genetically.engineered.to.suppress.the.expression.of.the.tomato.fruit.rip-ening. polygalacturonase. enzyme.. Using. a. portfolio. of. patents,. the. company. pro-tected.the.DNA.sequence.of.the.enzyme,.a.DNA.construct.to.suppress.the.enzyme.expression. in.plants,. the. transgenic. tomato. cell,. and.even,. the.overall.method. for.antisense-based. gene. suppression.of. this.or. any.other.gene.. In. the.United. States,.plant. and. utility. patents. are. awarded. by. the. United. States. Patent. and. Trademark.Office.(USPTO).

A.more.widely.employed.international.legal.system.to.award.rights.to.breeders.was.created.in.Europe.through.The.International Union for the Protection of New Varieties of Plants,.UPOV,.commonly.referred.to.as.Plant.Breeder’s.Rights.(PBRs).1,3.The.original.international.convention.that.established.UPOV.was.held.in.1961,.and.

TABLE 10.1 (continued)Overview of IP Protection Systems

Means of Protection

What Can Be Protected

How to Protect Term of Protection Rights of the Owner

Copyright Literary.works,.software,.dramatic.works,.music,.pictures,.sound.recordings,.architectural.works,.and.movies

Apply.for.federal.registration

Life.of.the.author.plus.70.years

Right.to.prevent.unauthorized.copying.or.public.performance

Trade.secrets

Any.technical.or.business.information.that.is.secret.and.that.gives.the.holder.an.advantage.over.a.competitor.who.does.not.have.the.information

Keep.secret;.no.registration.available

Unlimited.duration.as.long.as.the.subject.matter.is.secret

Right.to.prevent.unlawful.use

Sources:. Modified. from. Dodds,. J.. et. al.,. Plants,. germplasm,. Genebanks,. and. intellectual. property:.Principles,. options,. and. management,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices,. MIHR. (Oxford,. U.K.). and. PIPRA.(Davis,. CA),. 2007;. Dodds,. J.. and. Krattiger,.A.,. The. statutory. toolbox:.An. introduction,. in.Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices,.MIHR.(Oxford,.U.K.).and.PIPRA.(Davis,.CA),.2007.


the.treaty.has.been.revised.in.1972,.1978,.and.more.recently.in.1991..UPOV.provides.the. international. standardized. framework. in.which.each.national.government.can.establish.their.own.legal.system.

As.a.member.of.UPOV,.the.U.S..Plant.Variety.Protection.Act.offers.protection.of.sexually.propagated.or.tuber-propagated.plant.species..In.the.United.States,.PBRs.are.granted.through.Plant.Variety.Protection.(PVP).Certificates.issued.through.the.U.S..Department.of.Agriculture..UPOV.requires.that.a.plant.variety.meet.a.set.of.criteria.known.as.DUS.requirements;.distinct,.uniform,.and.stable..UPOV.provides.a.PVP-like.system,.which.can.offer.robust.protection.worldwide.

The. horticultural. industry. can. use. a. combination. of. plant. patents,. PVP,. and.PRBs.to.protect.and.market. its.varieties. in.most.parts.of. the.world..For.example,.the.University.of.California.relies.on.UPOV.and.U.S..plant.patents.to.manage.a.suc-cessful.Strawberry.Licensing.Program.in.domestic.and.international.markets..The.Strawberry.Licensing.Program.has.expanded.its.international.markets.in.countries.with.strong.IP.protection.and.the.UC.strawberry.cultivars.now.represent.up.to.60%.of.the.worldwide.production.4

Trade secrets.are.one.of.the.oldest.forms.of.IP.protection.and.are.used.to.pro-tect.confidential.business. information,.processes,.know-how,.data,.and.materials.5.However,. a. trade. secret. does. not. protect. against. competitors. reverse. engineering.a.product..In. the.horticultural.and.seed.industries,. trade.secrets.have.been.widely.used.to.protect.parent.inbred.lines.for.hybrid.seed.production.but.are.clearly.not.use-ful.for.the.protection.of.a.variety.that.can.be.directly.propagated.asexually.or.with.open-pollinated.seeds..Where.appropriate,.however,.trade.secrets.can.be.maintained.indefinitely.so.long.as.confidentiality.is.not.breached.

Another. form.of. IP. protection.of. limited. utility. in.horticulture. is. a.copyright..A copyright.protects. the.expressed. idea,.plant.variety.description.or.diagrams,.or.other.material.that.may.be.displayed.in.catalogs.or.seed.packages..This.form.of.IP.protection. is.weaker. than.others.because.anyone.is.free. to.use.the.actual.plant.or.depicted.idea..Also,.anyone.is.free.to.use.the.copyright.information.for.the.devel-opment.of.a.product..Copyright.use.in.plant.sciences.could.apply.to.plant.genome.information.in.electronic.databases.and.bioinformatics.software.and.hardware..For.example,.the.use.of.information.in.electronic.databases.can.be.limited.or.controlled.by.granting.access.to.databases.through.a.copyright.license.or.an.information.access.agreement..The.International.Center.for.Tropical.Agriculture.(CIAT),.for.example,.limits.the.ability.to.claim.intellectual.property.protection.over.any.data.or.informa-tion.in.its.databases.through.an.information.access.agreement.(http://isa.ciat.cgiar.org/urg/ita.do;jsessionid=9FEEC2C59FAB82D4EABF5052083238A3).

Trademarks. are.another. form.of. IP.protection.applicable. to.brand.names.of.horticultural.products;.however,.as.with.copyright,. it.does.not.protect. the.tech-nology. itself.. In. the. United. States,. trademarks. are. registered. at. the. USPTO..A searchable.database.of.trademark.registrations.is.available.on.the.USPTO.web-site.at.the.Trademark.Electronic.Search.System.(http://tess2.uspto.gov)..For.horti-cultural.crops,.trademarks.are.often.used.to.protect.any.word,.phrase,.symbol,.or.logo.that.represents.brand.varieties.or.products.such.as.Dole®.(a.registered.trade-mark. of. Dole. Fruit. Company),. Sunkist™,. and. Monsanto. Company’s. Roundup.Ready®.products.


Tangible property rights. reside.with. the.property.owner..MTAs.are. typically.used.to.transfer.possession.of.materials.but.not.ownership..The.party.who.transfers.the.materials. retains. full.ownership,.and. the. transfer. is. governed.by.a.contract,.typically.specifying.the.term.of.the.transfer,.how.the.materials.may.and.may.not.be.used,.and.other.related.issues,.such.as.confidentiality..MTAs.are.legal.contracts.and.are.also.referred.to.as.bailments,.which.is. the. legal. term.for.a.contract. that.transfers.physical.possession.but.not.ownership.of.personal.property..In.research,.MTAs.are.often.used.for.transfer.of.materials.for.research.use.only,.allowing.sci-entist.to.perform.academic.research..However,.there.is.typically.no.guarantee.or.promise. that. the. owner. will. give. rights. for. commercial. applications.. Also,. the.time.period.and.geographical.limitations.can.be.established.by.the.property.owner,.and,. thus,. unlike.patents,. limitations.on. the.use.of. tangible.property. typically.have. no temporal.or.geographic. limitations..Research-use.only.MTAs.are. typi-cally.easier.to.obtain,.especially.between.public.or.nonprofit.research.institutions,.but.could.represent.a.considerable.obstacle.downstream.if.the.materials.cannot.be.used.for.commercial.applications.

FREEDOM TO OPERATE

Navigating.the.complex.IP.landscape.of.a.research.project.in.horticultural.biotech-nology.requires.some.analytical.tools.and.specialized.analytical.capabilities.6.The.analysis.requires.both.legal.and.scientific.knowledge.as.well.as.access.to.both.patent.and.literature.databases.and.typically.takes.the.form.of.what.is.known.as.a.freedom.to.operate.(FTO).opinion..The.FTO.opinion.is.a.legal.assessment.about.whether.a.research.project.or.the.development.of.a.new.product.can.proceed.with.a.low,.or.tol-erable,.likelihood.that.it.will.not.infringe.existing.patents.or.other.types.of.IP.rights..It. is. important. to. note. that. the.FTO. determination. is.not. absolute. but. reflects. an.evaluation.of.risk.since.there.is.typically.some.uncertainty.around.the.interpretation.of.patent.claims.as.well.as.uncertainty.as.to.whether.new.IP.may.issue.or.be.dis-covered.at.a.later.date..The.FTO.opinion.may.lead.to.a.range.of.options:.identifying.in-licensing.targets,.considering.the.substitution.of.technologies.or.processes,.decid-ing.to.ignore.the.potential.infringement,.investing.in.work-around.technologies,.or.perhaps. deciding. to. abandon. the.project. all. together..Although,.private. firms. are.more.likely.to.engage.in.FTO.analysis.because.any.infringement.risk.may.directly.affect.their.ability.to.develop.new.products,.and.their.ultimate.profitability,.public,.and.not-for-profit.private.institutions.are.becoming.increasingly.aware.of.the.need.for.better.FTO.information..This.is.particularly.true.for.research.projects.undertaken.by.universities.or.not-for-profit.research.centers.for.the.specific.purpose.of.producing.new.crops.for.developing.countries..In.these.cases.where.deployment.is.anticipated,.it.is.critical.that.IP.considerations.be.taken.into.account.early.in.the.research.process.to.avoid.the.need.for.re-engineering.due.to.IP.constraints..Similarly,.horticulturists.should.address.IP.needs.to.ensure.that.commercialization.is.not.halted.due.to.a.lack.of.IP.clearance.

While.patents.are.the.most.common.type.of.IP.right.encountered,.a.thorough.FTO.analysis.needs.to.assess.all.types.of.existing.property.rights.for.the.likelihood.that.the.research. project. or. the. product. being. commercialized. infringes. terms. of. an. MTA..


Of particular.concern.are.tangible.property.rights,.such.as.cell.lines,.transgenic.plants,.germplasm,. and. plasmids..Because,. as.described.above,. the. transfer.of. tangible.property.often.occurs.under.the.terms.of.an.MTA.that.has.no.geographic.or.temporal.limitation..These.terms.can.be.particularly.problematic.and.directly.impact.FTO.

Enabling.technologies.for.plant.transformation.or.transformation.vectors.combine.several.components.such.as.promoters,.selectable.markers,.marker.removal.systems,.and.more..Because.of.the.fundamental.role.that.these.technologies.play,.they.have.been.extensively.patented..In.addition,.the.FTO.surrounding.enabling.technologies.related.to.plant.transformation.is.further.complicated.because.these.technologies.are.not.used.individually.but.are.combined.with.a.suite.of.related.enabling.technologies,.specific.trait.technologies,.and.deployed.in.many.different.plant.species..We.can.look.at.a.relatively.simple.example.of.a.single.component.of.a.transformation.vector.to.illustrate.the.elements.of.an.FTO.analysis.

The. target. technology.for. this.case.study.was.a. fruit-specific.promoter. from. the.tomato.E8.gene..The.E8.promoter.has.been.used.to.improve.fruit.quality,.extend.fruit.shelf.life,.and.to.express.edible.human.vaccines.specifically.in.ripening.tomato.fruit.7,8.The.first.step.in.an.FTO.investigation.is.to.clearly.define.the.target.technology..In.this.hypothetical.case,.the.fruit-specific.promoter.will.be.used.exactly.as.described.in.the.initial.publications.9,10.The.promoters.in.these.publications.are.virtually.identical.and.consist.of.about.2100.nucleotides.upstream.of.the.E8.structural.gene..Further.promoter.characterization.identifying.the.location.and.sequence.of.functional.elements.within.the.promoter.and.upstream.nucleotide.sequence.was.reported.in.Deikman.et.al.9.These.publications.draw.the.technical.boundaries.surrounding.the.target.promoter.technol-ogy.and.provide.important.prior.art.to.subsequently.filed.patents.

To.establish. the. relationship.of.publications.and.patents. that.describe.or.claim.the.E8.promoter,.a.patent.landscape.must.first.be.established..The.patent.landscape.should. include. patents. and. patent. applications. closely. related. to. the. technology..Keywords. and. authors. of. key. publications. are. used. to. search. for. patents. or. pat-ent.applications..A.separate.search.should. then.be.conducted. to. identify.patents.or. patent. applications,. which. referenced. the. scientific.publications.describing. the.technology..Additionally,.in.the.E8.case,.patented.DNA.and.protein.sequence.data-banks.were. searched.using. the. promoter’s. DNA.sequence. as. a.query.. The.patent.landscape.will.reveal.“family”.relationships.among.different.patents.and.published.patent.applications..Patent.families. include.later.patent.applications.that.claim.the.benefit.of.an.earlier,.related,.application,.or.later.patent.applications.that.arise.from.foreign.filings.of.the.parent.application..Figure.10.1.illustrates.a.patent.family.aris-ing.from.a.1989.patent.application.related.to.the.E8.promoter.filed.by.Agritope,.an.agricultural.biotechnology.company.

An.informative.way.of.analyzing.the.FTO.search.results.is.to.construct.a.timeline.of.scientific.literature,.patent.applications,.and.issued.patents.on.the.specific.tech-nology.and.on.potentially.overlapping.subject.matter..Ordering.the.patents.and.published.applications.according.to.their.priority.dates.(also.known.as.effective.filing.dates).reveals.important. relationships..For.example,. it. reveals.what.publica-tions. or. patents. are. prior. art. against. newer. patents.. Since. patents. may. only. be.granted.if.the.claims.are.both.novel.and.nonobvious.over.the.prior.art,.this.analysis.reveals.the.relative.dominance.of.earlier,.broader.patents.over.later,.narrower.patents..


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Figure.10.2.illustrates.the.IP.priority.timeline.for.the.E8.promoter..A.thorough.FTO.analysis.may.require.direct.contact.with.the.researchers,.and,.in.this.analysis,.it.was.learned.from.the.authors.of.the.Deikman.and.Fischer.publication10.that.they.did.not.apply.for.patent.protection.prior.to.their.publication..This.information.was.also.con-firmed.by.searching.patent.databases..Based.on.this.information,.it.was.presumed.that.the.basic.E8.promoter.technology.was.in.the.public.domain.based.on.the.pub-lications,.which.constituted.a.public.disclosure..However,.this.conclusion.required.thorough.review.and.documentation.of.the.published.literature.or.“prior.art”.relative.to.the.subject.matter.of.subsequent.patents.

As.shown. in. the.priority. timeline,. the.Deikman.and.Fischer10.and.Giovannoni.et al.11.publications. initially.describe. the.E8.promoter. technology..This.precluded.the.novelty.of.any.subsequent.patent.claims.on.the.E8.promoter.per.se.(for.example,.applications.filed.by.Agritope.and.Epitope)..Counsel.concluded.that.the.tomato.E8.promoter.constructs.per.se.and.as.described.in.the.scientific.publications.can.be.rea-sonably.considered.to.be.in.the.public.domain..However,.patent.issues.subsequently.may.limit.the.use.of.the.E8.promoter.to.drive.specific.heterologous.genes.or.to.use.in.combination.with.other. transcription.regulatory.elements.(see.Figure.10.2)..For.example,.Epitope’s.patent.U.S..5,859,330.contains.very.narrow.claims.to.the.use.of.the.E8.promoter.with.a.specific.gene,.S-adenosylmethionine.hydrolase..Therefore,.this.specific.E8.promoter-gene.combination.would.fall.under.the.scope.of.the.patent..

1985 19951990

Deikman and Fischer (1988)

Giovannoni et al. (1989)

Deikman et al. (1992)

Agritope448,095

12/12/1989

Agritope613,858

12/12/1990

Monsanto632,440

12/26/1990

Epitope 046,58304/09/1993

U.S. 5,723,746WO 94/24294

Agritope 255,83306/08/1994

U.S. 5,416,250 Epitope 261,67706/17/1994

U.S. 5,750,864

Epitope10/27/1994

U.S. 5,859,330

Agritope 360,97412/20/1994

U.S. 5,589,623

Patent and patentapplication timeline

Literature timeline

FIGURE 10.2 Timeline.of. tomato.E8.promoter. scientific.publications.and.United.States.(U.S.).and.World.(WO).patent.filing..(From.Fenton,.G..et.al.,.eds.,.Freedom.to.operate:.the.law.firms.approach.and.role,. in.Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices,.eds..A..Krattiger.et.al..2007,.available.online.at.www.ipHandbook.org)


Similarly,.other.patents.may.limit.the.use.of.the.E8.promoter.in.chimeric.constructs.that. contain. other. promoter. elements.. For. example,. another. of. Agritope’s. patent,.U.S..6,118,049,.claims.the.use.of.the.tomato.E8.promoter.fused.to.a.portion.of.the.tomato. E4. promoter.. These. examples. illustrate. the. complexities. in. analyzing. the.FTO.of.particular.DNA.elements..We.exemplify.that.FTO.may.differ.when.you.con-sider.a.DNA.element.by.itself.(i.e.,.promoters).or.in.combination.with.other.elements.(i.e.,.trait.genes).

This.example.provides.an.overview.of. the.data.and.information.that.should.be.considered.in.an.FTO.analysis..It.is.not.difficult.to.imagine.how.the.complexity.of.an.FTO.analysis.would.grow.dramatically.with.the.inclusion.of.multiple.enabling.technologies,.one.or.more.trait.technologies.and.proprietary.germplasm..This.is.one.of. the. challenges. of. understanding. IP. constraints. and. developing. FTO. strategies.in.plant.biotechnology.where.multiple.complementary.technologies.are.necessarily.integrated.to.develop.new.crop.varieties.

STRATEGIES FOR IP ACCESS

During. the. 1990s,. it. became. clear. that. universities. and. nonprofit. agricultural.research.institutions.were.constrained.in.their.historical.role.of.providing.many.new.agricultural.innovations—particularly.biotechnology-derived.innovations—directly.to.the.agricultural.industry.or.to.farmers..This.was.especially.apparent.for.specialty.or.horticultural.crops.that.do.not.individually.occupy.the.large.acreage.of.agronomic.crops.but.as.a.group.represent.a.high-value.agricultural.sector..While.there.are.many.contributing. factors,. access. to. the. suite. of. proprietary. technologies. required. to.produce.a.genetically.modified.crop.has.been. frequently.cited. as. a.barrier. to. the.commercialization.of.public.sector.agricultural.research.12.In.Europe,.a.similar.pic-ture.emerged.in.2000,.when.a.Swiss.scientist.developed.“Golden.Rice,”.genetically.modified. rice.with. elevated.provitamin.A,13.which. triggered. an. intellectual.prop-erty.audit..The.audit.revealed.that.70.proprietary.technologies.had.been.infringed.in.the.development.of.Golden.Rice.and.illustrated.the.complex.patent. thicket. that.surrounded.biotechnology.innovations.for.crop.improvement.14

The. complex. IP. environment. surrounding. agricultural. biotechnology. research.and.development,.exemplified.by.the.Golden.Rice.case.or.even.the.relatively.simple.FTO.opinion.described.above,.has.spawned.some.new.strategies.and.new.organiza-tions.committed.to.lower.the.IP.barriers.to.new.crop.developments.and.to.provide.more.open.access.to.patented.technologies..These.issues.are.critical.for.small.private.companies.attempting.to.enter.this.sector.but.can.also.be.important.for.public.or.not-for-profit.research.institutions..Most.plant.biotechnology.laboratories.routinely.use.patented.technologies.in.their.research.without.specific.legal.permissions..Although.patent.owners.have.rarely.been.concerned.about.academic.research.infringement.in.agriculture,.there.are.many.examples.where.fundamental.biomedical.research.has.been.challenged.because.of.IP.issues.15.In.addition,.projects.carried.out.in.public.or.not-for-profit.institutions.that.are.targeted.toward.the.development.of.crops.for.devel-oping.country.farmers.must.consider.the.IP.inputs.to.the.project..Increasingly,.the.sponsors.of.such.research.are.requiring.the.full.analysis.and.disclosure.of.strategies.to.ensure.global.access.to.all.IP.embodied.in.a.project.


To.develop.a.strategy.for.IP.access,. it. is.important. to.consider.both.the.IP.and.TP.rights..Most.researchers.are.still.relatively.unfamiliar.with.how.to.find,.under-stand,. and.utilize. IP. information,. including.published.patents.and.patent. applica-tions..Therefore,.it.is.important.for.scientists.to.seek.legal.expertise.to.understand.the.FTO.and.IPRs.of.the.components.and.processes.used.to.develop.agricultural.biotech.products..In.addition,.management.of.other.TP.agreements.(like.MTAs).is.crucial..Keeping.track.of.contractual.obligations.specified.in.sponsor.research.agreements,.employment. contracts,. and.MTAs. are. important. to. address. the. development.of. a.project.because.they.may.be.critically.important. in.the.downstream.deployment.of.a.biotechnology.product..For.example,.agreements.may.define.who.holds.patent.rights,.whether.a.technology.can.be.commercialized.or.transferred.to.third.parties,.or.if.the.license.is.for.“research-use.only.”.Therefore,.understanding.these.complex.IP.and.TP.issues.are.critical.in.order.to.develop.strategies.for.IP.access.

Scientists.worldwide.are.realizing.the.increasing.need.to.address.IP.and.TP.issues.in.order.to.deploy.their.biotechnology.discoveries..In.the.private.sector,.the.corporate.culture.prioritizes.preemptive.mitigation.of.IP.and.TP.issues,.and,.as.a.consequence,.biotechnology. companies. have. robust. legal. resources.. In. contrast,. public. sector.scientists.usually.do.not.have.access.to.the.legal.information.necessary.to.address.these. issues..In.2003,.several.public.sector.and.not-for-profit.agricultural. research.institutions.recognized.the.need.to.develop.an.organization.to.address.the.relative.inaccessibility.of.IP.information.and.to.provide.a.framework.to.ensure.that.IP.does.not.block.applications.of.agricultural.biotechnology.and,.in.particular,.to.facilitate.projects.that.can.have.broad.humanitarian.benefits.16.Numerous.universities,.includ-ing.the.University.of.California,.the.Donald.Danforth.Plant.Science.Center,.North.Carolina.State.University,.Ohio.State.University,.Boyce.Thompson.Institute.for.Plant.Research,.Michigan.State.University,.Cornell.University,.University.of.Wisconsin-Madison,. University. of. Florida,. the. United. States. Department. of. Agriculture,.Rutgers.University,.Texas.A&M. University,. and.Purdue.University.developed. the.Public.Sector.Intellectual.Property.Resource.for.Agriculture.(PIPRA;.www.pipra.org)..These.institutions.made.a.public.commitment.to.participate.and.promote.strat-egies.to.collectively.manage.public-sector.intellectual.property.in.support.of.both.U.S.. and. developing. country. agriculture.. This. initial. founding. group. of. PIPRA.members.has.grown. to.over.53. institutional.members. in.15. countries,. illustrating.the.widespread.concern.and.interest.in.working.collectively.to.remove.and.avoid.IP.barriers.that.might.slow.development.of.new.crops.

PIPRA.operates.on.several.levels.to.effectively.support.the.broad.application.of.agricultural.technologies.developed.in.public-non/profit.research.institutions..Its.pri-mary.strategy.has.been.to.adopt.a.highly.collaborative.program.built.on.consensus.views.of.its.members—all.of.whom.fundamentally.believe.that.intellectual.property.protection.is.an.important.tool.to.support.innovation..PIPRA.is.working.within.the.context.of.its.members.to.support.both.commercial.and.humanitarian.applications.of. technologies.and. to.develop.strategies.and.mechanisms.to.stimulate.even.more.innovation.globally.

A.number.of.strategies.have.been.implemented.to.enhance.FTO.using.public-sector.IP.for.agricultural.biotechnology.projects..For.example,.informed.decisions.regarding.dissemination.of.new.knowledge.via.open.publication.or.protecting.it.with.


a.patent.are.clearly.important.and.FTO.can.be.improved.if.public-sector.institutions.systematically.consider.how,.when,.and.if.to.use.the.patent.system.to.support.broad.innovation.17.Even.when.using.the.patent.system,.PIPRA.encourages.its.members.to.reserve.rights.to.use.their.newest.and.best.technologies.for.humanitarian.purposes,.particularly.when.they.issue.exclusive.commercial.licenses.18.For.U.S..agriculture,.it.is.also.important.to.use.licensing.strategies.that.promote.commercialization.of.technologies.for.both.large.and.small.acreage.crops..Thus,.for. technologies.with.potential.applications.in.many.crops,.instead.of.granting.an.exclusive.license.for.all.fields.and.in.all.crops,.it.may.be.prudent.to.license.for.a.specific.field-of-use.or.spe-cific.crop.and.retain.rights.to.use.the.technology,.or.to.issue.additional.licenses,.for.the.development.of.other.crops.that.may.not.be.within.the.commercial.interests.of..large.companies..The.anticipated.benefits.of.a.collective.IP.management.regime.are.to.enable.an.effective.assessment.of.FTO.issues.to.overcome.the.fragmentation.of.public-sector.IPR.and.re-establish.the.necessary.FTO.in.agricultural.biotechnology.for.the.public.good.and.to.enhance.private.sector.interactions.by.more.efficiently.identifying.collective.commercial.licensing.opportunities.

Among.PIPRA’s.core.activities.is.working.with.scientists.and.funding.agencies.on.a.project-specific.basis.to.understand.the.underlying.FTO,.IP,.and.TP.necessary.to.develop.and.deploy.agricultural.biotechnology.projects..Based.on.this.informa-tion,.PIPRA.works.with.scientists.and.technology.managers.to.design.and.implement.strategies.to.access.and.negotiate.third.party.rights..PIPRA.offers.services.to.develop.patent.landscapes.in.particular.technology.spaces,.for.example,.gene.suppression.in.plant.biotechnology.19

Delmer.et.al..described.other.initiatives.designed.to.play.roles.in.enabling.access.to. IPRs. for. the. development. of. crops,. including. horticultural. crops,. the. African.Agricultural. Technology. Foundation,. and. CAMBIA.20. The. African. Agricultural.Technology.Foundation. focuses.on.negotiating. access. to.proprietary. technologies.for.the.benefit.of.smallholder.farmers.in.Sub-Saharan.Africa.(www.aatf-africa.org).21.CAMBIA. offers. a. number. of. resources. and. initiatives. to. promote. innovation.(www.cambia.org).

In.particular,.foundations.with.an.interest.in.supporting.broad.innovation.as.well.as.corporations.and.corporate.foundations.whose.future.depends.on.the.broad.accep-tance.of.biotechnology.in.both.agronomic.and.horticultural.crops.are. likely.to.be.important.sponsors.of.activities.to.enable.access.to.IPRs.

REFERENCES

. 1.. Kesan,. J.P.,. The. statutory. toolbox:. Plants,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices. 2007,. MIHR.(Oxford,.U.K.).and.PIPRA.(Davis,.CA).

. 2.. Dodds,.J.,.A..Krattiger,.and.S.P..Kowalski,.Plants,.germplasm,.Genebanks,.and.intel-lectual. property:. Principles,. options,. and. management,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices.2007,.MIHR.(Oxford,.U.K.).and.PIPRA.(Davis,.CA).

. 3.. Lesser,. W.H.,. Plant. breeders’. rights:. An. introduction,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices.2007,.MIHR.(Oxford,.U.K.),.and.PIPRA.(Davis,.CA).


. 4.. Bennett,.A.B..and.M..Carriere,.The.University.of.California’s.strawberry.licensing.program,.in.Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices.2007,.MIHR.(Oxford,.U.K.).and.PIPRA.(Davis,.CA).

. 5.. Dodds,. J.. and. A.. Krattiger,. The. statutory. toolbox:. An. introduction,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices.2007,.MIHR.(Oxford,.U.K.).and.PIPRA.(Davis,.CA).

. 6.. Fenton,. G.,. C.. Chi-Ham,. and. S.. Boettiger,. eds.. Freedom. to. operate:. The. law. firms.approach. and. role,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices,. eds..A.. Krattiger. et. al.,. 2007,. available.online.at.www.ipHandbook.org.(accessed.January.28,.2011).

. 7.. Sandhu,.J.S..et.al.,.Oral.immunization.of.mice.with.transgenic.tomato.fruit.expressing.respiratory.syncytial.virus-F.protein.induces.a.systemic.immune.response..Transgenic Res.,.9(2),.127,.2000.

. 8.. Sheehy,.R.E.,.M..Kramer,.and.W.R..Hiatt,.Reduction.of.polygalacturonase.activity.in.tomato.fruit.by.antisense.RNA..Proc. Natl. Acad. Sci. USA,.85(23),.8805,.1988.

. 9.. Deikman,.J.,.R..Kline,.and.R.L..Fischer,.Organization.of.ripening.and.ethylene.regula-tory.regions.in.a.fruit-specific.promoter.from.tomato.(Lycopersicon esculentum)..Plant Physiol.,.100,.2013,.1992.

. 10.. Deikman,.J..and.R.L..Fischer,.Interaction.of.a.DNA.binding.factor.with.the.5′-flanking.region.of.an.ethylene-responsive.fruit.ripening.gene.from.tomato..EMBO J.,.7(11),.3315,.1988.

. 11.. Giovannoni,.J.J..et.al.,.Expression.of.a.chimeric.polygalacturonase.gene.in.transgenic.rin.(ripening.inhibitor).tomato.fruit.results.in.polyuronide.degradation.but.not.fruit.soft-ening..Plant Cell,.1(1),.53,.1989.

. 12.. Graff,.G.D..et.al.,.Accessing.intellectual.property.for.biotechnological.development.of.horticultural.crops..Calif. Agric.,.58,.122,.2003.

. 13.. Ye,.X..et.al.,.Engineering.the.provitamin.A.(beta-carotene).biosynthetic.pathway.into.(carotenoid-free).rice.endosperm..Science,.287(5451),.303,.2000.

. 14.. Kryder,.R.D.,.S.P..Kowalski,.and.A.F..Krattiger,.The.intellectual.and.technical.property.components.of.pro-vitamin.a. rice. (GoldenRice™):.A.preliminary. freedom-to-operate.review..ISAAA.Briefs,.20,.pp..1–56,.2002.

. 15.. Marshall,.E.,.Intellectual.property..DuPont.ups.ante.on.use.of.Harvard’s.OncoMouse..Science,.296(5571),.1212,.2002.

. 16.. Atkinson,.R.C..et.al.,.Intellectual.property.rights..Public.sector.collaboration.for.agricultural.IP.management..Science,.301(5630),.174,.2003.

. 17.. Boettiger,. S.. and. C.. Chi-Ham,. eds.. Defensive. publishing. and. the. public. domain,.in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices,. eds..A.. Krattiger. et. al.,. 2007,. available. online. at.www.ipHandbook.org.(accessed.January.28,.2011).

. 18.. Bennett,. A.B.,. Reservation. of. rights. for. humanitarian. use,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices.2007,.MIHR.(Oxford,.U.K.).and.PIPRA.(Davis,.CA).

. 19.. Chi-Ham,.C.L.,.K.L..Clark,.and.A.B..Bennett,.The.intellectual.property.landscape.for.gene.suppression.technologies.in.plants..Nat. Biotechnol.,.28(1),.32,.2010.

. 20.. Delmer,.D.P..et.al.,. Intellectual.property. resources. for. international.development. in.agriculture..Plant Physiol.,.133(4),.1666,.2003.

. 21.. Boadi,. R.. and. M.. Bokanga,. eds.. The. African. agricultural. technology. foundation.approach. to. IP. management,. in. Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices,.eds..A..Krattiger.et.al.,.2007,.available.online.at.www.ipHandbook.org.(accessed.January.28,.2011).

233

11 Structuring University–Private Partnerships for Developing and Commercializing Transgenic Horticultural Crops

Gordon Rausser and Reid Stevens

INTRODUCTION

Horticultural. research. is. conducted. primarily. in. the. public. sector,. with. private.research.institutions.playing.a.relatively.minor.role..As.a.result,.research.gaps.emerge.between.the.basic.research.generated.by.public.institutions.and.the.applied.research.needs.of.industry..This.gap.can.be.reduced.by.forming.public–private.partnerships.(PPPs). that. allow.academic. researchers. access. to. commercialization. technologies.(such.as.gene.expression.profiles.and.genome.maps).and.give.private.firms.access.to.new.research.and.innovation.

However,. significant. obstacles. hinder. the. formation. of. successful. research.partnerships..Both.parties.in.a.partnership.face.substantial.risks..These.risks.are.rooted.in.the.conflict.between.a.university’s.academic.objectives.and.the.private.

CONTENTS

Introduction............................................................................................................. 233Operational.Framework.......................................................................................... 235

Stage.1:.Setting.the.Bargaining.Space.and.Negotiating.the.Contract................ 235Stage.2:.Decision.Making.through.Bargaining.................................................. 238Stage.3:.Is.There.a.Shock?................................................................................. 238

General.Guidelines.................................................................................................. 239PPPs.and.Public.Good.Research........................................................................ 239Two.Leading.University–Private.R&D.Partnerships......................................... 241

PPPs.in.Horticultural.Research...............................................................................244References...............................................................................................................246


firm’s. corporate. incentives.. One. critical. risk. is. the. potential. co-opting. of. the.academic.research.agenda.by.private.interests..University.researchers.risk.the loss.of. academic. freedom. and. integrity. while. industry. risks. the. loss. of. investment.capital,.privacy,.and.proprietary.information..Differences.between.the.university’s.educational.objectives.and.the.private.sector’s.goals,.as.well.as.differences.in.the.cultures,. institutional. incentives,.and.time.frames,.can. lead.to.a.clash.of.values..Intellectual.property. (IP). rights. issues. are. also. a. frequent. source.of. contention..Given.these.risks,.partnerships.must.be.based.on.carefully.structured.contracts.to.protect.the.interests.of.the.private.and.public.partners.

Though.there.has.been.much.discussion.of.public–private.research.partnerships.in.both. the.popular.press.and.academic.community,. there. is.no.consensus.on. the.optimal.contract.structure.for.these.partnerships..We.have.developed.a.three-stage.framework.for.evaluating.PPP.contracts..This.framework.is.unique.because.it.takes.into. account. the. type. of. economic. good. the. partnership. produces. as. well. as. the.incomplete.nature.of. contracts. that. deal.with. sponsored. research..Partnerships. in.horticultural. research. deal. with. the. management. and. provision. of. impure. public.goods.*.The.optimal.structure.of.a.PPP.depends.on.the.degree.of.impurity,.and,.if.a.PPP.produces.a.good.that.is.impure.to.some.extent,.its.contract.should.differ.from.that.of.a.PPP.that.produces.a.purely.private.good..The.contracts.that.govern.these.partnerships. and. assign. ownership.of. the. impure. goods. they.produce. are. incom-plete. in. the. sense. that. there. is. a. set. of. events. that. can. influence. the. partnership,.which.cannot.be.enumerated.in.the.contract..These.events.could.be.unanticipated.research.opportunities.that.arise.during.the.research.process.or.could.be.an.unex-pected.discovery.that.falls.outside.of.the.contract’s.scope..An.optimal.contract.will.take.into.account.the.uncertainty.inherent.in.the.research.process.and.assign.control.in.the.case.of.unanticipated.events.in.a.manner.that.does.not.pervert.the.partner’s.incentives.

Contracts.for.PPPs.in.horticultural.research.and.their.associated.control.and.prop-erty. rights. come. in.many. forms,. ranging. from. large,.multiproject,.multiyear. alli-ances.to.small-scale.projects..Our.three-stage.framework.to.analyze.these.contracts.is.based.on.the.contractual.assignment.of.control.in.the.case.of.unanticipated.events..In.Stage.1,.the.public.and.private.partners.negotiate.to.determine.the.allocation.of.the.front-end.control.rights.and.the.back-end.property.rights.†.The.front-end.control.rights. determine. the. nature. and. scope. of. the. research. activities. that. the. partner-ship.will.undertake.as.well.as.decision-making.authority.over.those.activities.while.back-end.property.rights.determine.how.any.benefits.generated.by.the.research.will.be. distributed. among. the. partners.. The. partners. also. make. relationship-specific.investments.according.to. the.contract. in.Stage.1..In.Stage.2,. the.partners.bargain.over.management.decisions.with.bargaining.power.determined.by.the.contract.rights.

*.Impure. public. goods. are. goods. that. are. either. nonrival. or. nonexcludable.. A. nonrival. good. can. be.consumed.by.one.person.without.preventing.simultaneous.consumption.by.others. (e.g.,. intellectual.property)..A.good.is.nonexcludable.if.it.is.not.possible.to.prevent.anyone.from.consuming.the.good.(e.g.,.a.lighthouse).

†. In.this.chapter,.we.will.use.control.rights.to.refer.to.the.authority.to.make.decisions.during.the.research.process.(the.“front-end”).and.property.rights.to.refer.to.ownership.of.the.research.produced.by.the.partnership.(the.“back-end”).

235Structuring University–Private Partnerships for Commercialization

and.investments.made.in.Stage.1..The.outcome.of.this.bargaining.is.a.decision.that.lies.between. the.noncooperative.bargaining.solution.(where.each.partner.unilater-ally.exercises. their.control. rights).and. the.cooperative.bargaining. solution. (where.the.partners.maximize.their.joint.benefit)..In.Stage.3,.there.may.be.an.unanticipated.“shock.”.For.our.purposes,.a.shock.is.an.event.that.affects.the.partnership.over.which.there.are.no.explicit.contract.provisions..Such.a.shock.may.cause.the.players.to.revisit.their.control.and.property.rights..Depending.on.the.nature.of.the.shock,.the.partners.may.engage.in.renegotiation.that.reassigns.the.control.and.property.rights.(and.begin.again.at.Stage.1).or.they.may.conclude.the.partnership.

The.remainder.of.the.chapter.is.organized.as.follows:.in.the.“Operational.framework”.section,. we. present. the. three-stage. framework. for. evaluating. public–private. research.contracts;.in.the.“General.guidelines”.section,.general.guidelines.for.university–private.partnerships. in. research. and. development. (R&D). are. provided;. and. in. the. “Public–private.partnerships.in.horticultural.research”.section,.we.visit.the.implications.of.our.framework.for.PPPs.in.horticultural.research.and.more.specifically.for.the.development.and.commercialization.of.transgenic.horticultural.crops.

OPERATIONAL FRAMEWORK

There.is.a.considerable.body.of.literature.related.to.PPP.theory,.and.the.reader.inter-ested. in.developing.a.greater.depth.of.knowledge. in. this.area. is.directed. to. these.selected.citations.1–14

In.this.section,.we.describe.an.operational.framework.that.can.be.used.as.a.con-ceptual.lens.to.analyze.PPP.contracts.in.horticultural.research.that.includes.the.terms.and.conditions.used.to.assign.front-end.control.rights.and.back-end.property.rights..The.following.three-stage.framework.provides.a.lens.that.will.allow.us.to.evaluate.PPP.contracts.

StaGe 1: SettInG the barGaInInG Space and neGotIatInG the contract

In.Stage.1,.the.partners.negotiate.a.contract.and.make.investments..The.public.insti-tution. should. begin. this. process. with. a. self-assessment. to. identify. their. primary.objectives.in.seeking.out.private.partners,.their.strengths.and.assets,.and.the.desired.complementarities..This.assessment.is.essential.to.form.an.efficient.partnership.

Though. the. order. in. which. partnership. negotiations. proceed. is. not. of. much.consequence,.it.is.vital.for.the.public.institution.to.be.deliberately.early.in.the.pro-cess,.when.seemingly.innocuous.decisions.ex-ante.may.severely.limit.its.control.or.flexibility.at.crucial.junctures.ex-post..At.each.point.in.a.relationship,.it.is.important.for.the.public.institution.to.consider.the.long-term.consequences.of.all.relationship-related.decisions.

Consider.the.importance.of.the.public.institution’s.method.for.finding.and.select-ing.a.partner..Often.firms.seek.government.contracts.and.make.specific.offers,.leaving.public.institutions.the.passive.role.of.waiting.to.be.approached.and.evaluating.one.partnership.at.a.time..Alternatively,.the.public.institution.can.take.a.proactive.role..Given. the. results. of. the. self-assessment,. the. public. institution. can. seek. out. well-matched.partners.that.complement.their.strengths..Although.deliberately.seeking.out.


partners,.rather.than.waiting.to.be.approached.with.a.proposal,.requires.more.effort.up.front,.it.gives.the.public.institution.the.greatest.degree.of.control.over.the.selec-tion.of.partners,.which.implicitly.define.the.control.that.the.public.institution.will.have.over.the.remainder.of.the.structuring.process..In.addition,.by.actively.approach-ing.potential.partners.in.the.private.sector,.a.public.institution.can.form.a.consortium.with.a.group.of.specialized.partners.if.that.better.suits.the.public.institution’s.objec-tive..This.active.approach.can.substantially.broaden.the.public. institution’s.choice.set..Likewise,.a.proactive.approach.on.the.part.of.a.private.firm.can.increase.their.control.in.the.bargaining.process.

Once. a. partner. is. selected,. the. public. and. private. institutions. engage. in.nego-tiations.that.result.in.a.contract.that.allocates.to.each.of.the.partners.a.share.of.the.front-end.control.rights.and.back-end.property.rights..The.front-end.control.rights.enumerate.the.resources.committed.by.both.partners.and.give.the.partners.decision-making. power. over. the. partnership’s. R&D. processes,. and. the. back-end. property.rights. specify. the.manner. in. which. the.partnership’s. assets. will. be.distributed. at.the.conclusion.of.the.partnership..At.the.end.of.this.stage,.the.partners.invest.in.the.partnership.according.to.the.contract.

In.this.negotiation,.each.partner.works.to.minimize.its.share.of.input.while.mak-ing.sure.the.combined.resources.will.be.sufficient.for.a.successful.joint.effort..The.commitment.of.resources.in.the.front-end.is.fairly.transparent;.however,.the.implica-tions.of.choosing.particular.governing.structures.for.the.partnership.are.less.trans-parent..Given.the.diversity.of.assets,.it.is.difficult.for.potential.partners.to.balance.their.respective.asset.contributions..These.assets.can.be.tangible,.as.with.financial.assets.or.equipment,.or.intangible.“knowledge”.assets.15.Unlike.tangible.assets,.the.value.of.intangible.assets.is.not.easily.defined.as.it.relies.on.many.factors.such.as.the.nature.of.the.assets.and.the.degree.of.complementarity.

Knowledge.assets.may.be.tacit.(e.g.,.know-how).or.codifiable..Tacit.knowledge.draws.on.skills.and. techniques.and. is. transferred.by.demonstration,.apprentice-ships,. personal. instruction,. and. provision. of. expert. services.. Codifiable. knowl-edge.can.be.reduced.to.messages.and.is.easily.transferred..Knowledge.assets.can.assume.the.form.of.a.nonrivaled.or.inexhaustible.good..In.other.words,.sharing.this.information.will.not.reduce.the.amount.available.to.others..Although.nonrivaled,.these.assets.are.not.necessarily.public.goods.since.it.may.be.possible.to.exclude.access.by.others..This. is.certainly.true.of.patents,.copyrights,.and.trade.secrets..To.be.sure,.exclusive.control.over.a.specified.set.of.knowledge.assets.creates.rents.for. its. holders.. A. further,. important. distinction. is. between. generic. and. special-ized.assets.16,17.Generic.assets.are.useful.for.most.research,.such.as.basic.scientific.knowledge.concerning.biotechnology.research..In.contrast,.specialized.assets.are.suited.to.a.narrow.set.of.specific.applications.that.can.be.more.easily.commercial-ized..In.the.area.of.plant.R&D,.these.assets.can.be.further.characterized.as.input-.(e.g.,.herbicide-resistant.transgenic.seed).or.output-trait.assets.(e.g.,.high-nutrient.content.seeds).

Identifying.these.aspects.of.the.partner’s.assets.is.important.to.create.complemen-tarities.among.the.different.assets.held.by.the.public.and.private.partners,.and.when.negotiating.over.the.contributions.each.partner.will.make.to.the.relationship..Private.institutions.are.likely.to.have.more.access.to.funding,.state-of-the-art.scientific.tools,.


experience. in. commercialization,. and. marketing. expertise.. Public. institutions,. in.return,.offer.preferential.access.to.academic.resources.and.assistance.in.navigating.bureaucracies..The.objective.of.the.contract.is.to.combine.each.partner’s.assets.in.the.most.productive.combinations.

The.governance.structure.of.the.partnership.must.be.determined.in.the.first.stage..Fundamentally,. it. defines. each. partner’s. front-end. control. rights. and. back-end.property. rights..This. assignment.of. control.will. determine.how. the.partners.will.interact,.make.decisions,.resolve.conflicts,.and.terminate.the.agreement.if.necessary..The.most.important.consequence.of.the.governance.structure.is.that.it.determines.how.the.project.will.be.evaluated,.and.the.governance.structure.specifies.how.the.agreement.will.be.administered.and. if. the.scope.will.be.changed.(i.e.,. the.agree-ment.extended.or. terminated)..At. the.conclusion.of. the.partnership,. the.back-end.options.in.the.agreement.determine.how.benefits.are.disseminated.and.the.process.for.establishing.ownership..Each.of.these.issues.is.crucial.in.determining.how.both.the.pecuniary.and.nonpecuniary.benefits.of.the.project.are.shared.by.the.partners.and.by.the.public.

In. the. case. of. partnerships. that. involve. research,. licensing. options. are. impor-tant.when. the.PPP.generates. a.new,.marketable.product..Currently,. it. is. common.for. the. industry. partner. to. be. given. a. first-to-negotiate. licensing. option. for. some.subset.of.the.innovations.generated.under.the.partnership..Generally,.these.options.must.be.exercised.within.a.specified.time.period,.or.else.the.option.is.extended.to.third.parties.. In. response. to.public.outcry.over.poorly. structured. agreements. and.concern.about.blocking.patents,.right-of-first-refusal.options.evolved. into.right-to-negotiate.options..If.the.industry.partner.is.granted.the.more.limited.option.of.right-to-negotiate,.a.public.institution.should.have.greater.control.over.licensing.rights.and.can.prevent.blocking.patents.from.being.awarded.

Other.aspects.of.licensing.agreements.receive.less.attention.but.are.also.critical..One. such. aspect. is. the. percentage. of. the. total. innovation. for. which. the. indus-try.partner.holds.an.option.to.negotiate.an.exclusive.license.or.access.option..For.example,. in. some. partnerships,. the. private. partner. can. exercise. this. option. for.an.“allowable.percentage”.of.patents,.equal.to.the.percentage.of.the.funding.that.came. from. private. partner.. Under. other. agreements,. the. industry. partner. holds.this.option.for.all.patented.discoveries.generated.by. the.agreement..Third-party.options.are.also.a.critical.aspect.of.licenses.options..These.options.are.the.rights.that.parties.outside.the.agreement.have.to.innovation.generated.by.the.agreement..In.some.agreements,.third.parties.hold.open.options.on.patents.not.included.in.the.allowable.percentage.and.on.patented.innovations.either.covered.by.nonexclusive.license,.or.for.which.the.first-to-negotiate.option.has.expired.for.private.partner..In. this.case,. the.private.partner.has.no. recourse.once. their. licensing.option.has.expired,.and. the.public.partner. is. free. to.enter. licensing.negotiations.with. third.parties..In.contrast,.other.agreements.give.third.parties.only.a.conditional.option..The.private.partner.has.right-of-first.refusal.on.any.licensing.arrangement.between.the.public.institution.and.third.parties,.even.if.their.original.licensing.option.has.expired..Thus,.private.partner.is.guaranteed.an.option.of.first.refusal.on.any.third.party.offers.made.to.the.public.partner..This.severely.limits.the.options.available.to.third.parties.


StaGe 2: decISIon makInG throuGh barGaInInG

In.Stage.2,.the.partners.jointly.manage.the.partnership.by.making.decisions.based.on.a.two-person,.two-phase.bargaining.game.*.In.the.first.phase,.the.public.partner.and.the.private.partner.decide.what.threats.to.invoke.if.no.agreement.is.reached,.where.the.threat.strategies.are.chosen.to.maximize.their.pay-off.while.minimizing.effort.and.are.based.on.the.control.rights.and.property.rights.assigned.in.Stage.1..These.threat. strategies. determine. the. disagreement. point. and. are. taken. as. given. in. the.second.phase..These.strategies.need.not.actually.be.carried.out.and.may.not.even.be.explicit..All.that.is.required.is.the.potential.of.threat..In.this.stage,.the.partners.will.achieve.an.efficient.outcome,.in.which.the.partner.with.control.rights.implements.a.decision,.and.a.pay-off.is.exchanged.between.the.partners..The.partner.holding.the.control.rights.is.aware.of.the.noncontrolling.partner’s.reaction.function.and.unilat-erally.selects.an.action.that.maximizes.the.controlling.partner’s.objective.function.given. the.noncontrolling.partner’s. reaction.pattern.13.The.reaction.function.allows.the.noncontrolling.partner.to.influence.controlling.partner’s.decision.

Beyond. their. choice. of. threat. strategy,. each. partner. exercises. control. over. the.resources.dedicated.to.the.relationship.through.its.choices.in.Stage.1..For.example,.if.a.public.institution.is.approached.by.a.single.firm.and.considers.only.their.offer,.it.has.a.very.limited.choice.set.and.is.likely.to.have.little.leverage.over.that.firm’s.resources..In.contrast,.if.a.public.institution.considers.multiple.offers.from.partners.with.varied.assets,.its.choice.set.is.broader.

StaGe 3: IS there a Shock?

In.the.final.stage.of.the.agreement.(Stage.3),.the.partners.deal.with.unanticipated.shocks..When.there.is.a.shock,.the.partners.have.two.options:.(1).they.can.conclude.the.partnership.and.exercise.their.back-end.property.rights.over.the.partnership’s.property,.or.(2).they.can.renegotiate.the.control.rights.and.property.rights.assigned.and.begin.again.at.Stage.1..If.the.partners.choose.to.renegotiate,.the.allocation.of.bargaining.power.in.renegotiation.may.be.different.from.the.allocation.in.the.pre-vious.stages..By.this.stage,.a.partner.might.find.themselves.in.a.more.vulnerable.position.due.to.the.nature.of.the.shock.or.relationship-specific.investments..This.potential.for.changes.in.relative.bargaining.power.could.lead.to.a.reassignment.of.control.rights.

After.Stage.3,. the.partners.assess. the.outcome.of. their.partnership.and.con-sider. whether. to. renew. the. agreement.. Although. many. public. institutions. have.developed.policies.to.evaluate.partnerships,.there.is.no.generally.accepted.method.for. formal. review.of.partnerships.with.private. institutions..These.methods. rely.mainly.on.anecdotal.feedback.from.involved.personnel.to.measure.the.merits.of.specified.projects.and.to.monitor.unintended.consequences..The.informal.reviews.and.vague.impressions.of.both.partners.are.coupled.with.more.tangible.outcomes,.such.as.the.project’s.revenue,.in.assessing.whether.a.partnership.was.successful.or.not.

*.See.Rausser.et.al.18.for.an.extension.of.this.analysis.to.a.multi-person.bargaining.game.


A.key.policy. issue.is.developing.concrete.measures.of.PPP.productivity..Much.of.the.literature.on.PPPs.focuses.on.developing.proper.incentives.for.behavior.cov-ered.within.the.scope.of.an.individual.agreement,.but.little.consideration.is.given.to.incentives.that.fall.outside.a.specific.agreement..Because.many.of.these.agreements.are.up.for.renewal.once.completed,.there.are.incentives.for.the.public.institution.to.make.sure.that. the.private.partner. is.satisfied.with.the.outcome.of. the.agreement,.and,.under.increasing.financial.pressure,.this.may.affect.behavior.within.a.current.agreement.. In.other.words,. these.agreements. are.not.necessarily.one-shot.games,.but.might.be.a.single.round.of.a.repeated.game..As.such,. there.are.incentives.for.the.public.institution.to.develop.a.certain.reputation.so.that.the.private.partner.will.support. a. renewed. relationship..This. speaks. to.one.of. the.primary. concerns.with.these. agreements. that. public. institutions. will. fail. to. look. for. funding. from. other.sources,.will.become.dependent.on.renewing.these.agreements,.and.will.therefore.lose.their.ability.to.walk.away.from.negotiations,.and,.as.a.result,.will.sacrifice.much.of.their.bargaining.power..If.recognized,.these.issues.may.be.addressed.by.choosing.a.partner.with.which.there.is.strong.incentive.alignment.as.well.as.safeguards.in.the.agreement.

GENERAL GUIDELINES

In.this.section,.we.will.apply.our.operational.framework.to.structure.general.guide-lines.and.evaluate.the.control.rights.for.two.leading.university–private.partnerships.in.R&D..These.case.studies.provide.insights.into.structuring.horticulture.university–private.partnerships.

pppS and publIc Good reSearch

As.public. funding.of. scientific. research.has.declined,.and.knowledge. inputs.have.played.an.increasingly.important.role.in.industrial.processes,.universities.and.other.public.research.institutions.have.looked.to.private.sources.to.increase.their.research.budgets..Many.lessons.have.been.learned.as.public.criticism.and.scrutiny.of.these.research.partnerships.have.evolved.19.Issues.such.as.conflict.of.academic.and.indus-try. interests,.ownership.of,.and.access. to,. IP.(e.g.,. issues.of.hold-up.and.blocking.patents),. and. publication. delays. have. fueled. the. current. debate. and. often. present.insurmountable.obstacles.to.forming.research.partnerships.

A. host. of. external. forces. have. shaped. the. current. environment. in. which. pub-lic.researchers.are.seeking.to.actively.engage.with.private.firms..Among.these.are.diminishing. federal. and. state. funds. for.public.good. research. and. increased. state.funding. incentives. for. private–public. research.. In. addition,. legislation. (e.g.,. the.Bayh–Dole.Act),.the.restructuring.of.many.large.life.sciences.firms,.and.an.align-ment. of. private. and. public. research. incentives. have. contributed. to. this. trend.20.Moreover,. the. traditional. research.paradigm,.which.presumes. that. there. is.a.one-way. flow. from. basic. science. conducted. in. public. institutions. to. applied. research.and. commercialization. undertaken. by.private. industry,. has. begun. to.be. replaced.by.a.chaotic.R&D.feedback.loop.paradigm.13,20.Increasingly,.public.universities.and.


private. companies. are. engaging. in. joint. research,. establishing. relationships. with.exchange.and.collaboration.in.all.stages.of.research.

The.potential.benefits.from.university–industry.partnerships.are.clear..Comple-mentarities.between.scientific.and.practical.knowledge.have.the.capacity.to.generate.rapid. and. far-reaching. innovation.. Under. the. best. of. circumstances,. each. partner.is.seeking.attributes.and.assets.in.prospective.partners.that.complement.their.own.abilities.and.resources..Industry.is.interested.in.combining.its.knowledge.of.markets.with.information.on.new.research.and.innovation.in.order.to.identify.those.devel-opments. that. are. likely. to. lead. to. commercial. applications.. This. motivation. may.be.obvious,.but. industry. is.also. interested. in.more.subtle.assets.such.as.access. to.academic.expertise,.networks,.and.first-hand.information.about.up-and-coming.sci-entists.(current.graduate.students)..And.while.universities.are.very.clearly.interested.in.financial.capital,.they.are.also.seeking.intellectual.capital,.cutting-edge.research.technologies,21. proprietary. research. tools. (e.g.,. databases),. and. in. many. instances.enabling.IP.22.Access.to.these.research.assets.enhances.a.university’s.ability.to.pro-vide.first-rate.education.to.its.graduate.students.

Although.the.potential.benefits.of.research.partnerships.are.reasonably.transpar-ent,.the.potential.risks.to.both.parties.are.opaque..These.risks.pose.serious.obstacles.to. the.successful.formation.of.public–private.research.partnerships..In.addition.to.the.uncertainty.inherent.in.any.research.process,.the.differences.between.university.educational.objectives.and.corporate.goals.are.an.important.source.of.risk.in.these.relationships..Recent.data.show.that.almost.70%.of.research.in.universities.has.been.categorized.by. the.National.Science.Foundation. as.basic,.while. the. proportion. is.reversed. in. industry.. In. 2000,. while. universities. accounted. for. only. 14%. of. total.R&D. funding. in. the. United. States,. they. performed. about. 50%. of. the. total. basic.research.23. With. private. financing. comes. the. concern. that. the. traditional. orienta-tion.of. the.academic. research.agenda. toward.basic,.public.goods.research.will.be.directed.toward.more.applied,.appropriable.research.that.serves.the.objectives.of.the.private.partner,.and.that.this,.in.turn,.will.result.in.a.loss.of.academic.integrity..Not.only.research.direction.but.research.results.from.sponsored.studies.might.be.biased.toward.sponsors’. interests..Bekelman.et.al.,. for. instance,. show. that. in.biomedical.research.there.is.a.statistically.significant.association.between.industry.sponsorship.and.proindustry.conclusions.24

Industrial. sponsors. may. also. impose. constraints. on. communication. between.grantees. and. other. colleagues,. which,. in. turn,. may. hinder. research. progress. and.increase. research. costs.23. Planning. horizons. tend. to.differ;. university. researchers.focus.on.long-term.research.while.companies.often.seek.quick.turn-around.projects..In.addition,.the.cultures.and.values.of.research.partners.may.simply.clash,.creating.insurmountable.blocks.to.a.continuing.relationship..Furthermore,.the.incentives.to.secure.a.renewal.or.extension.of.an.existing.contractual.agreement.may.adversely.influence.university.scientist.behavior.under.a.current.collaboration.

Rights.to.IP.are.especially.contentious..Hold-up.and.background.rights.are.of.pri-mary.concern.to.an.industry.partner.interested.in.commercializing.the.products.of.a.research.partnership..Researchers.at.universities.and.other.public.institutions.often.use. proprietary. or. enabling. IP. research. tools. in. their. research. without. obtaining.rights..They.are.sometimes.blocked,.however,.for.any.research.discoveries.that.have.


commercial.value..Generally,.one.researcher. in.a.university.institution.may.freely.access.another.researcher’s.patented.research.tool.for.academic.study..This.oppor-tunity.does.not.typically.extend.to.private.researchers.unless.a.formal.agreement.is.forged..Thus,.a.private.company.looking.to.partner.with.a.particular.researcher,.for.example,.may.experience.hold-up.at.the.commercialization.stage.because.the.public.research.partner.did.not.obtain.formal.rights.to.all.research.inputs.(i.e.,.background.rights). from. some. other. private. company.. Note. also. that. if. numerous. university.researchers.and.graduate.students.are.involved.in.a.research.project,.industry.risks.loss.of.privacy,.and.protection.for.proprietary.information.

The.interests.of.parties.outside.a.research.agreement.(i.e.,.third.parties).are.also.at.risk.under.public–private.research.agreements..If.an.agreement.is.not.effectively.structured.with.regard.to.patenting.and.licensing.rights,.a.third-party.interest.in.hav-ing.access.to.research.products.and.innovations.may.not.be.adequately.represented..In.fact,.blocking.patents.can.and.do.arise.22

In.summary,.the.cooperation.between.universities.and.industry.opens.a.series.of.questions..Does.the.profit-driven.sponsor.shift. the.university’s.mission.away.from.basic.research?.Does.industry’s.desire.to.exploit.IP.rights.interfere.with.communica-tion.within.and.between.universities.to.an.extent.harmful.to.open.science?23.These.conflicts. are. an. inevitable. consequence. of. a. fundamental. clash. between. a. public.system.that.encourages.openness.in.science.and.an.industrial.system.that.gives.finan-cial.rewards.based.on.secrecy..In.the.end,.this.all.boils.down.to.one.question:.can.a.university–industry.partnership.be. socially.beneficial. or,.more.precisely,. Pareto.improving?*

Scotchmer23.argues.that.a.public–private.venture.is.justifiable.for.big.science.proj-ects..For.certain.large.projects,.the.public.sector.may.face.the.problem.of.choosing.the.right.investments.(those.with.high.probability.of.success).and.making.sure.the.funds.are.used.as.intended;.the.private.sector,.on.the.other.hand,.has.the.expertise.needed.to.screen.likely.successful.projects.but.sometimes.cannot.reap.unappropri-able.social.benefits,.thus.is.unable.to.recover.the.cost.of.research..In.this.situation,.a.PPP.can.help.solve.the.duality.problem..If.the.partners.do.not.have.access.to.the.same.information,.however,.industry.may.manipulate.the.public.sector.into.subsidiz-ing.its’.privately.profitable.projects.

two leadInG unIverSIty–prIvate r&d partnerShIpS

The.University.of.California. formed.a.partnership.with. the.Novartis.Agricultural.Discovery.Institute,.Inc..(NADI).in.1998.that.allowed.Berkeley.to.retain.control.of.an.open.research.agenda..Initially,.an.open.call.is.put.out.to.participating.faculty.for.research.proposals.with.neither.the.University.nor.NADI.defining.the.type.of.project.proposals.that.would.be.considered..Further,.the.committee.that.allocates.funding.to.each.project.(all.proposed.projects.receive.some.amount.of.funding).is.made.up.of.three.Berkeley.University.faculty.members.and.two.members.representing.NADI..In.addition,.the.criteria.used.for.ranking.project.include.the.quality.and.intellectual.

*.A.partnership.is.Pareto.improving.if.at.least.one.partner.is.made.better.off.by.joining.and.no.partner.is.made.worse.off.by.joining.


merit.of.the.proposed.research,.potential.advancement.of.discovery,.and.the.past.and.present.productivity.of. the.research..As.a. result,. the.direct.value.of.any.potential.projects.to.NADI.is.not.considered.

An. alternative. structure. governs. a. biological. research. agreement. between.Washington.University.(WU).in.St..Louis.and.the.agricultural.biotechnology.com-pany,.Monsanto..An.advisory.committee.solicits.proposals.that.conform.to.research.areas.specified.by.the.committee..In.this.case,.the.committee.is.equally.split.with.three.university.members.and.three.representatives.from.Monsanto..Therefore,.the.interests.of.Monsanto.have.more.weight,.both.in.defining.the.choice.set.of.research.proposals.that.will.be.considered.by.the.committee.and.in.selecting.which.of.those.proposals.are.funded..Moreover,. the.agreement.specifically.directs. the.committee.to.identify.and.fund.projects.that.not.only.have.exceptional.academic.merit.but.also.serve.the.research.interests.of.Monsanto.

Depending.on.the.mission.of.the.university.and.its.role.in.the.community,.both.of.these.alternative.governance.structures.have.merits..Because.the.research.interests.of. the.private.partner. carry.more.weight,. the.WU/Monsanto.agreement.may.be.more.likely.to.generate.innovations.that.result.in.commercial.applications,.meeting.the.objective.of.serving.the.community.with.successful.technology.transfers..On.the.other.hand,.the.Berkeley/NADI.agreement.more.adequately.protects.the.academic.freedom. of. participating. faculty.. What. is. important. is. that. the. public. institutions.make.conscious.decisions.about.where.they.are.comfortable.on.this.spectrum.of.con-trol.over.the.research.agenda.and.that.they.are.fully.aware.of.the.implicit.trade-offs.contained.in.the.related.contract.language.

The.primary.interest.of.universities.is.to.share.their.research.results.with.colleagues.as.rapidly.as.possible,.through.publications.and.presentations.at.conferences,.with.the.hope.that.scientific.knowledge.and.research.will.be.advanced..This.academic.mission.conflicts.with.the.interest.of.the.private.partner.to.appropriate.innovation.and.techno-logical.advancements,.thus.requiring,.for.a.certain.amount.of.time,.that.research.results.are.kept. from.competing. interests.until. the.private.partner.establishes. rights. to. the.innovation..As.such,.the.publication.delay.provisions.of.research.agreements.usually.come.under.considerable.scrutiny..In.fact,.guidelines.issued.by.the.National.Institutes.of.Health.recommend.a.delay.of.30–60.days.25

A.more.relevant.question.concerns.control.of.the.option.for.terminating.the.delay.period.rather.than.the.specified.maximum.length.of.this.period..For.example,.under.the.Berkeley/NADI.agreement,.NADI.has.an.initial.30.day.delay.during.which.they.must.decide.whether.an.innovation.has.the.potential.to.be.patented..If.they.decide.that.the.parties.should.proceed.with.a.patent.application,.publications.can.be.delayed.only.up.until.the.time.the.patent.application.is.filed.or.90.days—whichever.is.shorter..And.Berkeley.has.right.to.file.the.patent.application.at.any.time..The.filing.process.can.be.expedited,.with.an.initial.application.filed.in.a.day.or.so..Therefore,.under.this.agreement,.although.the.maximum.publication.delay.is.120.days,.Berkeley.has.complete.control.to.end.the.delay.(past.the.initial.30.day.period).

In.contrast,.in.the.WU/Monsanto.agreement,.important.issues.concerning.patent-ing.of.innovations.that.are.generated.from.the.partnership.include.who.is.responsible.for.filing.for.the.patents,.the.right.not.to.file.for.a.patent,.control.of.patent.litigation,.the.right.to.“know-how”.transfer,.and.ownership.of.the.core.technology..Who.holds.


the.option. to.file.a.patent.application. is. important.not.only.because.of.control.over. the. publication. delay. period. but. also. because. the. content. of. the. application.defines.who.is.responsible.for.generating.the.innovation.as.well.as.the.nature.of.the.innovation..Both.of.these.parameters.can.be.defined.broadly.or.more.conservatively;.this.will.have.significant.implications.for.who.controls.the.licensing.rights.and.how.strong.these.rights.turn.out.to.be..In.some.instances,.the.university.partner.is.respon-sible.for.filing.the.patent.applications,.but. the.degree.to.which.the.private.partner.provides.guidance.and.is.involved.in.patent.litigation.is.varied.

A. more. subtle. issue. is. whether. a. university. is. obligated. to. file. for. a. patent. if.requested. to. do. so. by. the. industry. partner,. or. whether. it. has. some. discretion..A university.partner.may.wish.to.avoid.expending.the.effort.required.to.patent.inno-vations.if.it.does.not.foresee.that.it.will.be.applied.commercially..For.example,.under.the.Berkeley/NADI.agreement,.Berkeley.can.elect.not.to.file.for.a.patent.that.NADI.does.not.intend.to.commercialize..In.other.words,.Berkeley.can.make.sure.that.inno-vation,. or. know-how,. that. would. not. otherwise. be. commercialized. remain. freely.available.to.the.public.and.that.limited.administrative.resources.are.not.diverted.to.pursue.meaningless.patents.

Of.vital.importance.for.industry–university.research.agreements.is.the.nature.of.the.licensing.options..Currently,.it.is.common.for.the.industry.partner.to.be.given.a.first-to-negotiate.licensing.option.for.some.subset.of.the.innovations.generated.under.the.research.agreement..Generally,.these.options.must.be.exercised.within.a.specified.time.period,.or.else.the.option.is.extended.to.third.parties..In.response.to.public.out-cry.concerning.previous,.poorly.structured.agreements,.such.as.the.Sandoz/Scripps.agreement,*. and. concern. about. blocking. patents,. right-of-first-refusal. options.evolved.into.right-to-negotiate.options..In.theory,.if.the.industry.partner.is.granted.the.more.limited.option.of.right-to-negotiate,.a.university.has.greater.control.over.licensing.rights.and.can.prevent.blocking.patents.from.being.awarded..Other.aspects.of.licensing.agreements.receive.less.attention.but.are.also.critical..One.such.aspect.is.the.percentage.of.the.total.innovation.for.which.the.industry.partner.holds.an.option.to.negotiate.an.exclusive.license.or.access.option..For.example,.under.the.Berkeley/NADI.agreement,.NADI.can.exercise.this.option.for.an.“allowable.percentage”.of.patents,.equal.to.the.percentage.of.the.research.funding.that.came.from.NADI;.there-fore,.NADI.has.limited.access.options..Under.other.agreements,.the.industry.partner.holds.this.option.for.all.patented.discoveries.generated.by.the.agreement.

Third-party.options.are.also.a.critical.aspect.of.licenses.options..These.options.are.the.rights.that.parties.outside.the.agreement.have.to.innovation.generated.by.the.agree-ment..Under.the.Berkeley/NADI.agreement,.third.parties.hold.open.options.on.patents.not.included.in.the.allowable.percentage,.and.on.patented.innovations.either.covered.by.nonexclusive.license.or.for.which.the.first-to-negotiate.option.has.expired.for.NADI..NADI.has.no.recourse.once.their.licensing.option.has.expired,.and.Berkeley.is.free.to.

*.In.1993,. the.publicly.funded.Scripps.Research.Institute.agreed.to.form.a.research.partnership.with.Sandoz.Pharmaceuticals..Sandoz.agreed.to.provide.$300.million.in.funding.for.research.over.10.years.in.return.for.a.worldwide.license.for.all.of.discoveries.made.by.researchers.at.Scripps..This.controver-sial.agreement,.which.would.have.given.Sandoz.licensing.rights.to.nearly.$1.billion.of.research.funded.by.the.federal.government,.was.restructured.after.the.government.threatened.to.cut.off.funding.for.the.institute.


enter.licensing.negotiations.with.third.parties..In.contrast,.under.the.WU/Monsanto.agreement,.third.parties.hold.only.a.conditional.option..Monsanto.has.right-of-first-refusal.on.any.licensing.arrangement.between.the.university.and.third.parties,.even.if.Monsanto’s.original.licensing.option.has.expired..Thus,.Monsanto.is.guaranteed.an.option.of.first.refusal.on.any.third.party.offers.made.to.WU..This.severely.limits.the.options.available.to.third.parties.and.ill.serves.the.interests.of.the.university.

PPPs IN HORTICULTURAL RESEARCH

The.horticultural.research.industry.is.composed.primarily.of.small-.to.medium-sized.enterprises.with.small.markets.for.individual.products.26.Even.though.these.firms.are.relatively.small,.they.have.demonstrated.a.capacity.to.apply.new.discoveries.for.pos-sible.commercialization..However,.when.it.comes.to.genetically.engineered.crops,.these. small.firms.generally.do.not.have. the.assets. to.develop.new.products..As.a.result,.research.funds.in.horticulture.are.sourced.primarily.from.the.public.sector.26,27

The.reluctance.of.major.biotechnology.R&D.companies.to.dedicate.funds.to.hor-ticultural. research. is,. in. part,. because. technological. advances. in. horticulture. are.not.viewed.as.“low-hanging.fruit.”.The.commercial.value.is.not.nearly.as.attractive.as.that.of.annual.agronomic.crops.grown.on.large.acreages..In.addition,.consumer.acceptance.of.genetically.modified.foods.is.considered.a.major.obstacle.to.the.adop-tion.and.commercialization.of.agricultural.biotechnology..These.constraints.on.the.application. of. modern. biology. to. the. market. for. horticultural. crops. are. the. most.important. impediment. to. the. formation. of. public–private. research. partnerships..Hence,.public.institutions.might.be.best.served.by.forming.partnerships.with.private.associations.or.consortia.(i.e.,.Western.Fruit.Growers’.Association).rather.than.single.firms..Such.partnerships.are.more.likely.to.“crowd-in”. in.contrast. to.“crowd-out”.public.good.research.at.land-grant.universities.13

Groups.of.horticultural.firms.have.expressed.interest.in.funding.research.for.crop.and.seed.improvement.in.response.to.the.recent.global.food.shortages.27.These.firms,.attracted.by.the.research.expertise.of.university.faculty,.have.begun.working.with.universities.to.develop.effective.new.technologies..Some.firms.have.indicated.that.partnerships.with.universities.have.the.added.advantage.of.improving.the.image.of.genetically.modified.crops.by.allowing.for.increased.public.exposure.to.the.benefits.of. their.work..Though.many.firms.have.expressed. interest.and.some.partnerships.have.been.formed,.there.is.potential.for.many.more.research.partnerships.

Public.research.institutions.have.much.to.gain.by.forming.partnerships.with.con-sortia.composed.of.small.private.firms,.nurseries,.or.grower’s.associations..Currently,.these.groups. sponsor. relatively. little.horticultural. research. in. the.United.States.28.A. model. similar. to. Australia’s. check-off. funding. organizations*. could. be. used.to. increase. private. sector. sponsorship. of. public. agricultural. research. institutions..One.check-off.organization,.Horticulture.Australia.Limited.(HAL),.invests.over.$80 million.annually.in.more.than.1200.research.partnerships..All.HAL.members.are.required.to.donate.a.percentage,.determined.by.an.external.council,.of.their.revenue.

*.A.check-off.program.levies.a.small.tax.on.the.producers.of.a.horticultural.product.and.uses.these.funds.to.sponsor.research.at.public.institutions.


to.HAL,.which.is.distributed.to.a.broad.range.of.research.programs.in.response.to.the.horticulture.industry’s.current.needs..The.establishment.of.similar.organizations.in. the. U.S.. horticulture. industry. (see. Figure. 11.1),. with. mandatory. contributions,.would.dramatically.increase.funding.for.public.good.research.

Domestic.and.international.public–private.research.partnerships.in.horticulture.are.considered.especially.important.for.developing.economies.29.Dixon26.notes.that.successful.entrepreneurs.in.horticulture.maintain.a.continuous.dialogue.with.scientists;.partnerships. are. one. approach. for. guaranteeing. this. dialogue.. Dixon. also. notes.that.linkages.between.research.and.industry.(public.and.private.relationships).have.improved.“where.levy.funding.systems.have.been.established.to.support.scientific.endeavors.”.In.other.words,.more.formal.financial.arrangements.between.partners.are.likely.to.yield.a.superior.exchange.

The.most.relevant.partnership.model.for.the.horticulture.industry.is.that.of.less.formal,. single,. or. multiple-project. partnerships. (sponsored. project. and. informal.arrangements)..Private.horticulture.institutions.should.seek.to.align.research.incen-tives.and.form.consortia.of.small.and.medium.firms.with.parallel.research.interests.to.concentrate.intellectual.and.financial.resources..These.consortia.are.organized.by.crop.or.pest.type.(or.other.research.interests).to.facilitate.networking,.identify.key.researchers.at.public.institutions,.and.propose.specific.research.projects..A.propor-tional-contribution.burden.sharing.scheme.between.consortia.members.is.likely.to.be. the.most. effective. self-governing.approach.given. the.public.nature.of. research.leads.and.outcomes.

The. university. should. accept. or. reject. these. proposals. based. on. the. research.synergy. and. embedded. options.. Although. all. universities. share. a. common. set. of.core.principles.that.guide.their.decisions,.different.institutions.emphasize.different.

Firms Universities

Commerical discoveries

Nurseries

Grower adoption

Packaging and processing

Consumers

Wholesale

Public institutions

Food serviceRetail

FIGURE 11.1 Sources.of.discovery.and.commercialization.


objectives;.the.private.partner.should.consider.the.university’s.research.culture.when.considering.research.partners..Given.the.nature.of.research.objectives.at.universi-ties,.the.horticulture.industry.partner.should.propose.research.projects.that.are.more.basic,.have.longer.time.frames,.and.are.not.adequately.addressed.by.current.private.research.efforts..These.partnerships.are.more. likely. to.be. successfully.negotiated.if. the. industry. partner. understands,. ex. ante,. the. need. for. academic. freedom.. On.the.back-end,.university.guidelines.and.policy.usually.constrain. its. researchers. to.specific. conditions. for. patenting. research. and. licensing. and. disseminating. results.(publication.delays)..Although. there. is. some.variation,. these.constraints.are. fairly.common.among.research.universities.

Both. partners. should. establish. links,. so. that. industry. can. effectively. utilize.public.research,.and.universities.can.secure.access.to.research.funding.and.com-plimentary. enabling. technologies.. These. collaborations. can. serve. as. stepping.stones.to.more.formal,.long-term.agreements..Alternatively,.once.initial.consortia-university.research.partnerships.are.established,.more.enterprising.members.of.the.consortia.can.capitalize.on.the.research.contacts.and.pursue.firm-specific,.applied-research. partnerships.. The. primary. obstacle. to. forming. these. research. partner-ships.is.high.transaction.costs..The.process.of.identifying.appropriate.researchers.as.potential.partners.can.involve.significant.search.costs..And.once.the.potential.partners.have.been.selected,.the.time.and.effort.involved.in.negotiating.a.research.agreement,.especially.given.the.differing.objectives.of.public.versus.private.insti-tutions,.can.be.substantial..The.consortium.approach.is.a.strategy.for.sharing.these.costs..If.the.consortia.are.not.well.structured,.however,.reduced.external.transac-tion.costs.may.face.higher.internal.costs.of.organizing.and.maintaining.the.con-sortia..Inequitable.benefits.sharing.within.a.consortium.may.also.be.a.source.of.conflict..And.although.this.approach.is.intended.to.serve.the.needs.of.medium-to-smaller-sized.firms,. the.smallest.enterprises.may.still.be.excluded.(especially.in.subsequent.partnerships).

Dramatic.improvement.in.the.development.and.commercialization.of.transgenic.horticultural.crops.can.come. from. increased. involvement. from. the.private. sector..Though.the.potential.benefits.from.university–industry.partnerships.in.the.field.of.agricultural. biotechnology. are. obvious,. the. private. sector. will. not. increase. their.investment.in.horticultural.R&D.without.active.solicitation.by.the.public.sector..As.soon.as.the.public.sector.is.able.to.form.horticultural.PPPs,.whether.they.are.com-posed.of.a.single.firm.or.a.consortia,.formal.or.informal,.or.in.developing.or.industri-alized.nations,.and.demonstrate.their.value.to.the.private.sector,.there.will.be.fewer.obstacles. to.forming.PPPs. in.the.future,.and. those.partnerships.are.likely. to.have.more.favorable.terms.for.the.public.sector.

REFERENCES

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. 15.. Rausser,.G.,.Ameden,.H.,.and.Simon,.L..Public–private.alliances.in.biotechnology:.Can.they.narrow.the.knowledge.gaps.between.rich.and.poor?.Food Policy,.25,.499,.2000.

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249

12 Why Are Regulatory Requirements a Major Impediment to Genetic Engineering of Horticultural Crops?

Steven H. Strauss

Regulations.in.the.United.States.and.in.most.other.countries.treat.all.plants.produced.using.recombinant.DNA.methods.(genetic.engineering.or.genetic.modification).as.illegal.for.use.in.the.environment.or.in.commercial.products.until.their.safety.and.acceptability.has.been.specifically.authorized..The.costs.of.complying.with.regula-tions. and. the. legal. risks. of. not. complying. place. severe. constraints. on. the. use. of.recombinant.DNA.breeding.methods.at.both. research. and.commercial.phases.. In.particular,.the.limitations.to.gene.release.in.the.environment.pose.severe.constraints.for.required.field.research,.development,.and.commercial.applications.for.most.hor-ticultural. crops,. a.problem. that. is. exacerbated. in.many.cases.by. their. incomplete.

CONTENTS

Need.for.Biotechnology.Scientists.to.Be.Informed.and.to.Inform.Regulations.....250Consequences.of.Failure.to.Comply.with.Regulations.Are.Large.......................... 252A.Multitude.of.Regulations.Exist.at.National.and.International.Levels................. 253Regulatory.Compliance.Is.Especially.Problematic.for.Horticultural.Crops........... 253Deregulated.Horticultural.Varieties.Do.Not.Provide.General.Models....................254Causes.of.Our.Stringent.Regulatory.System..........................................................254

Presumption.of.Harm.from.Transgenic.Methods............................................... 255Environmental.Concerns.Prompt.Strong.Regulations.......................................256Familiar.Genes.Meet.Same.Regulatory.Review................................................ 256

Environmental.Studies.Are.Seriously.Compromised.by.Regulations.................... 258Presumption.That.Stasis.Is.Desirable..................................................................... 258Consequences.of.Simple.Definitions.of.Clean.and.Green...................................... 259Conclusions.............................................................................................................260References...............................................................................................................260


domestication.and.wild.or.feral.relatives..This.chapter.explores.the.direct.and.indi-rect.causes. for. the.stringent.regulatory.system.in.place,.discusses.the.opportunity.costs. they. impose,.and.proposes.some.alternative.regulatory.concepts.. I.maintain.that.until.regulatory.systems.incorporate.a. tier. that.provides,.at. the.outset.of.field.research,.exemptions.or.workable.tolerances.for.adventitious.presence,.the.ability.to.use.transgenic.approaches.for.horticultural.breeding.will.be.severely.limited,.thus.foreclosing.a.number.of. important.options.for. improving.pest.management,.stress.tolerance,.and.product.quality.

It.is.common.to.see.lay.discussions.of.the.social.controversies.and.the.potential.of.genetic.engineering.(GE)*.virtually.ignore.the.federal.regulatory.gauntlet.that.GE.products.must.get.through..Those.who.tend.to.be.in.favor.of.GE.crop.solutions.often.assume.that.regulations.are.well-crafted.and.essential. to.protect.the.public.safety;.their.efficacy,.cost,.and.what.products.might.have.been.discouraged.even.before.they.are.created.are.rarely.considered..In.contrast,. those.against.GE.argue.that.regula-tions.are.not.strong.enough,.as.evidenced.by.the.very.existence.of.GE.products.with.the.absence.of. full. scientific.certainty.about. their.effects..Because.of. the.esoteric.nature.of.regulations,.it.often.seems.to.be.only.the.practitioners.of.GE.who.really.understand.the.implications.of.regulations.in.practice..Who.else.would.know.what.it.costs.in.time.and.labor.to.conduct.a.regulated.field.trial.apart.from.those.conducting.the.trials?.Or.of.what.it.costs.to.bring.a.product.to.market,.other.than.public.sector.institutions.or.companies.that.have.sought. to.do.so?.The.goal.of.this.chapter. is.to.discuss.the.costs.and.impediments.to.research.and.development.of.transgenic.horti-cultural.crops.from.the.perspective.of.a.public.sector.biotechnologist.who.works.on.ornamental.and.forest.trees..In.addition.to.my.own.experience,.this.article.is.moti-vated.by.the.apparent.absence.of.any.new.horticultural.transgenic.crops.in.the.public.sector.pipeline.(an.observation.based.on.discussions.with.many.colleagues).in.spite.of.a.rather.large.number.of.field.trials.that.have.been.conducted.during.the.past.two.decades.(see.http://www.isb.vt.edu/cfdocs/fieldtests1.cfm.for.listings)..This.suggests.that.regulatory.costs.and.obstacles,.in.combination.with.market.risks,.are.severely.impeding.transgenic.variety.development.

NEED FOR BIOTECHNOLOGY SCIENTISTS TO BE INFORMED AND TO INFORM REGULATIONS

Few.scientists.or.students.who.are.drawn.to.plant.science.or.to.its.practical.applica-tions.such.as.horticulture.and.forestry.like.the.idea.of.studying.government.regula-tions..It.sounds.about.as.exciting.as.reading.the.United.States.Internal.Revenue.tax.code,.and.about.as.enticing.as.a.trip.to.the.dentist.to.have.your.teeth.drilled..As.a.scientist,.I.fully.share.these.sentiments,.but.my.work.over.the.years.with.field.trials.of.genetically.modified.trees1,2.(Figure.12.1),.and.the.small.part.I.have.played.in.writing.

*.Throughout.this.chapter,.which.specifically.addresses.the.products.of.genetic.engineering.or.genetic.modification,.I.use.the.terms.“biotechnology”.or.“GE”.or.“GM”.as.shorthand..I.am.referring.to.crops.produced.using.methods.where.plants.are.modified.by.asexually.induced,.specific.genetic.modification.and.regeneration.of.the.modified.cells.into.plants.

251Regulatory Obstacles to Transgenic Horticultural Crops

FIGURE 12.1 (See color insert.).USDA-APHIS.authorized.field.trial.of.transgenic.poplars.in.Oregon.(United.States).during. its.first. (top).and.second. (bottom).growing.seasons..The.population,. part. of. a. gene. discovery. program. using. a. method. called. “activation. tagging”.(where. genes. are. randomly. upregulated. by. insertion. of. a. gene. expression. enhancer),. was.being.screened.for.novel.morphologies.under.field.conditions..The.trees.had.to.be.removed.prior.to.the.desired.long-term.nature.of.this.experiment.because.of.regulatory.costs.associ-ated.with.long-term.containment,.monitoring,.reporting,.and.removal.costs.for. large.trees..There.is.no.obvious.scientific.basis.for. intensively.regulating.such.trees.while.interspecies.hybrid.poplar.trees,.and.those.produced.through.non-transgenic.forms.of.mutagenesis,.are.essentially.unregulated.throughout.the.world.


about.regulatory.reforms3,4.and.taking.part.in.national.and.international.workshops.designed.to.inform.or.influence.regulations,5.have.shown.me.how.important.they.are..I.now.often.argue.that.every.plant.scientist.who.works.in.and.understands.the.poten-tial.benefits.of.transgenic.plant.biotechnology.needs.to.understand.regulations,.and.play.a.role.in.improving.them..The.goal.is.to.craft.regulations.that.more.effectively.target.and.limit.very.high.risk.applications,.while.minimizing.encumbrances.to.field.research.on.safe.and.highly.valuable.applications..At.least.for.now,.regulations.and.their.implementation.are.still.evolving,.providing.an.opportunity.for.influence.from.scientists.6. In. addition,. all. applications. for. permits. and. petitions. for. deregulation.(USDA). and. registration. of. GE. pest-tolerant. plants. (EPA). have. required. periods.of open.public.comment;.the.high.quality.science-based.or.data-based.input.that.is.often.received.from.biotechnology.scientists.is.valued.by.regulatory.agencies.

CONSEQUENCES OF FAILURE TO COMPLY WITH REGULATIONS ARE LARGE

Regulations.are.informed.by.science,.but.mainly.they.embody.the.overall.“attitude”.of.a.society.about.a.technology.7.Emotions,.perceptions,.economics,.and.politics.generally.dwarf.the.influence.of.science.in.developing.regulatory.policies..Regulations.can.be.writ-ten.with.a.tone.of.aversion.and.extreme.caution.when.society.senses.risk.and.harm.rather.than.direct.benefit—as.we.see.today.with.plant.biotechnology..Or,.they.can.be.written.with.a.sense.of.optimism.and.encouragement,.as.we.tend.to.see.today.with.respect.to.wind.power.and.related.technologies..Because.regulations.have.the.force.of.law.behind.them,.even.minor.violations.can.have.significant.penalties. including.heavy.fines.and.even.imprisonment..Thus,.they.have.a.power.and.gravity.very.different.from.research.procedures.or.recommendations,.such.as.those.followed.in.molecular.biology.laborato-ries.in.the.United.States.under.the.National.Institute.of.Health.(NIH).recombinant.DNA.research.guidelines..The.risks.and.costs.of.complying.with.regulations—or.being.unable.to.comply—often.determine,.not.just.inform,.scientific.and.business.strategies.

As.seen.with.the.StarLink.GE.maize.debacle8.and.with.the.ongoing.multimillion.dollar.lawsuits.over.accidental.infusion.of.USDA-approved.GE.rice.that.harmed.U.S..exports,9.the.consequences.of.getting.the.regulations.about.gene.dispersal.(often.called.adventitious.presence.[AP].or.low-level.presence.[LLP]).wrong,.even.in.small.detail,.can.be.enormous.for.companies.and.for.the.entire.agricultural.sector..In.addition,.recent.successful.lawsuits.over.USDA.decisions.on.herbicide.resistant.sugar.beets,.alfalfa,.and.creeping.bentgrass.have.set.new.precedents.for.use.of.the.National.Environmental.Protection.Act.. Its. requirement. for.Environmental. Impact.Statements. in. regulatory.decisions.on.crop.biotechnologies.has.brought.the.courts.into.the.regulation.of.crop.biotechnology. in.a.major.way10. that. requires. far.more.work.and. legal.detail. to. the.process.in.order.to.increase.the.likelihood.that.Animal.and.Plant.Health.Inspection.Service.(APHIS).decisions.can.withstand.legal.challenges..In.addition,.given.the.broad.interpretation. of. what. National. Environmental. Policy. Act. (NEPA). covers. in. these.cases—which.include.economic.damages.to.organic.and.conventional.farmers.from.AP—it.is.unclear.whether.the.preparation.of.Environmental.Impact.Statement.(EISs).will.improve.the.quality.of.scientific.analysis.of.the.underlying.biological.issues.


A MULTITUDE OF REGULATIONS EXIST AT NATIONAL AND INTERNATIONAL LEVELS

The.problems.with.AP.and.consequent.trade.disruptions.described.above.also.remind.us.that.we.have.not.one,.but.a.multitude.of.national.regulatory.regimes.that.can.vary.widely.by.country,.as.well.as.an.overarching.international.regulatory.policy.in.the.Cartagena.Protocol.of.the.Convention.on.Biological.Diversity.(CBD).11.The.CBD’s.provisions.must.be.addressed.if.living.agricultural.products.such.as.seeds.are.traded,.or.if.pollen,.seed.or.vegetative.propagules.can.move.across.country.boundaries..Such.rules.are.critical.for.trade.in.many.horticultural.crops,.where.the.products.are.often.living.(e.g.,.nuts,.fruits,.horticultural.varieties),.and.where.wind.and.insect.vectors.often. can.move. pollen,. fruits,. and. sometimes. small. seeds. many.kilometers..This.network.of.regulations.means.that.making.changes.to.regulations.is.truly.a.glacial.process;. it. involves.seeking.coordinated.changes.in.the.attitudes.of.highly.diverse.societies,.as.well.as.through.fractious.and.highly.political.bureaucracies.such.as.the.United.Nations..Given.the.negative.attitude.inherent.in.most.regulatory.regimes.con-cerning.crop.biotechnology,.it.is.not.hyperbole.to.state.that.the.regulatory.challenges.facing.horticultural.biotechnology.are.both.global.and.monumental.

REGULATORY COMPLIANCE IS ESPECIALLY PROBLEMATIC FOR HORTICULTURAL CROPS

For.most.horticultural.crops,*. the. implications.of. the. stringent. regulatory.system.are.even.more.grave.than.for.field.crops..This.is.because.the.high.regulatory.costs12.per.gene.insertion.event.tend.to.be.spread.over.a.smaller.variety.base,.with.a.smaller.economic.return,.and.with.a.longer.time.for.the.return.to.be.manifest..This.results.because.these.crops.are.far.more.diverse.in.their.genetics.and.geography,.transfer-ring.approved.biotech. traits. into.new.varieties. through.breeding. is.slow.due. to.a.longer. generation. time. and. biological. limits. to. inbreeding,. and. because. valuable.genotypes.tend.to.be.cloned.rather. than.sexually.propagated..Thus,.it. is.expected.that. individual. transgenic.events. from.elite.clones,.not.progeny. from.deregulated.or.registered.events,.will.each.require.separate.regulatory.dossiers.and.decisions.3

Moreover,.these.crops.as.a.category.tend.to.be.less.domesticated.and.thus.can.more. readily. mate. with. wild. or. feral. relatives,. and. spread. directly. via. seed. or.vegetative.propagation.in.wild.or.feral.environments..Because.of. their. large.size.and. potential. for. wide. pollen. or. seed. dispersal. by. wind,. insect,. or. animal. vec-tors,.containment.when.plants.are.old.enough.to.flower.and.are.producing.fruit.can.be. very. difficult,. costly,. and.often. impossible. to. assure.. This. creates. a. situation.where.gathering.needed.regulatory.data.on.environmental.effects,.under.the.very.strong.confinement.mandated.by.regulations,.poses.a.kind.of.“Catch-22” situation.(i.e., where.the.required.information,.at.a.high.level.of.scientific.rigor.and.ecologi-cal.relevance,.is.nearly.impossible.to.obtain.while.assuring.full.containment)..Even.if. the. data. could. be. obtained,. the. required. depth. of. analyses. (e.g.,. of. nontarget.

*.Throughout.this.chapter.my.focus.is.on.woody.fruit,.shade,.and.ornamental.horticulture.species.


effects,.fitness,.potential.for.spread,.effect.on.endangered.species).is.very.costly.and.by. their.nature. imprecise,. requiring,. for. reasonable.estimates,. large.experiments.and.years.of.study.over.many.environments..For.pest-tolerant.crops.(i.e.,.those.with.plant.incorporated.protectants,.[PIPs]),.the.required.analyses.by.EPA.are.expected.to.be.even.more.costly.and.complex..Rarely.are.the.paybacks.to.developers.from.improved.horticultural.crops.sufficient.to.cover.all.of.these.large.up-front.costs.

DEREGULATED HORTICULTURAL VARIETIES DO NOT PROVIDE GENERAL MODELS

The.very.few.woody.horticultural.crops.that.appear.to.have.successfully.navigated.the. regulatory. maze. have. special. characteristics,. and. thus. provide. few. general.lessons..They.are.trees.that.have.genes.that.protect.against.a.major.viral.pest.and.make.no.actual.novel.pest-toxic.compound.(papaya.and.plum:.they.invoke.the.natu-ral.RNA.interference.mechanism),.and.also.cannot.spread. in. the.wild. to.any.sig-nificant.degree..The.GE.cold-tolerant.and.male-sterile.eucalypt,.now. in.extensive.field.trials.and.part.of.a.petition.for.deregulation,.is.also.dependent.for.its.approval.to.allow.flowering.and.commercial.planting.on.its.presumed.sterility.or.inability.to.spread.13. It. is.as.yet.unclear. if,. in.a.practical.and.affordable.way,.normally.fertile.horticultural.varieties.that.have.wild.or.feral.relatives.can.comply.with.regulations.and.obtain.regulatory.approval.for.commercialization.

CAUSES OF OUR STRINGENT REGULATORY SYSTEM

How.have.we,.in.the.Unites.States,.produced.a.regulatory.environment.that.appears.so.hostile.to.transgenic.innovation.in.horticultural.crops?.The.political.and.legal.his-tory.of.our.regulatory.framework.is.well.known,6,14.and.there.are.a.number.of.very.significant.political.issues15.that.appear.to.have.played.a.major.role.in.shaping.the.negative,.or.at.least.highly.divided,.public.view.of.crop.biotechnology..Major.sources.of.controversy.include:

. 1..The. relatively. new. and. major. roles. for. strong. patents. in. crop. breeding,.which. provide. no. breeder’s. rights. to. the. use. of. genetic. material. and. no.limits.on.ownership.of.genes.and.transgenic.plants.when.they.move.in.the.environment..This.appears.to.be.considered.an.overstep.or.an.outright.ethi-cal.transgression.to.many.

. 2..The.growing.role.of.multinational.corporations. in.biotechnology..This. is.in.no.small.part.due.to.the.costly.intellectual.patent.and.regulatory.land-scapes.discussed.above..Negative.attitudes.toward.these.corporations.and.their.dominance.in.the.development.of.commercial.biotech.crops.are.also.a. result.of. the. legacy.of. the.production.and.marketing.of.pesticides,.and.of.divisive.products.such.as.recombinant.bovine.somatotropin.(rBST),.by.these.companies.or.their.predecessors.

. 3..The. lack.of.direct.benefits. to.consumers.and. food.production/service.com-panies,.in.the.face.of.perceived.risks.to.people.or.retail.chains,.from.use.of.herbicides.and.pesticidal.molecules.in.the.current.major.transgenic.varieties.


. 4..Divided.scientific.advice.on.the.risks.versus.benefits.of.GE.in.relation.to.the.stringency.of.regulations..The.large.majority.of.ecologists.I.have.met.with,.including.during.services.on.National.Research.Council.panels,.show.a. strong. negative. attitude. toward. GE. of. crops,. whereas. most. breeders,.agronomists,.and.biotechnologists.seem.to.view.them.positively..The.con-cern.expressed.by.ecologists.is.prompted.in.no.small.part.by.the.commonly.made.analogy.between. transgenic.and. invasive.exotic.species..Moreover,.many.serious.invasive.plant.species.are.the.result.of.intentional.introduc-tions. from.the.horticulture. industry..Thus,.although. there. is.only. limited.biological.homology.between.a.novel. invasive.organism.and.introduction.or.modification.of.one.or.a.few.genes.in.a.familiar.organism,.the.legacy.of.exotic.species.problems.creates.a.climate.that.dictates.extreme.precaution.and.concern.

. 5..The.growing.popularity.of.organically.certified.forms.of.agriculture.and.its.strong.direct.and.indirect.campaigns.against.transgenic.breeding.methods.and.varieties.as.dangerous.and.“unnatural.”

. 6..Waning. trust. in. government. and. government. organized. science. pan-els.to.make.wise.judgments.about. the.safety.of.novel.genes.in.foods.and.environment.

. 7..The.strong.political.and.legal.pressures.for.stringent.regulations.from.well-funded.nongovernmental.organizations.that.are.opposed.to,.or.highly.con-cerned.about,.GE.crops.

All.of. these. are. clearly.major.problems. for. any.efforts. to.produce.what.GE.crop.developers.would.view.as.more.balanced.science-based.regulations..However,.I.will.discuss.what.I.see.as.deeper,.more.foundational.issues.that.I.believe.have.contributed.to.making.the.regulatory.system.such.a.difficult.barrier.to.progress.in.horticultural.biotechnology.

preSumptIon of harm from tranSGenIc methodS

Thomas.Jefferson.is.widely.quoted.as.having.said.that.“the.greatest.service.which.can.be.rendered.any.country.is.to.add.a.useful.plant.to.its.culture.”*.Clearly,.some-thing. has. changed. since. the. era. of. transgenic. biotechnology. began.. Whereas. all.products.of.traditional.breeding.are.considered.generally.regarded.as.safe.(GRAS),.all.varieties.produced.using.transgenic.methods.are.in.effect.considered.the.opposite,.that.is,.hazardous.until.“proven”.safe.†.This.is.despite.the.common.scientific.knowl-edge,. and.FDA. rulings,. that. the. transgenic.method.per. se. is.not.more. risky. than.conventional.breeding.methods.such.as.inbreeding,.wide.hybridization,.and.muta-genesis..Moreover,.the.established.legacy.of.plant.breeding.includes.importations.of.exotic.plants.that.can.spread.widely;.enabling.agriculture.and.humans—arguably.the.

*.Thomas.Jefferson,.“A.Memorandum.of.Services.to.My.Country,”.September.2,.1800.(PTJ,.32:124)..Polygraph. copy. at. the. Library. of. Congress.. http://wiki.monticello.org/mediawiki/index.php/Useful_plant_%28Quotation%29

†. It.is.not.in.fact.possible.to.prove.the.absence.of.any.risk.


most.environmentally.destructive.forces.on.the.planet—to.migrate.around.the.globe.as.plants.have.been.and.are.bred.for.adaptation.to.new.regions..Clearly,.the.distinct.regulatory.treatments,.which.impose.such.a.striking.double.standard.of.strong.regu-lation.versus.the.absence.of.regulation,.are.a.legacy.of.history..It.is.fair.to.say.that.if.conventional.breeding.were.forced.to.undergo.the.same.scrutiny.as.does.GE,.much.of.it.would.not.be.legally.permissible.today..At.a.minimum,.conventional.breeding.would.all.be.subject.to.much.higher.costs.and.long.delays,.with.inestimable.penalties.for.yield.and.product.quality.improvement..It.is.also.very.likely.that.environmental.impacts.of.agriculture.would.be.far.greater,.as.the.amount.of.output.per.unit.area.of.land,.water,.and.fertilizer.would.certainly.be.far.lower.in.the.absence.of.vigorous.plant.breeding.programs.

envIronmental concernS prompt StronG reGulatIonS

The. pressing. environmental. problems. facing. society. are. another. motivation. for.strong.regulations.of.this.new.agricultural.technology..Whether.one.considers.climate.change,.non-point-source.pollution,.soil.erosion,.or.water.quality,.there.is.clearly.a.pressing.need.to.reduce.the.environmental.footprint.of.agriculture..However,.is.the.intensive. regulation.of.all. forms.of. transgenic.biotechnology,.and.only. transgenic.biotechnology.among.breeding.methods,. a. sensible.means. for.doing. this?.Such.a.practice.seems.especially.specious.in.that.the.environmental.benefits.of.transgenic.crops.have,.on.the.whole,.been.strongly.positive.to.date.(primarily.in.the.form.of.tillage.and.pesticide.ecotoxicity.reductions),16.yet.many.crops.with.similar.expected.benefits.have.not.made.it.to.market.at.all.12.Some.of.the.most.notable.examples.of.transgenic.crops.that,.though.developed.and.field.proven,.have.not.made.it.to.mar-ket,.are.horticultural.crops..These.include.virus-resistant.berries,.disease-resistant.apples,.and.disease-.and.insect-resistant.potatoes..All.of.these.would.have.reduced.pesticide.applications..Although.business.and.market.factors.also.contributed,.some-times.substantially,.to.decisions.not.to.commercialize.such.varieties,.the.overarching.hostile.regulatory.environment.made.the.business.proposition.marginal.at.the.outset,.especially. for. public. sector. breeders. and. smaller. companies.. It. is. not. difficult. to.argue.that.the.stringent.regulation.of.plant.biotechnology.has.had.the.opposite.envi-ronmental.consequence.of.what.was.intended.

famIlIar GeneS meet Same reGulatory revIew

Unfortunately,. the. “guilty. until. proven. innocent”. framework. applies. not. just. to.biotechnologies.that. impart.novel.properties,.such.as.new.kinds.of.pest.resistance.proteins.or.metabolites,.but.it.applies.to.all.cases.where.a.transgenic.method.is.used..Thus,.it.is.the.method,.not.the.actual.biological.novelty.of.the.new.gene.that.trig-gers.the.regulatory.system..As.a.result,.we.scrutinize.all.changes.from.the.method,.not. just. the. novel. property. imparted,. presuming. all. changes. are. hazardous. until.“proven”. otherwise.. This. means. that. mutagenesis. due. to. the. gene. insertion. pro-cess.is.intensely.scrutinized—though.mutagenesis. in.various.forms.has.been.long.applied.in.conventional.breeding..The.nature.of.the.insertion.site.and.any.changes.in.general.plant.chemistry.are.studied. in.detail,.not. just. transgene.expression.and.


its.associated.phenotypes..Regulatory.agencies.apply. similar. scrutiny.even.where.genes. from.sexually.compatible.or.closely. related. species. are. transferred,. or.nor-mally.expressed.genes.are.attenuated,.shut.down,.or.mutated.(often.called.cisgenics.or.intragenics)..Incremental.changes.to.existing.phenotypes.such.as.cold.hardiness,.reduced.rate.of.ripening,.and.pest.resistance—even.when.due.to.modified.expression.of.native.genes—are.treated.as.ecologically.novel.traits.if.GE.is.involved..Canada.has.attempted.to.put.in.place.a.method-neutral.regulatory.system.that.covers.GE.as.well.as.conventional.breeding,.called.the.“plants.with.novel. traits”.system.(http://www.inspection.gc.ca/english/sci/biotech/gen/terexpe.shtml).. In. practice,. however,.it.appears.to.regulate.all.forms.of.GE.crops.similarly.to.method-based.systems.in.the.United.States,.while.upsetting.conventional.breeders.when.their.new.varieties.come.under.regulation.for.the.first.time.

The. intense. scrutiny. compelled. by. the. GE. method. creates. serious. legal. and.epistemic.problems..How.can.we.prove.safety.when.the.variances.for.the.system.we.regard.as.GRAS.are.so.extraordinarily.wide?.Food.is.known.to.contain.“tox-ins”.and.contaminants.whose.concentrations.vary.widely.and.can.cause.adverse.effects.in.high.dosage.tests,.and.breeders.often.make.crosses.with.wild.relatives.that. have. not. been. widely. consumed. for. food. and. may. even. be. poisonous.. For.example,. if.a.modified.crop.has.chemical.components.whose.levels.are.elevated.but.are.still.within.the.enormous.range.of.variation.seen.among.conventional.vari-eties,. hybrids,. and. environments. (e.g.,. of. a. natural. alkaloid. or. terpenoid),. such.changes.might.not.be.considered.safe.or.desirable.from.a.toxicological.viewpoint.(i.e.,. in. light.of. the.known.biochemical.actions.of. those.compounds)..How.such.cases.would.fare.under.legal.scrutiny.in.the.EU.where.the.Precautionary.Principle.prevails,.or.under.legal.challenge.in.the.United.States.where.FDA.could.declare.such.changes.as.adulteration.if.supported.by.toxicological.science,.is.unclear..In.addition,.because.it.is.logically.impossible.to.prove.the.absence.of.a.risk,.it.is.very.difficult.to.scientifically.declare.safety.for.the.whole.organism,.especially.for.crops.or.where.gene.products.that.do.not.fit.the.standard.toxicology.model.(i.e.,.where.they.have.complex.phenotypic.changes,.and.thus.simple.dose–response.tests.per-formed.in.the.laboratory.are.not.meaningful)..This.has.led.to.continued.political.debate.over.how.safe.is.safe.enough,.including.over.whether.“substantial.equiva-lence”.is.a.satisfactory.regulatory.attribute.

This.indiscriminant.system.also.means.that.gene.transfers.from.related.species,.such.as. the. transfer.of.a.pest. resistance.gene.from.a.wild.relative,.faces. the.same.regulatory.system..Why.should.a.gene.introduced.through.hybridization.from.a.wild.relative,.with.its.usual.linkage.drag,.be.considered.less.risky.than.the.same.gene.iso-lated.and.introduced.using.GE.methods.and.accompanied.by.a.well-studied.vector.and.associated.sequences?.In.other.words,.why.are.they.regulated.at.all,.when.the.same.or.a.similar.result.can.be.produced.with.conventional.breeding,.though.with.less. precision?. Although. it. seems. likely. that. regulators. will. require. less. data. for.low.novelty.transfers.compared.to.wide.phylogenetic.transfers.or.newly.synthesized.genes,.just.by.entering.the.highly.politicized.regulatory.arena—where.agencies.sim-ply.respond.to.each.case.as.they.come.in.the.door—the.costs,.delays,.and.outcomes.are.unpredictable,.and.thus.can.result.in.costly.delays.or.roadblocks..The.unpredict-ability.of.the.regulatory.process.is.a.very.serious.problem.for.companies,.investors,.


and.grant.agencies.choosing.among.research.and.technology.transfer.options..They.have.little.idea.what.the.cost.and.time.delays.will.really.be,.and.agencies.provide.no.guarantees.up.front.

ENVIRONMENTAL STUDIES ARE SERIOUSLY COMPROMISED BY REGULATIONS

From. an. environmental. viewpoint,. the. presumption. of. harm. creates. even. larger.problems.and.regulatory.obstacles..As.discussed.above,. it. is.very.difficult. to.pre-dict. ecological. impact. from. small. studies. that. are. performed. under. containment..Although. simple. extrapolations. are. possible. when. the. toxicology. model. applies.(as.with.a.pest.toxin.whose.effects.on.wild.species.can.be.roughly.estimated.in.the.greenhouse.or.short-term.field.study),.even.this.simple.case.is.fraught.with.difficulty..Such.studies.say.little.about.the.effect.of.such.genes.under.varying.abiotic.and.biotic.environments.in.the.field,.and.they.cannot.predict.in.any.meaningful.way.what.might.happen.in.a.future.dominated.by.climate.change,.nor.can.assess.how.biological.com-munities.will.adapt.and.evolve.in.response.to.the.new.gene.product.and.phenotype.

In.other.words,.under.current.regulatory.constraints.we.are.unable.to.adequately.answer.any.of.the.big.questions.about. transgene.impacts..For.example,.how.will.the.myriad. species. that.might.be. exposed. to. a.naturalized. transgene-expressing.plant.be.affected.over.time?.Can.the.novel.gene/toxin.have.so.strong.an.effect.as.to.drive.an.herbivorous.species.to.extinction,.or.will.most.species,.or.other.ecosys-tem.adjustments,.attenuate.such.effects.over.evolutionary.time?.Do.the.perturba-tions.matter.given.the.very.large.effects.of.agriculture,.breeding,.climate-induced.variation,. anthropogenic. change,. and. exotic. species. in. general?. How. often. will.genes.of.value.in.the.management.of.simple.agricultural.systems,.or.as.a.result.of.crop.domestication.for.human.tastes.in.food.and.fiber,.be.ecologically.powerful.in.diverse.wild.or.feral.systems?.The.point.is.that.while.the.goal.of.regulations.is.to.force.informed.and.wise.decisions,.the.reality.is.that.the.process.imposed,.with.its.high.costs.and.legal.risks,.appears.to.do.more.harm.than.good.by.impeding.most.forms. of. transgenic. research. and. development. with. horticultural. crops.. A more.efficient.option.might.be.to.exempt.the.transgenic.method.and.small.or.contained.field. trials. from. regulation,. but. require. substantially.novel. gene. products—such.as. phylogenetically. novel. and. broadly. effective. toxins,. or. pharmacologically.active.molecules.that.result.from.synthetic.biology.or. long.distance.phylogenetic.transfers—to. undergo. regulatory. review. prior. to. large. scale,. uncontained. field.research.or.commercial.use..We.have.provided.more.specific.recommendations.for.regulatory.reform.elsewhere.3,4,14

PRESUMPTION THAT STASIS IS DESIRABLE

The. USDA. regulations. for. transgenic. biotechnology. treat. all. transgenic. innova-tions.as. risks..The.benefits.of. transgenic.plants.are.not. formally.considered..This.framework. is.not. surprising.given. the. evolution.of. the. current. regulatory. scheme.from. a. plant. pest. oriented. system.17. The. framework. therefore. implicitly. assumes.that.crop.species.and.their.wild.relatives.that.might.receive.transgenes.via.gene.flow.


are.superior.in.their.present.form.to.what.they.would.be.with.the.modified.genes—unless.a.strong.case.can.be.made.otherwise..This.presumption.(and.the.underlying.conservation-oriented.value.that.supports.it).seems.reasonable,.until.one.considers.the.very.strong.barrier.it.also.poses.to.the.transgenic.use.of.pest.or.stress.resistance.genes.to.promote.the.health.of.horticultural.woody.plants.in.cases.where.they.have.wild.or.feral.relatives.(as.nearly.all.do)..If.the.gene.disperses,.the.genetic.diversity.and.fitness.of.wild.relatives.might.be.increased.to.some.degree..This.might.in.fact.be.beneficial.because.woody.horticultural.and.forest.species.are.often.foundational.members.of.terrestrial.ecosystems,.providing.much.of.the.structural.habitat.and.pri-mary.productivity..Thus,.some.increased.vigor.and.adaptability.would.generally.be.expected.to.be.ecologically.advantageous,.not.disadvantageous..In.addition,.many.woody.species.are.under.serious.threat.from.climate.change.and.the.emergence.or.invasion.of.newly.epidemic.and/or.exotic.pests,18.and.thus.could.benefit.from.genes.that.increased.their.resilience.or.pest/stress.tolerance..Of.course,.in.cases.where.a.wild.relative.is.already.a.problematic.exotic.species.that.is.having.a.strong.negative.environmental.impact,.such.improvements.of.vigor.would.not.be.considered.desir-able..Such.cases.could.be.specially.identified.and.disallowed.(e.g.,.by.presence.on.a.noxious.weed.list),.rather.than.imposing.a.blanket.preclusion.to.gene.flow.to.wild.relatives.as.a.result.of.the.transgenic.method.

The.core.regulatory.and.ecological.problem.is.the.extreme.difficulty.in.predicting.the.outcome.of.transgene.introductions.in.terms.of.their.ultimate.ecological.impact.in.advance,.without.actual.field.releases.and.monitoring.over.many.years.and.sites..This,.however,.is.very.costly,.especially.where.strong.containment.must.be.imposed.during. these. trials.. As. stated. above,. this. is. a. reasonable. requirement. for. species.with.high.risk.relatives.such.as.a.Johnson.grass.or.a.scotch.broom,.but.unfortunately.under.the.current.operational.“presumption.of.harm”.such.precaution.is.applied.to.all.transgenes.and.species..This.makes.commercialization.of.each.transgenic.prod-uct.a.multidecade.and.multimillion.dollar.undertaking,.even.when.pest.resistance.genes.from.related.plant.species.are.used,.and.appears.to.make.transgenic.solutions.prohibitive.except. in.special.cases. (e.g.,.American.Chestnut,. a.dominant. tree. that.was.driven.near.to.extinction.and.has.strong.private.and.public.foundation.support.for. the.use.of.biotechnology.for. its.restoration).19.Given. the.growing.pace.of.such.serious.threats.to.wild.and.cultivated.trees,.it.would.appear.that.new,.expedited.regu-latory.options—such.as.exemptions.for.species.in.crisis.and/or.genes.from.related.species—are.critically.needed.

CONSEQUENCES OF SIMPLE DEFINITIONS OF CLEAN AND GREEN

Finally,.a.major.impediment.to.the.use.of.transgenic.methods.appears.to.reside.in.the.blanket.manner.with.which.society.seems.to.categorize.technologies.as.good.or.bad..Organically.certified.food.is.currently.considered.by.much.of.the.public.to.be.greener,.safer,.and.thus.superior. to.conventionally.produced.food..Yet.when.scru-tinized.it.has.not.shown.any.consistent.advantages.for.food.safety.or.nutrition,.and.its.net.environmental.benefits.are.also.questionable.(e.g.,.when.full.life.cycle.stud-ies.of.nitrogen,.land.use,.runoff,.soil.erosion,.transport,.and.energy.consumption—and. even. pesticide. ecotoxicity. in. some. cases—are. considered).20–22. Nonetheless,.


perceived.economic.harms.to.marketing.organic.products.as.a.result.of.“contamination”.of.organic.food.by.GE,.even.when.at.very.low.levels,.have.prompted.successful.law-suits..The.courts.have.viewed.organic.agriculture.as.an.environmental.good,.whose.possible.harm.thus.requires.careful.consideration.via.an.environmental.impact.state-ment..Such.a.ruling.for.alfalfa.has.resulted.in.its.withdrawal.from.the.marketplace,23.and.a.similar.case. is.pending.for.sugar.beet..These. legal.precedents.and.the.high.costs. they. impose. are. likely. to. continue. to. slow,. and. in.many. cases. will. prevent.development.of.GE.crops..Unfortunately,.these.legal.decisions.appear.to.be.informed.by.a.popular,.rather.than.a.scientific,.view.of.the.relative.environmental.value.of.GE.versus.organically.certified.food..Simple.green.labels.that.presume.GE.is.bad.and.any.GE.“contamination”.of.“green”.products.is.bad—when.uncritically.accepted.by.courts.and.a.large.section.of.the.public—pose.considerable.challenges.to.revision.of.the.current.regulatory.system.

CONCLUSIONS

Regulatory. change. that. would. decriminalize. the. GE. process. is. needed. to. move.forward..But.how.can.that.happen?.It.could.be.motivated.by.growing.urgency.for.improved. food. production,. as. expanded. uses. of. crops. for. bioenergy,. and. climate.change-induced.crop.losses,.continue.to.drive.up.food.prices..Change.may.also.be.motivated.by.the.many.humanitarian.GE.projects.underway.for.the.developing.world,.of.which.Golden.Rice.is.the.best.known..A.single.major,.highly.publicized.success.could.shift.public.opinion.substantially..Change.may.also.be.motivated.by.informed,.popular,.and.powerful.thought.and.environmental.leaders,.such.as.Stewart.Brand.and.Michael.Specter,.who.have.embraced.the.benefits.and.debunked.the.myths.surround-ing.GE.crops.and.other.environmental.and.scientific.technologies.22,24.However,.as.discussed.above,.due.to.the.many.layers.of.national.and.global.regulations,.and.the.strong.political.influences.on.them,.the.timescale.of.change.may.be.on.the.order.of.decades.or.more.

For. change. to. ultimately. occur. scientists. must. play. a. key. role.. By. educating.decision.makers.and.the.public.in.understandable,.contextually.relevant,.and.gen-erationally. appropriate. forms,. and. by. taking. an. active. part. in. providing. public.input.to.regulatory.decisions,.biotechnologists.can.help.to.craft.a.new.era.of.intel-ligent,.discriminating,.science-based.regulations..Transgenic.biotechnology.is.too.powerful.a.tool.to.surrender..Our.precarious.world,.the.billions.of.needy.people,.and.threatened.nonhuman.species.need.it.to.become.a.potent.and.central.part.of.the.crop.technology.toolkit.

REFERENCES

. 1.. Li,. J.. et. al.,. Stability. of. herbicide. resistance. over. 8. years. of. coppice. in.field-grown,.genetically.engineered.poplars..Western Journal of Applied Forestry,.23,.89,.2008.

. 2.. Strauss,.S.H..et.al.,.Ten.lessons.from.15.years.of.transgenic.Populus.research..Forestry,.77,.455,.2004.

. 3.. Bradford,.K.J..et.al.,.Regulating.transgenic.crops.sensibly:.Lessons.from.plant.breeding,.biotechnology.and.genomics..Nature Biotechnology,.23(4),.439,.2005.


. 4.. Strauss,.S.H.,.Genetic.technologies:.Genomics,.genetic.engineering,.and.domestication.of.crops..Science,.300(5616),.61,.2003.

. 5.. Strauss,.S.H..et.al.,.Strangled.at.birth?.Forest.biotech.and.the.convention.on.biological.diversity..Nature Biotechnology,.27,.519,.2009.

. 6.. APHIS,.Programmatic.Environmental.Impact.Statement.(for.new.proposed.regulations)..http://www.aphis.usda.gov/publications/biotechnology/content/printable_version/fs_programmatic_eis.pdf,.2007..(Accessed.January.27,.2011).

. 7.. Devos,.Y..et.al.,.The.interplay.between.societal.concerns.and.the.regulatory.frame.on.GM.crops.in.the.European.Union..Environmental Biosafety Research,.5(3),.127,.2006.

. 8.. Bucchini,.L..and.L.R..Goldman,.Starlink.corn:.A.risk.analysis..Environmental Health Perspectives,.110(1),.5,.2002.

. 9.. Vinluan,. F.,. Genetically. modified. rice. leads. to. ruling. against. Bayer. CropScience..Triangle Business Journal,. December. 7,. 2009.. http://www.bizjournals.com/triangle/.stories/2009/11/30/daily70.html.(Accessed.January.27,.2011).

. 10.. Bryson,.N.,.APHIS.programmatic.EIS–Implications.for.crop.biotechnology.regulation..In.Farm Foundation Conference,.Washington,.DC,.January.16–17,.2008.

. 11.. Kinderlerer,.J.,.The.Cartagena.protocol.on.biosafety..Collection Biosafety Reviews,.4,.12,.2008.

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. 13.. APHIS,.ArborGen,. LLC;.Availability. of. an. environmental. assessment. for. controlled.release.of.a.genetically.engineered.Eucalyptus.Hybrid..74.Federal Register,.74,.26648–26719,.June.3,.2009.

. 14.. Strauss,.S.H..et.al.,.Far-reaching.deleterious.impacts.of.regulations.on.research.and.envi-ronmental.studies.of.recombinant.DNA-modified.perennial.biofuel.crops.in.the.USA..BioScience,.60(9),.729,.2010.

. 15.. Herring,.R.J.,.Opposition.to.transgenic.technologies:.Ideology,.interests.and.collective.action.frames..Nature Reviews Genetics,.9(6),.458,.2008..(Accessed.January.27,.2011).

. 16.. NRC,.Impact.of.genetically.engineered.crops.on.farm.sustainability.in.the.United.States..http://www.nap.edu/catalog/12804.html,.2010,.p..240..(Accessed.April.20,.2011).

. 17.. Medley,. T.L.. and. S.L.. McCammon,. Strategic. regulations. for. safe. development. of.transgenic.plants..In.Biotechnology,.H.-J..Rehm.and.G..Reed,.eds..Weinheim:.Federal.Republic.of.Germany,.1995.

. 18.. Chornesky,.E.A..et.al.,.Science.priorities.for.reducing.the.threat.of.invasive.species.to.sustainable.forestry..BioScience,.55(4),.335,.2005.

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263

13 Virus-Resistant Transgenic Horticultural Crops: Safety Issues and Lessons from Risk Assessment Studies

Jonathan E. Oliver, Paula F. Tennant, and Marc Fuchs

CONTENTS

Introduction.............................................................................................................264Pathogen-Derived.Resistance.and.Engineered.Resistance.to.Viruses.in.Plants.... 265RNA.Silencing.and.Engineered.Resistance.to.Viruses.in.Plants.......................265

Safety.Issues.and.Risks...........................................................................................266Transencapsidation.............................................................................................266Recombination................................................................................................... 267Gene.Flow.to.Free-Living.Relatives..................................................................268Effects.on.Nontarget.Organisms........................................................................268Allergenicity.and.Human.Health.Effects...........................................................269Durability.and.Specificity.of.Engineered.Resistance.to.Viruses.in.Plants......... 270

Breakdown.of.Engineered.Virus.Resistance................................................. 270Specificity.of.Engineered.Virus.Resistance................................................... 271

Examination.of.Risks.Associated.with.Commercialized.Transgenic.Horticultural.Crops................................................................................................. 271

Squash.Resistant.to.Cucumber Mosaic Virus,.Zucchini Yellow Mosaic Virus,.and.Watermelon Mosaic Virus ................................................................ 272

Background................................................................................................... 272Risk.Studies................................................................................................... 272

Papaya.Resistant.to.Papaya Ringspot Virus ...................................................... 273Background................................................................................................... 273Risk.Studies................................................................................................... 274

Tomato.and.Sweet.Pepper.Resistant.to.Cucumber Mosaic Virus...................... 275Background................................................................................................... 275Risk.Studies................................................................................................... 276


INTRODUCTION

Horticultural.crops.were.the.first.transgenic.crops.commercialized.in.the.United.States.in. the.mid-1990s..Among.the.first.of. these.were.virus-resistant. transgenic.summer.squash.(Cucurbita pepo.ssp..ovifera.var..ovifera.L.)..The.first.transgenic.squash.cul-tivars,.deregulated.in.1994,.were.resistant.to.Zucchini yellow mosaic virus.(ZYMV).and.Watermelon mosaic virus.(WMV).1–3.Other.transgenic.summer.squash.cultivars.resistant. to. ZYMV,. WMV,. and. Cucumber mosaic virus. (CMV). were. released. in.19962–4. followed.by.papaya. (Carica papaya.L.).genetically.modified.for. resistance.to.Papaya ringspot virus.(PRSV).in.1998.2,5,6.To.date,.virus-resistant.summer.squash.and.papaya.are.the.only.transgenic.horticultural.crops.that.are.commercially.released.in. the. United. States,. along. with. Bt. sweet. corn. (Zea mays. L.).7. Potato. (Solanum tuberosum.L.).resistant.to.Potato virus Y.(PVY),.Potato leafroll virus.(PLRV).and.the.Colorado.potato.beetle.were.released.in.1998,.but.were.withdrawn.from.the.market.almost.immediately.thereafter.due.to.anti-biotechnology.campaigns.and.international.trade. barriers.8. In. the. People’s. Republic. of. China,. transgenic. tomato. (Solanum esculentum.L.).and.sweet.pepper.(Capsicum annuum.L.).resistant.to.CMV.and.papaya.resistant.to.PRSV.have.also.been.released.9

The. virus-resistant. transgenic. horticultural. crops. commercially. available. have.been.developed.by.applying.the.concept.of.pathogen-derived.resistance.10.Pathogen-derived.resistance.refers.to.the.use.of.a.pathogen’s.own.genes.to.confer.resistance.in.a.host.to.that.pathogen..In.the.case.of.the.horticultural.crops.released.to.date.in.the.United.States,.engineered.virus.resistance.has.been.achieved.through.expression.of.viral.coat.protein.genes.3,6

The.development.and.release.of.transgenic.horticultural.crops,.in.particular.those.engineered. for. virus. resistance,. have. raised. potential. safety. issues. regarding. their.impact. on. the. environment. and. human. health.2,11–13. Similarly,. concerns. have. been.expressed.over.the.release.of.horticultural.crops.expressing.cry.toxin.genes.from.the.bacterium,. Bacillus thuringiensis. (Bt).14,15. Over. the. years,. a. significant. amount. of.research.has.been.done.to.address.safety.issues.and.examine.potential.risks..In.this.chapter,.we.provide.a.synopsis.of.transgenic.horticultural.crops,.in.particular.virus-resistant.transgenic.crops.with.the.major.emphasis.on.those.that.are.currently.available.in.commerce..We.also.examine.the.mechanisms.underlying.engineered.virus.resis-tance.and.discuss.potential.safety.issues.with.this.technology..We.then.examine.risk.assessment.research.by.focusing.on.commercial.crops.for.which.data.from.realistic.

Examination.of.Risks.Associated.with.Transgenic.Plum.Which.Is under Consideration.for.Deregulation................................................................. 276

Background........................................................................................................ 276Risk.Studies........................................................................................................ 277

Overview.of.Risk.Lessons.of.Transgenic.Horticultural.Crops............................... 278Perspectives............................................................................................................. 279

Introduction........................................................................................................ 279Future.Trends.....................................................................................................280

Conclusions............................................................................................................. 281References............................................................................................................... 281

265Virus-Resistant Transgenic Horticultural Crops

field. studies. are. available,. thereby. using. each. released. crop. as. a. case. study. in. our.examination.of.the.safety.issues.associated.with.transgenic.horticultural.crops..Finally,.we. summarize. lessons. from. risk. assessment. research. and. evaluate. whether. safety.issues.account.for.the.limited.number.of.horticultural.transgenic.crops.released.to.date.

pathoGen-derIved reSIStance and enGIneered reSIStance to vIruSeS In plantS

The. first. report. on. engineered. resistance. to. viruses. in. plants. was. published. in.1986.16. These. researchers. noted. that. tobacco. plants. expressing. the. coat. pro-tein. gene. of Tobacco mosaic virus. (TMV).exhibited.delayed. infection. following.mechanical. inoculation.with.TMV..This.observation.conformed. to. the.concept.of.pathogen-derived.resistance.that.had.been.postulated.earlier.10.Following.the.initial.breakthrough.by.Abel.et. al.,16.viral. coat.protein.genes. from.various.viruses.were.introduced.into.numerous.economically.important.crop.species.in.hopes.of.achieving.resistance.17.It.was.initially.believed.that.resistance.was.provided.by.the.viral.protein.itself.via.a.mechanism.involving.excess.plant-expressed.coat.protein.that.interfered.with.the.uncoating.step.in.viral.replication.18.However,.it.soon.became.apparent.that.resistance.could.be.achieved.in.transgenic.plants.producing.low.or.undetectable.lev-els. of. coat. protein.19. Further. observations. indicated. that. the. mechanism. involved.degradation.of.the.transgene-derived.messenger.ribonucleic.acid.(mRNA).into.small.fragments.in.a.sequence-specific.manner.20–23

rna SIlencInG and enGIneered reSIStance to vIruSeS In plantS

Extensive. research. has. shown. that. engineered. virus. resistance. in. transgenic. plants.works. primarily. through. the. antiviral. pathways. of. the. mechanism. known. as. RNA.silencing..This.mechanism.regulates.the.expression.of.genes.at.the.RNA.level.follow-ing.RNA.transcription.by.the.host.plant..RNA.silencing.is.triggered.by.the.replica-tion.of.genomic.viral.RNA.within. the.host.cell.18,21,24.Following.entry. into. the.cell,.most.viruses.form.double-stranded.RNA.(dsRNA).intermediates.during.their.replica-tion..These.dsRNA.are.recognized.by.host.RNase.III.Dicer-like.enzymes.and.cleaved.into.short.fragments.called.small.interfering.(si).RNA.(∼21.nts)..These.fragments.then.associate.with.the.RNA-induced.silencing.complex.(RISC),.which.proceeds.to.target.homologous.RNA.sequences.within. the.cell..Once. identified.by. the.RNA.silencing.machinery,.these.target.RNA.sequences.are.similarly.cleaved.18,21,23,24.In.this.way,.the.host.cell.severely.limits.or.entirely.prevents.viral.replication,.resulting.in.resistance..Though.originally.identified.in.plant–virus.interaction.studies,.RNA.silencing.has.been.shown.to.be.present. in.a.wide.range.of.organisms. including.humans,.plants,.nema-todes,.and.fruit.flies.and.is.believed.to.function.as.a.sort.of.primitive.immune.system.25

The.activation.of.these.cellular.viral.defenses.within.host.plants.is.believed.to.explain.the.long-recognized.phenomenon.of.recovery.from.viral.symptoms.seen.in.the.upper.leaves.of.some.infected.plants,.as.it.has.been.shown.that.a.silencing.sig-nal.can.move.systemically.within.the.plant,.activating.silencing.ahead.of.the.viral.replication.advance.26


Lending.further.support.to.the.importance.of.the.RNA.silencing.system.in.plants.is.the.discovery.that.most.virus.families.encode.one.or.more.proteins.that.suppress.silencing.21,24,27–29.Viral. suppressors.have.been.shown. to. interact. in.multiple.ways.with. the. RNA.silencing. machinery. to.prevent. effective. control.of. the. expression.of.the.viral.genome.within.the.host..One.such.example.is.the.HC-Pro.protein.from.PVY..This.protein.binds.the.siRNAs.produced.by.the.cell’s.RNA.silencing.machin-ery,. thereby. squelching. the. signal,. which. effectively. suppresses. RNA. silencing.within.the.host.cell.30.This.not.only.can.allow.for.continued.PVY.replication,.but.has.also.been.shown.to.be.primarily.responsible.for.the.observed.phenomenon.of.viral.synergism.where.two.unrelated.viruses,.when.co-infecting.the.host.plant,.can.exhibit. more. severe. symptoms. than. either. virus. on. their. own.. Experiments. with.PVY.and.Potato virus X.(PVX).have.shown.that.HC-Pro.of.PVY.can.suppress.host.defenses.and.allow.for.PVX,.which.very.weakly.suppresses.silencing.on.its.own,.to.replicate.and.produce.much.more.severe.symptoms.than.either.PVX.or.PVY.cause.on.their.own.13

The. fact. that.RNA.silencing. is. a.nucleotide. sequence-based. resistance.mecha-nism.has.unique.implications.for.risk.assessment.and.the.development.of.new.virus-resistant.transgenic.plants..This.feature.is.discussed.in.the.next.section.

SAFETY ISSUES AND RISKS

Due. to. the.expression.of.viral. gene. constructs. to.provide. resistance. to.viruses. in.transgenic.plants,.there.are.unique.safety.issues.associated.with.this.technology2,11–13.versus.many.of.the.other.commercialized.transgenic.crop.plants..Among.these.are.the. risks. of. viral. recombination. and. transencapsidation.. However,. not. all. of. the.potential.risks.associated.with.this.technology.are.unique.to.virus-resistant.transgenic.plants..Risks.also.associated.with.other.transgenic.plant.technology.including.gene.flow. to. free-living. relatives,. allergenicity. and. other. human. health. effects,. break-down.of.resistance,.and.effects.on.nontarget.organisms.also.need.to.be.assessed.with.regard.to.engineered.resistance.against.viruses..In.the.following.section,.we.examine.each.of.these.areas.of.concern.and.assess.their.scientific.merits.

tranSencapSIdatIon

One.of.the.concerns.associated.with.virus-resistant.transgenic.crops.is.the.potential.that.viral.capsid.proteins,.when.produced.in.a.transgenic.host.plant,.may.transencap-sidate.the.genome.of.a.challenge.virus.2,11–13.Since.viral.capsid.proteins.play.roles.in.such.diverse.processes.as.movement.within.the.host,.replication,.suppression.of.gene.silencing,.and.vector.transmission.specificity,31.it.is.plausible.that.a.transencap-sidated.virus.may.have.altered.properties..If.the.coat.protein.of.a.virus.vectored.by.aphids,.for.example,.were.transgenically.produced.within.a.host.plant,.it.is.conceiv-able.that.an.aphid.non-transmissible.virus.could.be.encapsidated.within.the.coat.pro-tein.derived.from.the.transgene..This.transencapsidated.virus.(with.the.coat.protein.from. the.aphid-borne.virus.and. the.genome.of. the.aphid.non-transmissible.virus).might.then.acquire.the.characteristics.of.an.aphid-borne.virus,.potentially.allowing.it.to.move.more.expeditiously.into.new.host.plants—possibly.even.to.plant.species.


that.the.parent.viruses.heretofore.had.not.come.into.contact.with..Transencapsidation.has. been. shown. experimentally.11. However,. many. of. the. concerns. with. respect.to. transencapsidation. have. been. alleviated. by. our. current. understanding. of. RNA.silencing..This.is.true.because.when.RNA.silencing.is.active.(as.is.expected.if.the.transgenic.plants.exhibit.viral.resistance),.the.expression.of.viral-derived.proteins.is.regulated.posttranscriptionally,.with.little.to.no.detectable.capsid.protein.produced..In. the. case. of. untranslatable. coat. protein. transgenes,. this. concern. is. alleviated.altogether..Also,.since.the.interactions.between.viral.proteins.or.between.capsid.pro-teins.and.viral.genomes.is.often.very.specific,31.it.seems.unlikely.that.interactions.necessary.for.a.viral.coat.protein.to.aid.in.the.replication.or.movement.of. its.cor-responding.viral.genome.would.take.place.in.coordination.with.genomic.material.or.proteins.from.a.heterologous.virus..Finally,.it.is.unclear.how.these.risks.are.substan-tially.different.from.the.risks.already.present.when.a.host.plant.is.infected.with.two.distinct.viruses—which.has.been.shown.to.lead.to.transencapsidation11—and.how.transencapsidation.would.be.any.more.likely.to.occur.in.the.transgenic.case.than.in.the.non-transgenic.multiple.infection.scenario.2,11,12

Even. if. transencapsidation.did.occur,. it. is.questionable.how. it. could.result. in.viruses.with.permanently.altered.properties,. since. it. is.not.conceivable.how. the.movement.of.a.transencapsidated.virus.to.a.new.host.would.be.anything.other.than.a.so-called.dead.end,.since.(due.to.the.lack.of.a.coat.protein.from.the.heterologous.virus. in. the.new.host).all.new.viruses.produced. in. the.new.host.plant.would.be.encapsidated.within.their.own.capsid.protein.2,11–13.An.exception.would.be.if.the.new.host.were.transgenic.and.expressing.the.coat.protein.gene.of.a.closely.related.heterologous. virus.. In. any. event,. if. problems. with. transencapsidation. did. arise,.the.problem.could.be.eliminated.by.ending.the.cultivation.of.the.transgenic.crop.in.question.25

recombInatIon

Another.potential.risk.of.virus-resistant.transgenic.crop.plants.expressing.viral.genes.is. that. of. recombination. between.viral-derived. transgene.mRNA.and. the.genomic.RNA.of.an.infecting.virus.2,11–13.It. is. thought.that.this.type.of.recombination.could.potentially.give.rise.to.a.new.virus.strain.or.new.virus.species.possessing.characteris-tics.different.from.those.of.the.parent.viruses..Recombination.may.involve,.for.example,.an. incoming.virus.containing.a.viral.coat.protein.gene.whose.product. is.defective in.its.ability.to.be.vectored,.and.transcripts.of.a.homologous.viral.transgene.sequence.possessing.a.functional.copy.of.the.coat.protein.gene..Recombination.between.these.sequences.may.then.restore.vector.transmissibility..Unlike.the.risk.of.transencapsida-tion,.recombination.cannot.be.as.easily.dismissed.as.an.evolutionary.dead.end,.since.viral.progeny.identical.to.the.recombined.strain.could.be.produced.in.a.new.host.2,12,13.Recombination. between. transcripts. of. a. viral. gene. construct. in. a. transgenic. plant.and.an.incoming.virus.has.been.shown.by.several.groups.32.If.resistance.were.effec-tive.through.RNA.silencing,.the.occurrence.of.recombination.is.less.likely.unless.the.incoming.virus.were.a.related,.but.divergent,.isolate.of.the.virus.from.which.the.trans-gene.is.derived..In.that.case,.it.is.conceivable.that.the.incoming.virus.would.replicate.to.the.same.extent.as.in.a.susceptible.plant,.providing.opportunities.for.recombination..


Nevertheless,.it.is.unclear.how.recombination.would.be.more.likely.to.occur.between.a.viral. transgene.mRNA.and.an. infecting.virus. than.between. two.viruses. that.are.coinfecting.the.same.host.(a.common.situation).2,33–35.It.is.likewise.unclear.how.recom-bination.is.any.more.likely.to.occur.in.the.transgenic.case.than.in.the.case.of.cross-protection,.an.accepted.method.used.in.controlling.viral.diseases..Cross-protection.relies.on.the.use.of.mild.virus.strains.to.protect.plants.from.economic.damage.caused.by.closely.related.severe.virus.strains.36,37.Therefore,.RNA.molecules.of.distinct.viral.strains.have.ample.opportunities.to.recombine.in.cross-protected.plants..Though.not.shown.to.have.emerged.in.cross-protected.plants,.recombinant.viral.strains.resulting.from. recombination. between. Arabis mosaic virus. (ArMV). and. Grapevine fanleaf virus.(GFLV).have.been.observed.38.The.use.of.recombinant.mild.strains.of.ArMV.and.GFLV.to.cross-protect.against.GFLV.is.an.accepted.experimental.control.method.for.GFLV39.despite.their.deliberate.dissemination.in.the.environment.

Gene flow to free-lIvInG relatIveS

Another.concern,.not.unique.to.virus-resistant.transgenic.crops,.is.the.risk.of.transgene.flow.2,12,13.Transgene.movement.from.a.transgenic.crop.species.to.a.free-living.relative,.through.pollen.flow,.can.be.a.significant.concern..If.transgenes.provide.a.selective.advan-tage,.it.is.conceivable.that.hybrids.between.transgenic.and.free-living.compatible.species.might.acquire.a.fitness.benefit.and.eventually.a.competitive.edge.over.free-living.plants.40.In. an. extreme. scenario,. a. transgenic. hybrid. may. outcompete. free-living. plants. and.thereby.lead.to.an.elimination.of.entire.species,.land.races,.or.varieties.of.non-transgenic.plants..In.the.case.of.virus-resistant.transgenic.plants,.it.is.postulated.that.a.hybrid.pos-sessing.a.transgene.conferring.virus.resistance.might.outcompete.compatible.free-living.plants.and.become.established.in.the.natural.environment..Though.this.is.unlikely.to.be.due.to.an.increase.in.weediness.potential.of.the.transgenic.crop.itself,.such.a.scenario.may.have.far-reaching.environmental.consequences.in.the.case.of.free-living.species.2

The. likelihood.of.outcrossing.between. transgenic.crop.plants.and. free-living.relatives.depends.on.numerous.factors,.including.pollen.phenology,.pollen.compat-ibility,.and.spatial.proximity.40.Each.of.these.factors.could.vary.significantly.between.any.two.given.transgenic.crop.species.and.environments..Therefore,.evaluations.of.the.likelihood.of.gene.flow.(and.its.effects).must.be.carried.out.on.a.case-by-case.basis..Even.if.gene.flow.from.a.virus-resistant.transgenic.crop.to.free-living.relatives.could.readily.occur,.it.is.not.obvious.what.effect.this.would.have..Gene.flow.from.domesticated.crop.species.developed.through.traditional.breeding.practices.has.also.been.shown.to.occur,40–44.but.in.the.case.of.traditional.breeding,.problems.arising.from. gene. flow. to. free-living. relatives. have. not. been. seen,. and. it. is. unclear. how.the.effect.of.transgenic.virus.resistance.genes.would.be.substantially.different.from.those.of.resistance.genes.derived.from.traditional.breeding.

effectS on nontarGet orGanISmS

An.additional.concern.regarding.transgenic.horticultural.crops.is.their.potential.to.have. negative. effects. on. nontarget. organisms,2,45. for. example,. organisms. that. are.not. intentionally. targeted.by. the.disease.or. pest.management. strategy.. Effects.on.


nontarget.organisms.can.be.difficult. to.evaluate,.and.even.when.an.effect. is.seen,.it. can. be. difficult. to. assess. the. significance. of. this. observation.. With. respect. to.virus-resistant.transgenic.crops,.it.is.not.easy.to.identify.a.mechanism.which.might.result.in.an.effect.on.nontargets,.because.the.titer.of.the.transgene.protein.product.is. likely.manyfold. lower. than. the. amount.of. the. corresponding.viral. protein. in. a.non-transgenic.virus-infected.plant.46,47.In.addition,.given.the.fact.that.the.resistance.mechanism.is.likely.to.be.RNA.silencing,.in.many.cases.little.to.no.protein.is.likely.produced.at.all,.especially.in.the.case.of.untranslatable.transgenes..Furthermore,.crop.plants.derived.through.traditional.breeding.practices.have.been.shown.to.produce.a.wide.range.of.allergens.and.toxins.with.clear.effects.on.animals,.plants,.insects,.and.nematodes.that.may.come.into.contact.with.these.plants.during.their.lifetime.48

allerGenIcIty and human health effectS

Another.area.of.concern.regarding.transgenic.horticultural.crops.is.the.possibility.of.the.introduction.of.allergenic.proteins.into.the.food.supply.and.the.introduction.or. increase.in. the.production.of. toxic.compounds.48–52.Although.this.concern.also.applies.to.crop.varieties.developed.using.conventional.breeding.methods,.transgenic.products.have.received.strict.scrutiny.presumably.because.of.the.nature.of.the.trans-gene.proteins..The.underlying.concept.of.safety.evaluations.of.genetically.modified.foods.was.proposed.in.the.early.1990s53.and.is.based.on.comparative.analyses.of.the.transgenic.crop.with.the.conventionally.bred.parent.that.has.a.history.of.safe.use.(i.e.,.substantial.equivalence).54.Additionally,.safety.testing.of.whole.foods.in.animals.is.used.to.determine.toxicity.and.allergenicity.of.genetically.modified.foods.as.well.as.toxicity.testing.of.individual.proteins..The.latter.tests,.in.combination.with.nutritional.analysis,.are.regarded.as.more.sensitive.and.accurate.55,56.While.postmarket.monitor-ing.of.transgenic.food.crops.provides.data.on.patterns.of.human.nutritional.exposure.and.may.be.useful.in.confirming.premarket.risk.assessment.and.the.detection.of.rare.unintended.effects.on.health,.the.evaluation.is.not.regarded.as.a.component.of.risk.assessment.and.is.not.a.substitute.for.thorough.premarket.risk.assessment.57

Based.on.this.approach,.a.number.of.transgenic.crops.expressing.protein.prod-ucts,. such. as. those. derived. from. Bt-derived. toxins. (cry). and. marker. transgenes.(nptII,.uidA),.have.been.found.to.present.little.to.no.risk.to.food.or.feed.safety.2,58–62.Further,.analysis.of.potential.pleiotropic.effects.on.inherent.plant.toxins.and.antinu-trients.of.transgenic.plants.and.their.progenitor.cultivars.(e.g.,.maize,.rape,.tomato,.potato,. and.soybean).has. shown.minor. to.perceptible.variations,.albeit.within. the.ranges. documented. in. literature,. in. the. contents. of. these. compounds.63. Natural.biological.variation,.including.nutrient.variation,.of.individual.plants.grown.under.the.same.conditions.is.expected,.given.the.influences.of.differences.in.plant.devel-opment,.metabolism,. and.biotic. factors.63,64.However,. the.differences.between. the.transgenic.and. the.progenitor. cultivar.can. also.be.attributed. to. somaclonal.varia-tion,. given. that. the. transformation. of. many. crops,. including. papaya,. involves. an.adventitious. regeneration. protocol,. and. in. some. cases,. 2,4-dichlorophenoxyacetic.acid,65–68. a.plant.growth. regulator.known. to. introduce.genetic.mutations,. is.used..Nonetheless,.backcrossing.to.the.original.parental.variety.(and.selecting.progeny.with.appropriate.traits).effectively.eliminates.composition.alterations.caused.by.tissue.


culture.methods.69.It.is.important.to.also.note.that.variation.in.composition.is.not.limited.to.transgenic.crops.generated.by.recombinant.DNA.technologies.and.tissue.culture.methods..Nutritional.variation.has.been.reported.for.a.number.of.plant.prod-ucts.derived.from.conventionally.bred.varieties,.and.ranges.for.most.of.the.compo-sitional.variables.are.available.in.the.literature.70.Variation.in.conventional.crops.is.attributed.to.genetics.as.well.as.preharvest.conditions,.maturity.at.harvest,.harvest-ing.methods,.postharvest.handling,.and.storage.conditions.

Although.numerous.animal.studies.have.been.conducted.with.transgenic.crops.carrying. cry. insecticidal,. cowpea. trypsin. inhibitor,. phytase,. and. snowdrop. lec-tin. genes,58. there. are. only. a. few. published. studies. on. the. safety. assessment. of.whole.foods.derived.from.transgenic.crop.plants.transformed.with.viral.coat.pro-tein.genes..Presumably.viral.coat.proteins.are.not.regarded.as.potential.allergens.or.toxins.given.the.physicochemical.and.structural.properties.of.the.proteins.and.the. low.exposure. levels.due. to. low.or.undetectable. transgene.protein.expression.(because.of.RNA.silencing)..For.transgenic.viral.proteins.expressed.in.commer-cialized.horticultural.crops,.sequence.relatedness.of.35%.(or.higher).or.a.continuous.stretch.of.eight.amino.acids.is.not.shared.with.known.allergens.71.Moreover,.resis-tance. to. digestion. under. acidic. conditions. has. not. been. demonstrated.2,72,73. It. is.important.to.bear.in.mind.that.many.of.the.crop.plants.available.in.commerce.contain.natural.toxins.and.allergens.48.Peanuts,.tomatoes,.soybeans,.kiwi,.and.potatoes.are.a.few.examples.

durabIlIty and SpecIfIcIty of enGIneered reSIStance to vIruSeS In plantS

The. issues. of. broad-spectrum,. durable. resistance. with. regard. to. virus-resistant.transgenic. horticultural. crops. do. not. conceivably. have. any. impact. on. the. envi-ronment.and.human.health..If.the.engineered.resistance.to.viruses.were.to.show.limitations. in. terms. of. durability. and. specificity,. it. would. likely. only. create. an.agronomic.problem.and.affect.growers.2.However,.these.issues.should.be.considered.in.light.of.an.effective.management.of.the.technology.

Breakdown of Engineered Virus ResistanceThe.risk.that.virus.resistance.may.break.down.or.not.prove.durable.is.not.unique.to.virus-resistant.transgenic.crops..It.is.a.risk.shared.by.conventional.crops.and.other.transgenic.technologies.including.the.pest.resistance.of.Bt.crops..The.durability.of.resistance.refers.to.the.ability.of.a.gene.conferring.resistance.to.hold.up.over.time.after.being.widely.deployed..In.the.case.of.virus.resistance,.however,.the.potential.mechanisms.for.resistance.breakdown.are.different..As.alluded. to.previously,.one.potential.breakdown.of.resistance.could.occur.if.virus.isolates.that.are.genetically.divergent.enough,.at.the.nucleotide.sequence.level,.from.the.transgene.are.not.rec-ognized.by. the.RNA.silencing.machinery.and.are.subsequently.capable.of. infect-ing.the.genetically.modified.host.plant..Another.potential.mechanism.of.resistance.breakdown. centers. on. the. virus’. ability. to. mutate. in. such. a. way. as. to. overcome.the. resistance. triggered. by. the. transgene.. A. third. potential. mechanism. for. resis-tance.breakdown.involves.infection.of. the.resistant.host.plant.with.a.heterologous.virus74,75. encoding. a. strong. suppressor. of. gene. silencing.. This. heterologous. virus.


might.suppress.the.host.resistance.provided.by.the.host.transgene,.thereby.allowing.the.host. to.be. infected.by. the.original.virus. toward.which. their. resistance.had.been.targeted.

Though.each.of.these.mechanisms.could.lead.to.a.potential.breakdown.in.host.resistance,. it. is. important. to. note. that. the. risk. of. resistance. breakdown. is. not.unique. to.virus-resistant. transgenic. crops.nor. is. it. unique. to. transgenic. crops..Resistance.breakdown.is.also.an.issue.associated.with.the.deployment.of.resis-tance.genes.derived.via.traditional.breeding..Pathogens.capable.of.overcoming.deployed. resistance. genes. have. been. extensively. documented;76. likewise,. the.deployment.of.new.resistance.genes.has.been.shown.to.alter.pathogen.popula-tions.to.overcome.that.resistance.76.Initial.infection.by.a.virus.not.targeted.by.a.traditional.breeding-derived.resistance.gene.has.also.been.shown.to.lead.to.a.break-down.of.the.resistance.to.the.virus.targeted.by.the.resistance.gene77—analogous.to.the.breakdown.of.resistance.due.to.a.co-suppressor.of.RNA.silencing..Therefore,.it. is.unclear.how. the.risks.of.breakdown.(with. transgenic.virus. resistance).are.substantially.different.from.those.associated.with.the.use.of.resistance.genes.in.traditional.breeding.

Specificity of Engineered Virus ResistanceAnother.concern.unique.to.virus-resistant.transgenic.technology.is.the.so-called.specificity. of. resistance. provided. by. the. transgene.. As. has. been. shown. previ-ously,78. the. resistance. provided. by. the. transgene. might. only. be. specific. to. the.virus.isolate.from.which.it.was.derived.and.a.few.closely.related.isolates..Though.previously.not.well.understood,.the.current.understanding.of.RNA.silencing.sug-gests.that.this.may.be.due.to.the.sequence.specificity.of.the.resistance.mechanism.itself..Since.the.RNA.silencing.mechanism.relies.on.the.alignment.of.cleaved.frag-ments.of.the.target.RNA.in.the.search.for.invading.RNA.sequences,.divergence.at.the.sequence.level.(over.∼10%).can.lead.to.an.apparent.breakdown.in.resistance.79.However,. this.has.not.been. shown. to.be. the. case.with.all. of. the.virus-resistant.transgenic.plants,80.as.a.single.transgene.is.able.to.confer.resistance.to.challenge.from. numerous. isolates. of. the. same. virus.81. Also,. it. is. important. to. note. that.similar.specificity.has.been.shown.with.resistance.genes.derived.from.traditional.breeding,82.and.it.is.unclear.how.the.resistance.provided.in.the.transgenic.case.is.substantially.more. specific. than. the. resistance.provided.by. the.resistance.genes.derived.by.traditional.breeding.

EXAMINATION OF RISKS ASSOCIATED WITH COMMERCIALIZED TRANSGENIC HORTICULTURAL CROPS

Once.safety.issues.associated.with.transgenic.horticultural.crops.are.identified,.how.are.risks.assessed?.How.does.one.examine.the.significance.of.risk.assessment.data?.When.is.there.enough.evidence.to.start.drawing.conclusions.on.the.safety.of.trans-genic.horticultural. crops?. From. risk. assessment. conclusions,. are. there. any. safety.issues.that.need.to.be.examined.further?.Or,.are.there.any.that.can.be.put.to.rest,.so.to.speak?.In.the.following.section,.we.address.these.questions.with.regard.to.virus-resistant.transgenic.horticultural.crops.


Given.the.tremendous.amount.of.transgenes.that.have.been.engineered.to.provide.virus.resistance.and.the.numerous.crops.that.these.genes.have.been.introduced.into,.there.is.a.staggering.amount.of.scientific.literature.on.resistance.to.viruses.in.trans-genic.crop.plants.17

To.focus.our.efforts.on.findings.that.we.believe.to.be.the.most.relevant,.we.have.chosen.to.examine.risk.assessment.of.virus-resistant.transgenic.horticultural.crops.already.commercialized. (e.g.,.papaya,. summer.squash,. tomato,.and.sweet.pepper).or.awaiting.deregulation.(e.g.,.plum)..In.our.examinations,.we.will.rely.primarily.on.realistic.field.studies,.which.may.provide.the.most.accurate.reflection.of.risks,.and.to.a.lesser.extent.on.laboratory.and.greenhouse.studies..We.will.look.at.the.conclusions.that.can.be.made.about.the.safety.of.virus-resistant.transgenic.horticultural.crops.by.analyzing.the.significance.of.risk.assessment.studies,.as.well.as.at.identifying.gaps.in.knowledge.where.further.experimental.evidence.may.be.needed.before.conclusions.can.be.drawn.with.respect.to.the.safety.of.these.crops.

SQuaSh reSIStant to cucumber mosAic Virus, zucchini Yellow mosAic Virus, and wAtermelon mosAic Virus

BackgroundThe.first.disease-resistant.transgenic.crop.to.be.commercialized.in.the.United.States.was.transgenic.summer.squash..This.squash,.which.possesses.resistance.to.ZYMV.and.WMV,.was.deregulated.in.1994.2,3.Another.summer.squash.cultivar.resistant.to.CMV,.ZYMV,.and.WMV.was.later.released.in.1996.2–4.Virus-resistant.transgenic.squash.possess.the.coat.protein.genes.from.each.virus..Early.testing.indicated.that.they. provide. high. resistance. to. viral. infection1,3,4. and. prevent. viral. epidemics. by.reducing.secondary.plant-to-plant.spread.83.No.similar.resistance.to.multiple.viruses.is.available.in.traditionally.bred.commercial.summer.squash.2

Squash.is.unique.among.commercial. transgenic.plants. in.that. it. is.monoecious.and.readily.outcrosses,44.emphasizing. the.significance.of.gene.flow.issues. for. this.crop..Since.the.center.of.origin.for.many.squash.species.is.in.the.southern.United.States. and. Mexico,42. the. commercialization. of. virus-resistant. transgenic. squash.marks.the.first.transgenic.crop.to.be.released.within.its.center.of.origin.

Risk StudiesAs.summer.squash.was.the.first.virus-resistant.crop.with.a.coat.protein.transgene.to.be.commercialized,. the.potential.allergenicity.and.impacts.on.human.health.were.considered.extensively.46.No.significant.difference.in.protein,.total.fat,.dietary.fiber,.carbohydrate,.calories,.vitamins.A.(and.its.precursor,.β-carotene).and.C,.calcium,.iron,.sodium,.ash,.moisture,.and.sugar.profiles.(fructose,.glucose,.sucrose,.maltose,.and.lactose).was.found.between.transgenic.and.non-transgenic.squash.46.Also,.an.exami-nation. into. human. consumption. of. virus-infected. non-transgenic. summer. squash.fruits.led.to.the.conclusion.that.there.was.likely.to.be.no.significant.negative.impact.on.human.health.beyond.those.of.virus-infected.traditionally.bred.squash.cultivars.with.which.consumers.have.a.long.history.of.exposure.to.without.any.clear.hazards.arising.46


Field.trials.relating.to.gene.flow.from.virus-resistant.transgenic.summer.squash.to.a.free-living.relative.(C. pepo.ssp..ovifera.var..texana).and.the.persistence.of.trans-genes.among.hybrids.of.transgenic.and.free-living.C. pepo.have.been.carried.out..Gene.flow.occurred.with.sympatric.populations.(populations.of.related.species.exist-ing.in.the.same.geographic.area).under.conditions.of.low.disease.pressure.84.Hybrid.plants.containing.transgenes.were.likely.to.produce.more.fruit,.seed,.and.be.more.vigorous. than.free-living.C. pepo. and.non-transgenic.hybrids.under.conditions.of.high.disease.pressure.85.This.was.not. true.under.conditions.of. low.virus.pressure,.where.free-living.plants.outperformed.the.transgenic.hybrids.85.These.results.clearly.indicated.the.advantage.that.the.transgenes.might.provide.under.conditions.of.high.disease.pressure,.although.it.is.not.clear.whether.this.poses.a.significant.risk.in.terms.of.population.dynamics..Surveys.of.free-living.C. pepo.for.viruses.in.areas.where.transgenic.summer.squash.had.not.yet.been.released.showed.an.extremely.low.inci-dence.of.viruses,.including.CMV,.ZYMV,.and.WMV.86.These.results.suggested.that.viruses.have.a.limited.effect.on.the.dynamics.of.free-living.C. pepo.populations.86.Also,. the. studies. on. gene. flow. and. its. consequence. in. squash. do. not. necessarily.indicate.any.risk.of.engineered.virus.resistance.beyond.that.of.conventionally.bred.resistance.genes,.as.far.as.free-living.populations.are.concerned.2

An.additional. study.by.Fuchs.et.al.4. investigated. the. likelihood.of. transencap-sidation. in. transgenic. squash,. tomato,.and.melon,. so.as. to.allow.the. transmission.of. an. aphid. non-transmissible. strain. of. CMV. by. aphids. through. interaction. with.the.coat.protein.transgene.derived.from.an.aphid.transmissible.strain.of.CMV..The.results.of.this.study.failed.to.demonstrate.that.transencapsidation.could.occur.over.two. consecutive. growing. seasons.4. However,. transencapsidation. of. an. aphid. non-transmissible.strain.of.ZYMV.likely.occurred.in.transgenic.squash.expressing.the.coat.protein.gene.of.an.aphid.transmissible.strain.of.WMV.at.a.very.low.rate.and.without.triggering.an.epidemic.87

Similarly,.virus-resistant.transgenic.summer.squash.had.no.effect.on.the.genetic.diversity.of.CMV.strains,. suggesting. that. these.plants.did.not. facilitate. the.emer-gence.of.recombinant.viruses.88

papaya reSIStant to PAPAYA ringsPot Virus

BackgroundAn.early.success.story.in.the.development.and.commercialization.of.virus-resistant.transgenic. fruit. crop. plants. is. the. case. of. papaya. resistant. to. PRSV.6. This. virus.causes.one.of. the.most.devastating.viral.diseases.of.papaya..There.is.no.practical.resistance.known.to.PRSV.in.Carica.germplasm;.therefore,.control.of.this.virus.has.relied.on.exclusion,.movement. to.new.growing. regions.where.PRSV. is.not. found.(often. involving. the.destruction.of.native. rainforest. habitats),. and. the.use.of.mild.isolates.of.PRSV.in.attempts.to.control.the.disease.via.cross-protection.6.PRSV.is.an.aphid-borne.potyvirus.and.can.readily.spread.over.long.distances.by.its.vectors..Papaya.is.grown.in.tropical.and.semitropical.regions.and.Hawaii.is.by.far.the.largest.producer.of.papaya.in.the.United.States..The.production.center.for.Hawaiian.papaya.is.in.the.Puna.district.of.the.island.of.Hawaii..Production.from.this.district.comprises.


over.95%.of.the.total.Hawaiian.papaya.production..In.1992,.PRSV.was.discovered.in.Hawaii’s.Puna.district5.and.within.3.years.nearly.all.of.the.plants.in.Puna.were.severely.affected..By.1997,.papaya.production.had.declined.by.36%.from.21,800.tons.(at.the.start.of.the.outbreak).down.to.14,000.tons.6

Transgenic. papaya. cultivars,. ‘SunUp’. and. ‘Rainbow,’. resistant. to. PRSV. were.released.in.1998.and.widely.planted.in.Hawaii.providing.effective.virus.control.and.allowing.for.increased.papaya.production.(from.the.low.in.1998).2,6.Since.the.release.of.the.PRSV-resistant.transgenic.papaya.in.Hawaii,.additional.papaya.cultivars.resis-tant.to.PRSV.containing.other.PRSV.coat.protein.transgenes.have.also.been.devel-oped.in.Australia,.Florida,.Brazil,.Taiwan,.Jamaica,.the.Philippines,.Thailand,.and.Venezuela.68,81,89–94.The.transgenic.varieties.are.at.various.stages.of.development.and. evaluation..Other. transgenic.papaya.purportedly. resistant. to.PRSV.have.also.been.developed.utilizing.the.viral.replicase.gene67.but.the.major.focus.here.will.be.on.PRSV-resistant.papaya.involving.the.coat.protein.gene.

Risk StudiesSince.the.development.and.commercial.release.of.transgenic.papaya.expressing.the.coat.protein.gene.of.PRSV,.numerous.experiments.and.field.trials.have.probed.the.potential.risks.associated.with.this.transgenic.technology.

As.one.of.the.first.widely.commercialized.fruit.crops,.there.has.been.much.inter-est.in.the.effects.of.the.coat.protein.transgene.on.human.health.and.potential.aller-genicity.as.well.as.investigations.into.compositional.changes.in.fruit.with.respect.to.nutrient.and.antinutrient.content..Descriptions.of. transgenic.papayas.developed. in.Hawaii.report.on.percent.soluble.solids.above.the.minimum.required.for.commercial.fruit.and.yields.of.almost.three.times.those.of.industry.averages.5.Comparable.values.for.vitamin.C.and.minerals.(potassium,.phosphorus,.calcium,.magnesium,.sodium,.iron,.copper,.zinc,.and.boron).for.the.transgenic.and.non-transgenic.cultivars.have.been.published.95,96.Also,.no.evidence.of.ill.effects.has.been.linked.to.the.consump-tion.of.transgenic.papaya.in.the.United.States.and.Canada.2

Another.study.on.transgenic.papaya.from.Thailand.reported.on.comparable.nutri-ent.composition.with.the.non-transgenic.counterpart.93.Recently,.the.levels.of.nutrients.(protein,.fat,.carbohydrate,.minerals).and.antinutrients.(oxalates,.hydrocyanic.acid,.and.benzyl.isothiocyanate).in.three.transgenic.papaya.lines.expressing.a.PRSV.coat.protein.gene.construct,.which.are.not.currently.available.on.the.market,.were.com-pared.to.those.of.the.commercial.papaya.cultivar.‘Sunrise.solo’.grown.under.the.same.conditions.in.an.experimental.plot.in.Jamaica.73.Since.papaya.is.a.climacteric.fruit,.three.stages.of.maturity.were.considered.to.facilitate.an.evaluation.of.the.changes.in.various.parameters.that.accompany.the.ripening.process.after.harvest..With.the.exception.of.one.transgenic.line,.no.significant.differences.were.observed.in.selected.nutrients.and.antinutrients.between.the.control.and.test.samples.at. three.stages.of.maturity,. although. a. few. random. variations. were. noted.73. Overall,. the. composi-tional.changes.over.the.three.maturities.were.as.expected.and.comparable.to.those.reported.97–99.Sugars,.vitamin.C,.and.carotenoids.followed.a.general.upward.trend,.whereas.slight.decreases.in.moisture,.ash,.and.fat.at.the.final.stage.of.ripening.were.observed.. Some. variability. in. the. concentrations. of. the. three. antinutrients. tested.was.observed,.but.the.values.were.within.the.range.of.concentrations.reported.for.


the.parental.variety.100.A.general.trend.of.decreasing.antinutrient. levels.was.noted.during.ripening.in.transgenic.and.control.fruits..Similar.nonsignificant.variations.in.selected.horticultural.traits.(water,.lipid,.nitrogen,.protein,.reducing.sugar,.vitamin.A.(and.its.precursor),.vitamin.C).were.obtained.with.transgenic.papaya.expressing.the.replicase.gene.of.a.PRSV.isolate.from.the.People’s.Republic.of.China.67,101

Recently,. Powell. et. al.102. evaluated. the. safety. of. transgenic. papaya. in. a. sub-chronic.feeding.study..A.diet.formulated.with.10%.transgenic.papaya,.the.equivalent.of.twice.the.average.daily.human.consumption.of.fresh.papayas.based.on.food.con-sumption.data.from.the.Caribbean.and.Latin.America,103,104.was.administered.to.rats.for.90.consecutive.days..For.comparison,.reference.non-transgenic.papaya.‘Sunrise.solo’,.from.which.the.transgenic.papaya.was.derived,.and.a.control.laboratory.rodent.diet.formulation.were.also.evaluated..Markers.of.general.health,. including.body.weight,. food. intake,. and.activities.of.plasma,. liver,. and.kidney. function. enzymes.(acid.and.alkaline.phosphatases.and.alanine.and.aspartate.transaminases).were.com-parable.for.the.test,.reference,.and.control.groups..No.significant.effects.were.observed.in.organ.weights.or.histopathology.102.Changes.in.the.liver.and.kidney,.the.sites.of.biotransformation.and.detoxification,.and.of.excretion.of.metabolic.waste.products,.respectively,.were.not.observed.102.Overall,.the.plasma.cholesterol.levels,.which.are.markers. of. cardiovascular. risk,. were. similar. to. the. control. as. were. triglycerides,.which.are.biomarkers.for.hepatotoxicity..Although.not.statistically.relevant,.varia-tions.in.the.values.of.the.parameters.monitored.with.the.control,.reference,.and.test.groups.were.observed.102.Based.on.literature.ranges,.the.variations.were.attributed.to.natural.biological.fluctuations.and.were.not.regarded.as.reflecting.a.toxicologically.meaningful.effect.

In.addition.to.looking.at.the.effect.of.transgenes.on.papaya.fruit,.other.researchers.have.focused.on.the.effects.of.transgenic.papaya.on.nontarget.organisms.including.soil.microbial.organisms..Hsieh.and.Pan,105.looking.at.populations.of.fungi,.bacteria,.and.actinomycetes.present.in.the.soil.from.field.plantings.of.both.transgenic.and.non-transgenic.fields,.found.highly.similar.(>80%).populations.in.both.soil.environments.(transgenic.vs..non-transgenic).as.well.as.in.upper.and.lower.soils.within.the.environ-ments..These.authors.concluded.that.the.planting.of.transgenic.papaya’s.effect.on.the.soil.microorganisms.is.limited.105.Minor.effects.on.nontargets.have.been.observed,106.but.these.effects.are.varied.and.not.consistent.across.all.virus-resistant.transgenic.crops..Another.study,.also.examining.the.effects.of.transgenic.papaya.on.the.soil,.relied.upon.polymerase.chain.reaction.to.determine.the.persistence.and.availability.of. transgenic. genes. that. may. be. released. by. transgenic. papaya. during. growth.107.Though. transgenic.DNA.was.detected.at. low.levels,.no.gene. transfer.events. from.soil.DNA.extracts.to.Acinetobacter.(a.bacterium.well.known.for.its.ability.to.uptake.foreign.DNA).were.observed.107

tomato and Sweet pepper reSIStant to cucumber mosAic Virus

BackgroundWith.regard.to.transgenic.tomato.and.sweet.pepper.engineered.for.CMV.resistance,.the. body. of. publications. is. much. smaller. than. for. the. aforementioned. transgenic.crops..Transgenic.tomato.and.sweet.pepper.containing.the.coat.protein.gene.from.


a.local.isolate.of.CMV.were.released.in.the.People’s.Republic.of.China.108.CMV.is.a.cucumovirus.that.is.transmitted.in.a.nonpersistent.manner.by.several.aphid.spe-cies.109.It.has.the.widest.host.range.of.any.known.plant.virus.with.1300.species.in.more. than.500.genera.of.over.100. families..Control.of.CMV.can.be.achieved.by.planting.resistant.crops.but.resistance.in.many.crops.species.is.often.not.available.to.a.broad.range.of.CMV.strains.109

Risk StudiesA. study. on. gene. flow. was. performed. with. transgenic. sweet. pepper. and. tomato.by. Ming. et. al.110. Seeds. and. pollen. of. many. different. plants. at. varying. distances.from.fields.of.transgenic.plants.were.examined.to.determine.whether.gene.flow.had.occurred..Selection.of.seedlings.on.antibiotic-containing.medium.and.polymerase.chain.reaction.were.used.to.monitor.transgene.movement,.but.transfer.of.transgenes.was.not.detected.110

For. assurance. of. food. safety,. transgenic. sweet. peppers. and. tomatoes. express-ing.the.coat.protein.gene.of.CMV.were.evaluated.in.animal.feeding.studies.108.The.animals.received.about.12,600.and.7100.times.the.average.daily.human.consump-tion.of.sweet.peppers.and.tomatoes,.respectively..Comparable.performance.of.rats.fed.transgenic.and.non-transgenic.diets.was.demonstrated.108.Significant.differences.were.not.reported.in.mean.weekly.body.weights,.body.weight.gains,.or.food.con-sumption.of. rats. fed. transgenic.or.non-transgenic. sweet.pepper. and. tomato.diets..Similarly,. significant. differences. were. not. observed. with. the. hematological. and.blood. biochemical. parameters. monitored. (including. cholesterol. and. triglyceride),.although.fluctuations.in.the.values.were.observed.108.In.another.study,.Cai.et.al.111.conducted.30-day.acute.toxicity.(LD50).experiments.with.male.and.female.rats.and.mice. using. gavage. administration. of. a. series. of. doses. ranging. from. 1. to. 10.g/kg.body.weight.of.dry.pepper.fruit.containing.the.coat.protein.gene.of.TMV.and.CMV..Abnormalities.in.body.weights,.organ.weights,.histopathology,.and.hematology.were.not.observed.111

EXAMINATION OF RISKS ASSOCIATED WITH TRANSGENIC PLUM WHICH IS UNDER CONSIDERATION FOR DEREGULATION

backGround

Though. not. yet. commercially. available,. the. transgenic. plum. cultivar. ‘Honey.sweet’.(aka.C5).resistant.to.Plum pox virus.(PPV).has.been.deregulated.by.the.U.S..Department.of.Agriculture’s.Animal.and.Plant.Health.Inspection.Service.(USDA-APHIS).and.registered.by.the.Environmental.Protection.Agency.(EPA).in.the.United.States.and.is.being.widely.tested.in.Europe.under.varying.growing.conditions..PPV.is.a.potyvirus.that.is.considered.the.most.important.pathogen.in.Prunus.by.the.U.S..and.E.C..agencies.112.Conventional.breeding.has.not.been.able.to.produce.any.trees.of.commercially.acceptable.varieties.with.high.resistance.to.PPV..Therefore,.control.of.PPV.has.relied.on.prevention.via.certified.planting.material,.quarantine.measures,.and.eradication.112.PPV.is.spread.by.multiple.aphid.species,.but.control.of.the.vector.is.not.feasible.for.both.efficacy.and.environmental.impact.reasons.113


Transgenic.plum.trees.containing.multiple.copies.of.the.PPV.coat.protein.gene.were.developed114.and.line.C5.was.shown.to.be.highly.resistant.to.PPV.infection.113.The.involvement.of.the.posttranscriptional.RNA.silencing.mechanism.in.the.resis-tant.line.C5.was.confirmed.115,116.Clone.C5.was.tested.extensively.in.the.field.in.the.Czech.Republic,.Poland,.Romania,. and.Spain. to.demonstrate. the.effectiveness.of.the.engineered.resistance..Transgenic.C5.trees.all.remained.free.from.PPV.infec-tion.even.6117,118.and.8.years.after.transfer.to.the.field,119.while.100%.of.the.control,.non-transgenic.trees.were.infected.with.PPV.after.these.periods..Though.these.data.indicate. the. durability.of. the. engineered. resistance. to. PPV,. trees. inoculated. with.PPV. via. chip. budding. exhibited. very. mild. symptoms. after. several. years,. though.these.symptoms.did.not.progress.to.severe.118,119

rISk StudIeS

Fruit.compositional.analyses. indicated. that.PPV-resistant. transgenic.C5. is. typical.for. P. domestica. plums. in. terms. of. protein,. total. fat,. antioxidant. capacity,. pheno-lics,.starch,.dietary.fiber,.ash,.moisture,.acidity,.carbohydrates,.sugar.profiles.(glucose,.sucrose,.lactose,.maltose,.and.fructose),.calcium,.magnesium,.sodium,.potassium,.iron,.and.vitamins.A,.B1,.B2,.B3,.and.C.120

The.potential.for.viral.recombination.between.transgene.transcripts.and.incoming.PPV.RNA.was.examined.in.transgenic.plums.as.well.as.the.effects.of.PPV-resistant.transgenic.plums.on.aphid.vector.populations.121.Utilizing.transgenic.European.plum.lines.as.well.as.non-transgenic.plums.from.an.experimental.orchard.and.Japanese.plums.from.an.external.control.plot,.85.PPV.isolates.were.collected.from.these.three.popu-lations.of.trees.and.their.genetic.diversity.was.compared..Looking.at.variable.regions.of. the. PPV. genome. including. the. coat. protein.gene,. no. significant.differences. in.genetic.variability.were.found.among.isolates.from.the.three.populations,.indicating.that.the.PPV.populations.were.not.being.selectively.altered.in.the.transgenic.trees.121.Subsequent.analysis.of.12.PPV.isolates.showed.no.detectable.recombinant.virus.121.Of.note.in.this.experiment,.C5.could.not.be.used.as.a.source.of.virus.isolates.for.testing.since.it.remained.free.from.infection.8.years.after.natural.exposure.to.PPV.populations..Recombination.in.C5.trees.therefore.would.have.been.impossible.due.to.lack.of.virus.infection.

For.assessing.the.impact.of.transgenic.plums.on.nontarget.organisms,.the.diver-sity.of.aphid.populations.visiting.transgenic.and.non-transgenic.plums.was.investi-gated.121.Aphids.were.captured,.identified,.counted,.and.their.viruliferous.potential.was.subsequently.characterized..These.comparisons.found.no.significant.differences.between.the.aphid.populations.from.either.transgenic.or.control.plums.in.terms.of.total.aphid.numbers,.aphid.species.distributions,.and.viruliferous.potential.over.the.2.year.period.of.the.study.121.To.test.the.effect.of.heterologous.viruses.on.the.stability.of.RNA.silencing.in.transgenic.plum.line.C5,.trees.were.graft-inoculated.with.differ-ent.combinations.of.PPV.and.either.Prunus necrotic ringspot virus.(PNRSV),.Prune dwarf virus.(PDV),.or.Apple chlorotic leafspot virus.(ACLSV).75.PNRSV,.PDV,.and.ACLSV.are.common.in.Prunus.sp..The.engineered.resistance.to.PPV.was.stable.and.was.not.suppressed.by.the.presence.of.heterologous.viruses.during.a.3.year.field.trial.in.Romania.and.Spain.75


OVERVIEW OF RISK LESSONS OF TRANSGENIC HORTICULTURAL CROPS

Based.on.the.existing.body.of.research.into.the.potential.risks.posed.by.virus-resistant.genetically.modified.horticultural.crops,.there.appears.to.be.a.significant.amount.of.evidence.that.these.crops.have.little.to.no.detrimental.impact.on.the.environment.and.human.health.beyond.those.of.conventional.horticultural.crops.

The.work.on.summer.squash.supports.the.aforementioned.claim.that.transencap-sidation.is.not.a.significant.environmental.risk.beyond.that.already.posed.by.multiple.infected.conventionally.bred.plants.4,87.This. is.also. true. for. the.risks.of.gene.flow.to.free-living.relatives84–86.and.human.health.effects,.specifically.on.allergenicity.46.Furthermore,.there.is.a.documented.safe.release.of.this.transgenic.crop.over.the.past.12.years.in.the.United.States.2

In. the.case.of.papaya,. the.results. from.the.allergenicity.and.nutritional.experi-ments. seem. to. very. strongly. refute. risks. to. human. health.73,93,102,111. The. threat. of.negative.nontarget.effects.appears.to.be.lessened.though.the.papers.published.on.this.topic.are.very.limited.in.scope.105–107.With.respect.to.the.other.areas.of.risk,.direct.experimental.evidence.does.not.appear.to.exist.in.sufficient.quantities.to.make.any.firm.conclusions.on.transencapsidation,.recombination,.and.gene.flow..However,.the.safe.commercial.use.of. transgenic.papaya.over.a.decade.and.evidence.from.other.transgenic.crops.may.speak.to.some.of.these.issues.enough.to.be.extrapolated.to.the.PRSV.papaya.case.2

The.work.on.plums.supports.the.fact.that.recombination.is.unlikely.to.facilitate.the.emergence.of.virus.species.with.altered.or.new.biological.properties.beyond.the.occurrence. in.conventional.plums.subjected. to.mixed.virus. infection.121.This.also.applies. to. the. risks. on. nontarget. organisms.121. It. is. doubtful. that. transencapsida-tion.could.occur.to.a.meaningful.level.in.transgenic.plum.line.C5,.because.of.unde-tectable.levels.of.PPV.coat.protein.115–117.This.also.casts.doubt.on.whether.the.coat.protein.is.expressed.at.a.meaningful.level.to.have.human.health.impacts.differing.significantly.from.those.seen.in.PPV-infected.non-transgenic.plums.

Based.on.the.information.from.the.transgenic.tomato.and.sweet.pepper,.there.is.further.evidence.to.support.the.claim.that.gene.flow.is.not.a.major.concern.in.this.case.109. Most. significantly,. the. animal. feeding. studies. give. further. support. to. the.safety.of.virus-resistant.transgenic.crops.expressing.viral.coat.protein.genes.108,111

Similar.conclusions.on.the.risk.assessment.of.other.transgenic.horticultural.crops.have.been. reported..The.only.Bt.horticultural. crop.commercially.available. in. the.United.Stated.is.Bt.sweet.corn,.although.other.Bt.transgenic.vegetables.(i.e.,.cauli-flower,.cabbage,.and.eggplant).are.being.considered.for.commercialization.in.India.62.Studies. have. shown. that. Bt. sweet. corn. provides. consistently. excellent. control. of.lepidopteran.pests..This. technology.also.substantially. reduces. insecticide.applica-tions.and.better.preserves.predators.of.the.European.corn.borer.than.commonly.used.broad-spectrum. insecticides.62. Bt. sweet. corn,. like. virus-resistant. summer. squash.and.papaya,.are.consumed.in.the.United.States.with.no.ill.effects.reported.

In.summary,.transgenic.horticultural.crops.have.become.important.components.of. disease. and. pest. management. programs. in. the. United. States. and. the. People’s.Republic.of.China..Their.adoption.rate.is.constantly.increasing.since.their.first.release.


in.the.mid-1990s..Safety.issues.have.been.expressed.during.their.development.and.release,.but.most.of.these.risks.are.the.same.or.similar.to.those.posed.by.tradition-ally.bred.plants.with.host.resistance..Since.the.commercialization.of.virus-resistant.transgenic.squash.in.1996,.considerable.data.have.been.gathered.from.many.parts.of.the.world.on.the.effects.on.the.environment.and.human.health.of.virus-resistant.and.Bt.horticultural.crops..From.these.studies,.some.general.trends.have.emerged..Commercialized.summer.squash,.tomato,.sweet.pepper,.and.papaya.expressing.viral.coat.protein.genes,.and.Bt.sweet.corn.have.effectively.controlled.viruses.and.species.of.Lepidoptera,.respectively..Also,.extensive.research.has.been.published.showing.that.these.transgenic.crops.have.little.to.no.impact.on.the.environment.and.human.health.beyond.those.of.virus-infected.plants.in.natural.settings,.in.traditional.agricul-ture,.and.exposed.to.conventional.pest.management.strategies.2,14,45,62,121,122

PERSPECTIVES

IntroductIon

Since.the.initial.discovery.of.engineered.resistance.to.viruses.via.expression.of.the.TMV.coat.protein.gene.in.plants,16.pathogen-derived.resistance10.and.coat.protein-mediated.resistance.have.proven.to.be.effective.tools.to.control.viruses.in.horticul-tural.crops.17.As.discussed,.PRSV,.ZYMV,.CMV,.and.WMV.have.been.effectively.controlled.in.commercial.settings.of.transgenic.papaya,.summer.squash,.tomato,.and.sweet.pepper.with.the.use.of.coat.protein.genes..In.addition,. in.2007.alone,.more.than.25.field.trial.permits.for.resistance.against.other.viruses.were.granted.by.the.USDA-APHIS.123.Target.viruses.included.Tomato spotted wilt virus,.Beet necrotic yellow vein virus,. Sorghum mosaic virus,. Grapevine fanleaf virus,. Grapevine leafroll-associated virus 2,. Grapevine leafroll-associated virus 3,. Citrus tristeza virus,.Cassava mosaic virus,.Sugarcane mosaic virus,.Sugarcane yellow leaf virus,.Papaya leaf distortion mosaic virus,.PPV,.and.PVY.123

In. recent. years,. new. knowledge. of. the. mechanism. behind. engineered. virus.resistance. has. been. gained.. This. has. greatly. expanded. the. potential. for. utilizing.the.antiviral.pathways.of.RNA.silencing.to.control.plant.viral.diseases..Though.all.the.currently.available.commercial.virus-resistant.transgenic.crops.utilize.the.coat.protein-mediated.resistance,.recent.publications,.patents,.and.field.trial.data.reveal.that. there. are. many. alternative. approaches. currently. in. the. pipeline. to. engineer.virus.resistance.in.plants..Transgenes.incorporating.short.fused.sequences.derived.from.different.viral.strains.have.been.engineered.successfully.to.provide.resistance.to.several.virus.species.124.Other.methods.which,.unlike.the.RNA.silencing-based.approaches,.do.seem.to.depend.on.protein.production.have.also.been.shown.to.have.some.promise,.including.the.use.of.defective.viral.movement.protein.and.replicase.genes.25.The.risks.that.these.gene.constructs.pose.are.not.yet.fully.determined.and.are.likely.different.from.those.risks.already.discussed.with.regard.to.the.viral.coat.protein.gene..Though.the.risk.assessment.of.the.coat.protein.technology.has,.as.dis-cussed,.alleviated.most.of.the.concerns.of.this.technology,.questions.remain.about.the.new.technologies.that.are.becoming.available..Do.they.alleviate.any.of.the.prob-lems.of.the.existing.technology?.Do.they.raise.new.safety.issues.that.may.pose.their.


own.potential.risks?.Do.they.alleviate.a.previous.concern.that.was.no.longer.seen.as.a.problem.only.to.raise.new.issues.that.are.potentially.more.difficult.to.assess?.In.the.following.section,.we.will.describe.some.of.the.latest.approaches.toward.virus.resistance.in.transgenic.plants.and.discuss.if.and.how.these.technologies.alleviate.some.of.the.existing.concerns.

future trendS

Given. the. discovery. and. elucidation. of. the. antiviral. pathways. of. RNA. silencing,.many.new.approaches.have.been.used.to.develop.transgenes.more.likely.to.stimu-late.RNA.silencing.via.the.design.of.sophisticated.transgenes..Since.RNA.silencing.theoretically.is.stimulated.in.a.plant.cell.by.the.presence.of.dsRNA,.many.of.these.transgene.constructs.attempt.to. transcribe.RNA.molecules.that.are.more.likely.to.form.dsRNA.structures..One.strategy.for.accomplishing.this.is.the.use.of.inverted.repeats,. which. involves. the. creation. of. a. transgene. containing. two. copies. of. the.viral.target.complementary.DNA.(cDNA).sequence.separated.by.a.spacer.DNA.of.some.length.25,125.After.transcription,.it.is.thought.that.the.resulting.RNA.will.form.a.hairpin.with.the.inverted.RNA.forming.a.double-stranded.structure..The.use.of.introns.is.a.similar.approach.in.that.two.inverted.regions.of.viral-derived.cDNA.are.separated.by.an. intron.126.Once. the. intron. is.spliced.by.host.machinery. following.transcription,.a.dsRNA.structure.is.formed..Another.approach.consists.of.producing.two.complimentary.pieces.of.RNA.which.can.then.form.a.dsRNA.from.bidirectional.promoters.127.These.new.strategies.have.been.shown.to.produce.a.significantly.higher.proportion.of.virus-resistant.transgenic.plants.than.the.use.of.full-length.coat.protein.transgenes.25,125,126.In.addition,.they.hold.a.seeming.advantage.over.a.full-length.coat.protein.gene.in.the.sense.orientation.as.they.are.generally.unable.to.produce.a.func-tional.protein,.alleviating.concerns.arising.from.the.presence.of.the.coat.protein.in.plant.material..Coat.protein.expression.can.also.be.prevented.by.using. transgenic.approaches.involving.a.transgene.that.produces.an.RNA.product.which.is.untrans-latable,.either.because. it. lacks.the.necessary.translation.start.codon.for.ribosomal.processing.or.because.it.is.oriented.in.antisense.directions..It.should.be.noted.that.the.use.of.introns,.often.derived.from.plants.themselves,.potentially.poses.the.risk.of. silencing. host. genes. from. where. the. intron. was.derived,. if. the.RNA. silencing.machinery.incorrectly.processes.the.transgene.RNA..A.similar.concern.applies.to.the.siRNA.technology.overall.128

Another.approach.utilizing.the.knowledge.of.viral.silencing.is.to.produce.resis-tance.by.using.modified.plant.microRNA.(miRNA).cistrons.to.produce.a.range.of.artificial.antiviral.miRNAs.129,130.The.durability.of. this.approach.compared. to. the.use.of.longer.dsRNA.approaches.has.not.been.demonstrated.131

Some. nonviral. sources. of. virus. resistance. have. also. been. investigated.. These.would.theoretically.alleviate.concerns.about.synergism,.recombination,.and.transen-capsidation..These.include.the.transfer.of.host.resistance.genes.against.viruses.into.other.hosts.via.genetic.engineering,.or.the.silencing.of.host.genes.that.are.necessary.for.viral.replication.25.The.use.of.plant-generated.antibodies.against.viruses,.which.failed.to.progress.for.many.years.though.initially.perceived.as.promising,.has.recently.been.shown.to.be.effective.in.controlling.viruses.in.plants.expressing.the.transgenes.


for. the.production.of. these.antibodies.132.The.potential.risks.of. these. technologies.remain.largely.undiscussed.and.untested.

CONCLUSIONS

To.date.only.a.handful.of.horticultural.transgenic.crops,.including.vegetable.crops.(summer.squash,.sweet.pepper,.tomato,.and.sweet.corn).and.one.fruit.crop.(papaya).are.available.commercially..Most.of.these.crops.have.been.engineered.for.virus.resis-tance.(summer.squash,.sweet.pepper,.tomato,.and.papaya),.while.sweet.corn.has.been.developed.for.insect.tolerance..Noteworthy,.the.majority.of.virus-resistant.transgenic.crops.were.released.over.a.decade.ago..Why.have.not.more.transgenic.horticultural.crops. been. released?. Though. a. virus-resistant. transgenic. plum. has. been. deregu-lated.in.the.United.States,.why.have.transgenic.horticultural.crops.not.been.released.recently?.Can.safety.issues.be.held.accountable.for.the.limited.number.of.transgenic.horticultural.crops.released.commercially?.As.discussed.in.this.chapter,.extensive.research.on.risk.assessment.of.transgenic.horticultural.crops.has.been.carried.out.in.various.environments.and.varied.conditions.of.disease.and.pest.pressure.2,14,45,62,133.This.wealth.of.information.implies.that.safety.issues.should.not.hinder.the.release.of.new.horticultural.crops.that.are.engineered.based.on.the.identical.or.similar.technol-ogies.to.those.used.for.the.development.of.the.transgenic.horticultural.crops.already.released..Factors.other.than.safety.issues.(e.g.,.institutional,.policy,.and.economical.factors). are. apparently. playing. more. important. roles. worldwide. in. stymieing. the.adoption.of.transgenic.horticultural.crops.134–136

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289

14 Molecular Approaches for Transgene Containment and Their Potential Applications in Horticultural Crops

Yi Li and Hui Duan

CHALLENGES TO TRANSGENIC HORTICULTURAL CROPS

Horticultural.crops.were.the.first.commercialized.transgenic.plants.in.the.United.States.. From. 1994. to. 1995,. Flavr. Savr. tomato,. Endless. Summer. tomato,. and.virus-resistant.squash.were.marketed.1,2.A. tomato.paste.(puree).produced.from.a. transgenic.processing. tomato.was. the.best-selling.paste. in.1999.and.2000. in.the.United.Kingdom..Despite.these.early.commercial.successes,.the.number.of.transgenic. horticultural. crops. currently. marketed. in. the. United. States. is. very.small,. limited. to.papayas,. sweetcorn,. squash,. and. carnations.1,2.Also,. the. eco-nomic.impact.of.transgenic.horticultural.crops.is.minimal..Except.papaya,.trans-genic.horticultural.crops.have.had.very.small.market.shares.3,4.The.well-known.estimate.that.70%.of.food.products.in.U.S..supermarkets.contain.transgenic.crop.ingredients.is.due.to.the.widespread.use.of.transgenic.corn,.canola,.and.soybean.products.in.virtually.all.processed.foods.1

CONTENTS

Challenges.to.Transgenic.Horticultural.Crops........................................................ 289Candidate.Molecular.Strategies.Addressing.Concerns.over.Transgenic Horticultural.Crops...............................................................................290

Male.and.Female.Sterility..................................................................................290Genesafe.Technologies....................................................................................... 293Parthenocarpy.Technology.................................................................................294Chloroplast.Transformation............................................................................... 295Gene.Deletor.Technology...................................................................................296

Prospectives............................................................................................................299Acknowledgments...................................................................................................299References...............................................................................................................299


There.are.a.number.of.challenges.for.commercialization.of.transgenic.horticultural.crops..The.biggest.obstacle.is.probably.high.costs.associated.with.food.and.environ-mental. safety. testings. required. by. federal. agencies.. While. traditionally. bred. crop.plants.are.not.subjected.to.any.testing.or.regulations,.testing.required.for.a.deregula-tion.approval.of.a.transgenic.crop.is.extensive,.and.can.cost.$10–20.million.per.novel.gene.in.a.crop.variety.5.Because.each.horticultural.plant.species.has.many.varieties.and.each.variety. represents.a.small.market.value,. recovering.high.costs.associated.with.the.required.testing.can.be.a.challenge..As.a.consequence,.commercial.horticultural.companies.are.not.enthusiastic.to.pursue.transgenic.horticultural.crops.1

Another.hurdle. for. commercialization.of. transgenic.horticultural. crops. is.envi-ronmental.and.food.safety.concerns.4.One.example.is.transgenic.glyphosate-resistant.creeping.bentgrass..The.herbicide-resistant.bentgrass.should.make.weed.control.more.effective.and.also.enhance.the.uniformity,.quality,.aesthetics,.and.playability.of.golf.course. turf.6. Studies. by. the. U.S.. Department. of. Agriculture. (USDA). Animal. and.Plant.Health.Inspection.Service.(APHIS).suggest.that.(1).there.appear.no.major.unin-tended.effects.resulting.from.the.introduction.of.the.glyphosate.resistance.gene.into.the.creeping.bentgrass.genome,.(2).the.transgenic.bentgrass.is.not.sexually.compatible.with.any.federally.acknowledged.threatened.or.endangered.species.or.with.any.spe-cies.on.the.federal.noxious.weed.list,.and.(3).the.transgenic.bentgrass.does.not.differ.in.pest.and.pathogen.susceptibility.or.resistance.from.its.parent.7.However,.Watrud8.reported.that.pollen.of.the.herbicide-resistant.transgenic.creeping.bentgrasses.trav-eled.up.to.21.km.and.the.pollen.could.fertilize.wild.bentgrasses.and.a.close.relative,.redtop. (Agrostis gigantean).. Reichman9. detected. nine. herbicide-resistant. creeping.bentgrass.plants.that.were.found.up.to.3.8.km.beyond.the.control.area,.and.they.there-fore.concluded.that.there.were.both.seed.dispersal.and.pollen-mediated.crossing.with.wild.creeping.bentgrass..Although.the.herbicide-resistant.creeping.bentgrass.displays.no.increase.in.vegetative.spread.or.relative.fitness,10.the.public.opposition.to.dereg-ulation.of. the.herbicide-resistant.bentgrass. is.strong.. It.appears. that. the. transgenic.herbicide-resistant.bentgrass.has.a.long.way.to.go.before.it.is.marketed.6

CANDIDATE MOLECULAR STRATEGIES ADDRESSING CONCERNS OVER TRANSGENIC HORTICULTURAL CROPS

Gene-based.technologies.that.can.reduce.environmental.and.food.safety.concerns.over.transgenic.plants.could.facilitate.commercialization.of.transgenic.horticultural.crops..A.number.of.transgene.containment.technologies.that.may.reduce.gene.flow.have.been.reported.11–15.Some.of.these.technologies.should.also.be.useful.to.address.food.safety.concerns.on.transgenic.horticultural.crops..In.this.chapter,.we.will.pro-vide.a.brief.review.on.some.of.these.molecular.technologies.and.discuss.their.poten-tial.applications.and.limitations.in.horticultural.crops.

male and female SterIlIty

Male.sterility.is.defined.as.the.inability.of.pollen.to.fertilize.the.ovum,.and.female.sterility. is.defined.as. the. inability.of. the. female.organ. to.be.pollinated.or. to.pro-duce.seeds.upon.pollination..In.both.cases,.sterility.can.be.caused.by.inadequacy.in.

291Molecular Approaches for Transgene Containment

structure.or.function.of.the.genital.organs..To.create.sterility.in.higher.plants,.male.and.female.organs.have.been.targeted.for.manipulation..Several.types.of.male.and.female.sterility.technologies.have.been.developed.14,16,17

The.first.group.of.sterility.genes. is.cytotoxin.genes..Cytotoxin.genes.used.for.creating. sterility. include. ribonuclease. genes. such. as. the. bacterial. barnase. gene.cloned.from.a.soil.bacterium,.Bacillus amyloliquefaciens,16,18–24.diptheria. toxin.A.chain. (DTA;. ADP-ribosyl-transferase),25,26. and. the. ribosome. inactivating. protein.(RIP).27.Mariani.et.al.23.used.a.tobacco.tapetum-specific.gene.promoter.(TA29).to.control.the.expression.of.the.bacterial.barnase.gene.in.tobacco.and.oilseed.plants.and. induced. male. sterility..Expression. of. the. barnase. gene. destroyed. the. tapetal.cells.and.successfully.prevented.pollen.formation,. thereby.producing.male-sterile.plants..Because. the.expression.of. the.barnase.gene.was.restricted. to. tapetum.tis-sues,.vegetative.growth.and.floral.development.of. the.transgenic.plants.were.nor-mal.. Similarly,. female. sterility. has. been. achieved. using. the. same. barnase. gene..Goldman28.used.a.stigmatic.secretory.zone-specific.gene.promoter.sequence,.the.5′.untranslated.region.of.the.STIG1.gene.cloned.from.tobacco.plants,.to.drive.the.expression.of.the.barnase.gene..The.pistils.of.transgenic.plants.that.expressed.the.STIG1::barnase.gene.underwent.normal.development.but.lacked.the.stigmatic.secretory.zone.and.therefore.became.female.sterile..The.pollen.grains.of.the.trans-genic.plants.could.germinate.on.the.ablated.stigmatic.surface.although.they.failed.to.penetrate.the.transmitting.tissue.of.the.style,.suggesting.that.pollen.was.normal..Using.a.developing.seed-specific.gene.promoter,. the.FBP7.gene.promoter.cloned.from. petunia,. to. control. the. expression. of. the. barnase. gene. in. tobacco. plants,.Colombo.et.al.29.were.able.to.generate.transgenic.plants.that.produced.no.ovules.or.seeds..The.FBP7.promoter.was.specifically.active.in.the.coat.of.developing.seeds.and. is.completely. silent. in. the.gametophytically.derived. tissues..Because.normal.seeds.were.produced.if.wild-type.plants.were.pollinated.with.transgenic.plants,.the.pollen.of.the.FBP7::barnase.plants.was.fertile.

A.large.body.of.experimental.evidence.has.demonstrated.that.the.bacterial.bar-nase.gene.is.highly.effective.to.produce.sterile.plants.when.expressed.in.reproductive.organs..However,.barnase.expression.sometimes.also. results. in.deleterious.effects.on.vegetative.growth.of.transgenic.plants.if.its.expression.is.leaky.and.affects.non-target.organs.30.The.bacterial.barstar.protein,.a.potent.inhibitor.of.barnase,.has.been.used. successfully. to. restore. fertility. of. barnase-mediated. sterility23. although. the.efficacy.of. the.barstar.gene.can.vary.30,31.Also,. there.are.concerns. that. the.bacte-rial.barnase.protein.may.be.toxic.to.humans.and.animals.because.barnase.can.be.a.poison.for.human.and.animal.cells.32–34.Even.though.it.is.unclear.whether.minute.amounts.of.the.bacterial.barnase.protein.present.in.transgenic.crops.can.be.of.harm.to.humans.and.animals,.the.use.of.a.barnase.gene.in.edible.horticultural.crops.should.be.avoided..For.nonedible.horticultural.crops.in.which.seeds.or.fruits.are.not.used.for.human.and.animal.consumption,.the.barnase.gene.would.provide.an.outstanding.tool.for.inducing.sterility.

A.second.group.of.genes.that.can.be.used.to.produce.sterility.is.one.involved.in.specific.metabolic.pathways. in.higher.plants..For.example,. inhibition.of.pyruvate.dehydrogenase.production.in.mitochondria.resulted.in.male.sterility.35.Tissue-specific.repression.of.an.extracellular.invertase,.Nin88,.caused.male.sterility.21.Disruption.of.


flavonoid.biosynthesis.through.manipulation.of.chalcone.synthase.gene.expression.could.lead.to.male.sterility.36,37.Overexpression.of.β-ketothiolase.via.integration.of.the.transgenes.into.the.chloroplast.genome.of.tobacco.plants.produced.male.sterile.phenotype.38.Expression.of.a.bacterial.enoyl-CoA.hydratase/lyase.enzyme,.which.led.to. re-routing. of. the. phenylpropanoid. pathway,. and. an. unedited. atp9. gene,. a. pro-tein.involved.in.the.proton.channel.of.ATP.synthase,.also.caused.male.sterility.39–43.Compared.to.the.use.of.the.barnase.gene,.food.safety.concerns.over.transgenic.plants.can.be.minimized.if.these.metabolic.genes.are.used.to.create.sterility..However,.the.effectiveness.of.using.metabolic.genes.to.create.sterility.is.much.lower.than.that.of.using.the.barnase.gene..Also,.the.efficacy.of.the.metabolic.genes.can.be.plant.spe-cies.dependent..Pilot.studies.to.test.the.effectiveness.of.a.chosen.metabolic.gene.to.produce.sterility.in.a.plant.species.of.interest.should.always.be.carried.out.before.it.is.used.as.a.sterile.gene.tool.in.that.particular.crop.

The.third.group.of.useful.genes.to.create.sterility.involves.hormone.biosynthesis,.catabolism.or.signaling.pathways.in.higher.plants..Plant.hormones.play.an.important.role.in.the.reproductive.organ.development.and.altering.expression.of.genes.involved.in. hormone. levels. or. response. in. reproductive. organs. often. can. lead. to. sterility..For. instance,. an. inhibition. of. expression. of. an. ethylene-forming. enzyme,. l-ami-nocyclopropane-l. carboxylate. oxidase,. in. pistils. was. effective. in. disrupting. ovule.development. and. therefore. caused. female. sterility.44. Transgene-mediated. reduc-tion. in. jasmonic.acid.concentration. in.anthers. led. to.male. sterility.due. to.defects.in. anther. and. pollen. development.45,46. Expression. of. the. rolC. gene,. cloned. from.Agrobacterium rhizogenes,. resulted. in.male. sterility. and. reduced. female. fertility,.presumably.due.to.the.RolC-mediated.changes.in.hormone.sensitivity.or.concentra-tion.in.reproductive.organs.47.Tissue-specific.overexpression.of.a.cytokinin.oxidase.(CKX1).gene.involved.in.cytokinin.degradation.in.transgenic.maize.(Zea.mays).also.produced.male-sterile.plants.48.Similarly,.expression.of.a.mutant.version.of.a.gibber-ellins.receptor.gene.(gai).in.anthers.and.pollen.of.tobacco.and.Arabidopsis.caused.the.abortion.of.these.reproductive.organs.48.Male.infertility.or.abnormal.male.organ.development.as.a.result.of.expression.of.the.CKX1.could.be.restored.by.applications.of.exogenous.cytokinins..Similar.to.the.Group.2.genes.discussed.earlier,.using.hor-mone-related.genes.should.be.more.acceptable.by.the.public.regarding.food.safety.issues..However,.the.effectiveness.of.these.hormone.genes.can.vary.from.one.plant.species.to.another,.and.may.be.further.influenced.by.environmental.factors.

A.fourth.group.of.genes.that.can.be.used.to.produce.sterility.is.homeotic.genes,.important.for.floral.organ.development..Genetic.and.molecular.studies.demonstrate.that.homeotic.genes.act.alone.and. together. to.specify. the.fate.of.floral.organ.pri-mordia.in.higher.plants.(see.reviews.by.Ferrandiz,49.Lohmann.and.Weigel,50,51.Ma,52.Weigel.and.Meyerowitz,53.and.Zik.and.Irish.54)..Sterility.can.be.achieved.by.ectopic.expression.of.homeotic.genes..For.example,.overexpression.of. the.Brassica napus AGAMOUS.gene,.which.regulates.the.development.of.stamens.and.pistils,.leads.to.the.conversion.of.stamens.into.carpel-like.structures.in.transgenic.tobacco.plants.55.Expression. of. a. tobacco. AGAMOUS. homolog,. TAG1,. resulted. in. the. replacement.of. stamens. with. petaloid. organs. and. the. conversion. of. pistils. to. nonreproductive.organs.56. In.both. cases,. the. transgenic.plants. produced.were.male. sterile..On. the.other.hand,.Mitsuda.et.al.57.used.a.Chimeric.REpressor.Gene-Silencing.Technology.


(CRES-T).to.repress.expression.of.four.transcription.factor.genes,.namely.APETALA3,.AGAMOUS,.LEAFY,.and.AtMYB26.that.are.involved.in.the.control.of.floral.mer-istem. and. organ. identities,. and. the. regulation.of. anther. dehiscence,. respectively..Transgenic.plants.expressing.each.chimeric.silencer.gene.construct.were.sterile,.and.resembled.the.loss-of-function.phenotype.of.each.corresponding.gene..One.advan-tage.of.using.these.floral.organ.genes.to.create.sterility.is.that.the.products.of.floral.organ. development. genes. have. no. toxic. effects. on. human. and. animal. health. and.therefore.there.should.be.few.concerns.if.used.in.edible.horticultural.crops..However,.ectopic.expression.of.floral.organ.development.genes.frequently.leads.to.alterations.in.floral.morphology..For.horticultural.crops,.alteration.in.floral.organ.morphology.can.be.desirable.or.undesirable.depending.on.plant.species.and.applications.

GeneSafe technoloGIeS

Genesafe. technologies,. also. called. genetic. use. restriction. technologies. (GURTs),.involve.the.use.of.genetic.switch.mechanisms.to.control.expression.of.a.toxin.gene.in.seeds.(see.review.by.Kausch.et.al.14)..It.is.an.extension.of.sterility.technologies.discussed.in.the.“Male.and.female.sterility”.section.of.this.chapter..The.best.example.of. the.genesafe. technologies. is. the.one. invented.by.Oliver.et.al.,58.many.called. it.the.“terminator”.seed.technology..The.strategy.uses.a. late.embryogenesis.specific.gene.promoter.to.control.the.expression.of.a.lethal.gene,.the.bacterial.barnase.gene..However,.the.barnase.gene.is.interrupted.by.a.short.blocking.DNA.sequence.so.that.it.is.inactive..The.blocking.DNA.sequence.is.flanked.by.two.loxP.sequences.in.the.same.orientation..LoxP.is.the.recognition.sequence.of.the.Cre.recombinase.from.the.bacteriophage.P1. loxP/Cre.DNA.recombination.system.59,60.The. interrupted. lethal.gene.is.introduced.into.one.parental.plant.line..The.Cre.gene.that.is.under.the.control.of.a.chemically.induced.gene.promoter.such.as.the.tetracycline.inducible.promoter.is.introduced.into.another.parental.line..Both.of.these.plant.lines.can.produce.viable.seeds.if.self-pollinated..If. the.lethal/barnase.line.and.the.Cre. line.are.hybridized,.their.progeny.should.contain.both.the.chemically.inducible.Cre.gene.and.the.inter-rupted.barnase.gene. that. is.under. the.control.of.a. late-embryogenesis.active.gene.promoter..If.hybrid.seeds.produced.from.the.cross.of.these.two.plant.lines.are.treated.with.the.chemical.inducer,.the.Cre.enzyme.will.be.produced.in.seeds.and.the.interrupt-ing.DNA.sequence.should.be.deleted..As.a.consequence,.excision.of.the.interrupting.DNA.sequence.should.make.the.barnase.gene.functional.in.mature.embryos.during.the.seed.development..If.these.resulting.seeds.are.used.for.planting,.when.the.plants.derived.reach.the.reproductive.stage,.the.lethal.gene.should.be.expressed.specifically.in.mature.embryos..The.barnase.gene.expression.in.mature.embryo.will.cause.the.death.of.embryo.and.thus.prevent.seed.germination.

Similar. to. the. “terminator”. seed. technology,. Kuvshinov. et. al.61. described. a.“recoverable.block.of.function”.(RBF).technology..A.lethal.gene,.also.the.barnase.gene,.was.driven.by.a.germination-specific.promoter,.and.the.barstar.(an.inhibitor.of.barnase).gene.was.placed.under.the.control.of.a.heat-shock.promoter..Under.natural.conditions,. the. barnase. gene. will. be. expressed. during. seed. germination. but. the.barstar.gene.will.be.inactive.because.the.heat-shock.promoter.is.not.induced..The.expression.of.the.barnase.gene.in.germinating.seeds.will.prevent.seed.germination..


However,. when. needed,. seed. germination. and. seedling. growth. can. be. restored.through.a.heat.shock.treatment.of.germinating.seeds..Thus,.the.RBF.system.could.be.a.useful.tool.to.reduce.seed-mediated.escape.of.transgenes.

As.represented.by.the.“terminator”.seed.and.RBF.systems,.genesafe.technologies.are.conditional.seed.sterility.technologies..For.the.“terminator”.seed.technology,.one.important.requirement.is.high.efficiencies.of.the.chemical-induced.removal.of.the.blocking.DNA.sequence.from.the.barnase.gene..On.the.other.hand,.high.efficiency.of. the. barstar. gene. in. the. RBF. system. is. not. critical.. Regarding. the. food. safety.concerns,.it.should.be.noted.that.the.barnase.gene.used.in.the.two.genesafe.systems.discussed.in.“Male.and.female.sterility”.section.may.be.replaced.with.a.gene.whose.product.is.nontoxic.to.humans.or.animals.

parthenocarpy technoloGy

Parthenocarpy.(seedlessness).is.the.production.of.normal-sized.fruits.without.fertil-ization.and.the.fruit.produced.contains.no.embryo.and.endosperm..Fruit.development.in.higher.plants.normally.requires.pollination,.fertilization,.and.seed.development.that.stimulate.cell.division.of.specific.floral.and.fruit.tissues..Traditionally,.parthe-nocarpic.fruits.are.produced.from.mutants,. triploid.plants,.or.flowers.treated.with.exogenous. growth. regulators.62. Transgenic. parthenocarpy. was. reported. in. auxin-overproducing. transgenic. petunia63. and. in. tobacco. plants64. using. the. iaaM. gene.cloned.from.Agrobacterium tumefaciens.63.With.ovary-.or.fruit-specific.gene.pro-moters. to. direct. expression. of. the. iaaM. gene. or. RolB. gene,. seedless. fruits. were.produced.from.eggplant,.tobacco,.tomato,.and.watermelon.65–69

Rotino69.reported.that.transgenic.tobacco.and.eggplants.that.expressed.the.coding.region.of.the.iaaM.gene.from.Pseudomonas syringae pv. savastanoi,.driven.by.an.ovule-specific.DefH9.gene.promoter.from.Antirrhinum majus,.produced.partheno-carpic.fruits..If.flowers.were.emasculated,.seedless.fruits.were.produced.from.these.transgenic.plants..If.pollinated,.the.plants.produced.fruits.containing.seeds..In.the.case.of.eggplant,.the.expression.of.the.iaaM.transgene.enabled.fruit.set.and.growth.under. environmental. conditions.where.no. fruit. setting. was. observed. in. the.wild-type.control.plants..Under.normal.growth.conditions,.the.transgenic.plants.produced.marketable.fruits.from.pollinated.or.unpollinated.transgenic.flowers,.but.the.control.plants.produced.fruits.of.marketable.size.only.if.flowers.were.fertilized.69

We. have. used. two. types. of. gene. promoters. to. control. the. expression. of. the.Agrobacterium iaaM.gene..One.is.the.auxin.inducible,.ovary.active.GH3.promoter.cloned.from.soybean70,71.and.another.is.the.ovary.specific.AGL5.promoter.72.Using.these.two.iaaM.fusion.genes,.we.produced.transgenic.Arabidopsis,.tobacco,.tomato,.and.watermelon.plants.68.Transgenic.plants.that.expressed.the.GH3.promoter::iaaM.or. AGL5. promoter-iaaM. gene. were. capable. of. producing. seedless. fruits.. Also,.because.overproduction.of.auxin.in.the.transgenic.plants.was.restricted.to.the.ovary.and.developing.fruit,.no.obvious.side.effects.were.evident..The.seedless.tomato.and.watermelon.fruits.produced.from.the.transgenic.plants.contained.similar. levels.of.acids.and.sugars,.vitamins,.and.other.nutrients.relative.to.the.seeded.fruits.produced.from.control.plants..Furthermore,.the.seedless.tomatoes.and.watermelons.were.nor-mal. in. size. or. sometimes. were. significantly. larger. than. those. produced. from. the.


wild-type.control.plants.grown.under.identical.conditions,.although.fruit.productivity.per.plant.was.not.significantly.altered.

Seeds.can.be.produced.from.many.naturally.or.transgenically.produced.seedless.fruit.plants.if.pollination.is.enforced..To.have.a.total.sterility.of.seedless.fruit.plants,.we.have.also.constructed.a.number.of.gene.cassettes.that.can.lead.to.both.male.and.female.sterility.and.also.production.of.normal.sized.fruits.with.no.seeds.67.We.used.reproductive.organ-specific.gene.promoters.to.control.the.expression.of.a.cytotoxin.gene,. the. bacterial. barnase. gene. for. the. initial. testing,. to. achieve. both. male. and.female. sterility..We. used.an. ovary-specific. gene. promoter. to. control. overproduc-tion.of.auxin.for.seedless.fruit.production.68.With.a.combination.of.the.male-.and.female-sterility.gene.and.the.seedless.fruit.gene,.we.have.demonstrated.that.trans-genic.tobacco.and.tomato.plants.are.totally.sterile.(both.male.and.female.sterile).and.also.capable.of.producing.normal.sized.fruit.with.no.seeds.67

Nonhormonal. genes. have. also.been. shown. to. be. capable. of. producing. seedless.fruits..For.example,.Yao.et.al.73.have.shown.that.the.loss.of.function.mutation.in.the.MdPI.MADS-box.transcription.factor,.a.homolog.of.Arabidopsis.mutant.pistillata,.con-fers.parthenocarpic.fruit.development.in.apple.plants..Ampomah-Dwamena74.reported.that. repression.of. a. tomato.MADS.box. gene,.TM29,. using. either. cosuppression.or.antisense.techniques,.resulted.in.infertile.stamens.and.ovaries.and.subsequently.led.to.the.production.of.parthenocarpic.fruits..However,.these.transgenic.tomato.plants.also.produced.aberrant.flowers.with.morphogenetic.alterations.in.the.organs.of.the.inner.three.whorls.. Ito.and.Meyerowitz75. reported. that.overexpression.of. the.cytochrome.P450.gene.could.lead.to.production.of.parthenocarpic.fruits..Wild-type.Arabidopsis.fruits.developed.to.normal.size.only.if.the.ovules.were.fertilized..When.expression.of. the. cytochrome. P450. gene. was.hyperactivated,. fruits. grew. without. fertilization.and. reached. nearly. normal. size.. When. wild-type. pollen. was. used. to. pollinate. the.cytochrome.P450.overexpressing.plants,.the.pollinated.fruits.became.more.than.10%.longer.and.40%.wider.than.wild-type.fruits.but.they.produced.very.few.seeds.

Seedless.fruit.technologies.can.be.used.to.reduce.seed-mediated.transgene.escape.from.horticultural.crops.if.seeds.of.these.plant.species.are.of.little.value..Since.seeds.can.be.produced.from.seedless.fruit.plants.if.pollination.is.enforced,.seedless.fruit.technologies.can.be.used.in.both.vegetatively.and.sexually.propagated.crops..Further,.the.use.of.a.seedless.fruit.technology.should.lead.to.improvement.of.fruit.quality.and.productivity.68,69,76

chloroplaSt tranSformatIon

Chloroplasts.of.higher.plants.are.semiautonomous.organelles.with.a.small,.highly.polyploid. genomes. and. their. own. transcription–translation. machinery.77. In. most.flowering.plants,.chloroplasts.are.inherited.from.their.female.parents.although.for.some.plant. species. such.as. pines. their. chloroplasts. are. inherited. from. pollen.78,79.For. the.plant. species. whose. chloroplasts. are. inherited. only. from. the. female,. if.transgenes.are. intergrated.into.the.chloroplast.genome,.pollen.produced.from.the.resulting.transgenic.plants.should.be.transgene.free..In.the.case.of.tobacco.plants.with. transgenes. inserted. into. chloroplast. gemone,. for. instance,. only. three. out. of.1,000,000.pollen.grains.contain.transgenes.78


Chloroplast. transformation.offers.several.advantages.77,80–82.First,.each.plant.cell.contains.a.large.number.of.chloroplasts,.with.some.having.up.to.100.chloroplasts.per.cell.83.Also,.each.chloroplast.contains.50–100.or.more.copies.of.its.own.genome84..As.a.consequence,.if.a.trait.gene.is.incorporated.into.the.chloroplast.genome,.the.gene.can.be.enriched.up.to.100,000.copies.per.each.cell..High.copy.number.of.transgenes.per.cell.leads.to.high.expression.levels..For.example,.47%.leaf.total.proteins.were.the.Bt.cry2Aa2.protein.if.the.Bt.cry2Aa2.gene.was.introduced.into.the.choloroplast.genome.of. tobacco.85. Second,. transgene. expression. via. chloroplast. transformation. is. more.stable.because.gene.silencing.that.can.affect.expression.of.nuclear.transgenes.appears.to.have.little.effect.on.transgenes.that.are.introduced.into.the.chloroplast.genome.86.Third,.for.plant.species. in.which.chloroplasts.are.inherited.exclusively.through.the.female.line,.chloroplast.transformation.can.provide.an.effective.means.of.preventing.transgenes.from.transferring.to.non-transgenic.crops.or.wild.relatives.through.pollen.

In. spite. of. the. technical. challenges. in. transforming. chloroplasts,. stable. incor-poration.of.trait.genes.into.chloroplast.genomes.has.been.reported.in.Arabidopsis thaliana,.cotton,.poplar,.rice,.and.soybean.87,88.Also,.chloroplast.transformation.has.been.described.in.a.number.of.horticultural.crops.such.as.cabbage,89.cauliflower,90.carrot,91.egg.plant,92.lettuce,93,94.bladderpod,95.petunia,96.tobacco,97–100.potato,101.and.tomato.102–104.Genetic.transformation.of.chloroplasts.should.provide.an.effective.tool.to.reduce.pollen-mediated.transgene.flow.in.these.horticultural.species.whose.chlo-roplasts.are.inherited.from.female.parents..On.the.other.hand,.problems.associated.with.the.seed-mediated. transgene.escape.will.not.be.addressed.with.a.chloroplast.transformation.strategy.

Gene deletor technoloGy

Gene.deletor.technology.refers.to.techniques.that.can.eliminate.all.transgenes.in.a.particular.organ.or. in. the.entire.plant.when. transgene.functions.are.completed.or.the.transgene.presence.is.of.concern.105–108.The.principle.of.the.gene.deletor.technol-ogy.is.illustrated.in.Figure.14.1..All.transgenes,.such.as.trait.and.selection.marker.genes,.are.placed.within.the.two.loxP-FRT.hybrid.sites..In.addition,.the.FLP.or.Cre.recombinase.gene.under.the.control.of.a.conditionally.active.(e.g.,.organ-.or.tissue-specific,.or.developmental.stage.specific).gene.promoter.sequence.is.inserted.into.the.two.loxP-FRT.sites..If.a.pollen.and.seed-specific.gene.promoter.such.as.PAB5.from.Arabidopsis109. is.used. to.control. the.expression.of. the.FLP.gene,. the.FLP.protein.will.be.produced.only.in.pollen.and.seed..We.have.shown.that.transgenic.plants.have.transgenes.present.and.functional.in.all.organs.during.the.plant.life.cycle.except.in.seed. and. pollen.. Because. of. pollen-. and. seed-specific. FLP. expression,. all. trans-genes.except.an.86-bp.nonprotein.encoding.loxP-FRT.fusion.sequence.are.excised.from.pollen.and.seed..The.excised.DNA.sequence,.consisting.of.one.loxP-FRT.site,.the.trait.gene,.the.selection.marker.gene,.and.the.FLP.gene.should.be.destroyed.by.nonspecific.nucleases.in.the.cell.110,111

Site-specific.recombination.systems.(e.g.,.bacterial.phage.Cre/loxP.and.Saccha-romyces cerevisiae FLP/FRT). function. through. interactions.of.a. recombinase.with.its.specific.recognition.sites.112–115.Recombinase-mediated.excision.between.directly.oriented.recognition.sites.results.in.removal.of.the.intervening.DNA.leaving.one.


recognition.site. intact;116–120.FLP/FRT.and.Cre/loxP. function. through. interactions.of.a.recombinase.with.its.specific.recognition.sites.116–120.Traditionally,.site-specific.DNA.recombination.systems,.such.as.bacterial.phage.Cre/loxP.and.Saccharomyces cerevisiae FLP/FRT. systems,. have. been. used. to. excise. marker. genes. or. short.spacer.sequences.in.higher.plants.114,121.Recombinase.recognizes.these.flanking.rec-ognition.sites.and.excises.any.intervening.DNA..Further,.in.addition.to.DNA.recom-bination.systems,.other.site-specific.DNA.excision.systems.can.also.be.used.in.the.gene.deletor.technology.

With.a.combined.use.of.loxP.and.FRT.sequences.as.the.flanking.sites.for.FLP.or. Cre. recombinase,. we. have. observed. high. efficiency. for. deleting. all. functional.

Non-transgenic plant Transformation using

trait genes and agene deletor cassette

Integration of genedeletor system andtrait genes into host

plant genome

All transgenes includingthe gene deletor systemare deleted from pollen

and seed if FLP isexpressed in pollen and

seed speci�cally

All transgenes includingthe gene deletor system

are deleted from theentire plant if FLP is

conditionally expressedin all cells

Organ-speci�c orconditionally inducible

expression of FLP

Transgenic plant hosting traitgene and a gene deletor system

Host plant genome

Host plantgenome

Host plantgenome

Deletion of all transgenesin cells where FLP is

expressed

Excised transgenesdestroyed in the cell

Host plant genomewith transgenes deletedin FLP expressed cells

Pollen and seedreverted back tonon-transgenic

Entire plantreverted back tonon-transgenic

loxP-FRT loxP-FRT

FIGURE 14.1 (See color insert.).Schematic.illustration.of.the.principle.of.the.gene.deletor.technology..On.the.left,.an.application.of.the.gene.deletor.technology.to.generate.non-transgenic.pollen,.seed,.or.plant.from.a.transgenic.plant.is.illustrated..The.schematics.presented.on.the.right.show.an.FLP-mediated.transgene.deletion.at.the.DNA.level..Any.transgenes,.such.as.trait.genes,.selection.marker.gene,.and.FLP.or.Cre.recombinase.gene.that.have.been.inserted.into.the.two.loxP-FRT.sites.(86.bp.in.length),.will.be.deleted.from.any.cell,.in.which.the.FLP.recombinase.is.expressed..When.a.pollen-.and.seed-specific.gene.promoter.is.used.to.control.recombinase.expression,.all.functional.transgenes.are.deleted.from.these.specific.organs..If.a.conditionally.inducible.gene.promoter,.such.as.chemically.or.high-temperature.inducible,.is.used.to.control.recombinase.expression,.the.system.will.delete.all.transgenes.throughout.the.plant.upon.induction..(Modified.from.Luo,.K..et.al.,.Plant Biotechnol. J.,.5,.263,.2007;.Moon,.H.S..et.al.,.Trends Biotechnol.,.28,.3,.2010.)


transgenes.from.pollen.and/or.seed.of.transgenic.tobacco.plants.105.Based.on.analysis.of.more.than.25,000.progeny.for.each.representative.transgenic.event,.efficiencies.for.automatic.deletion.of.all. transgenes.from.pollen.and/or.seed.are.as.high.as.100%.under. greenhouse. conditions.. Also,. we. have. demonstrated. that. the. high. excision.efficiency.trait.remained.stable.in.vegetatively.propagated.progeny.plants..The.cur-rent.version.of.the.gene.deletor.technology.is.readily.applicable.to.address.the.pollen-.and.seed-mediated.transgene.flow.problems.of.vegetatively.propagated.plants. that.are.important.to.the.agricultural,.forestry,.bioenergy,.ornamental,.and.paper/pulping.industries..This.technology.can.also.be.used.directly.in.sexually.propagated.crops.if.artificial.seeds.(i.e.,.no.pollination.or.fertilization.is.needed).are.used.for.the.propa-gation.of.these.crops..Artificial.seeds.have.been.produced.from.many.major.agricul-tural.crops.122

A.modified.version.of.the.current.gene.deletor.technology.could.be.used.in.sexu-ally.propagated.horticultural.crops.such.as.tomato,.lettuce,.eggplants,.etc..One.pos-sible.strategy.is.to.introduce.a.chemically.inducible.RNAi-FLP.gene.cassette.into.the.gene.deletor.system..Application.of.a.chemical. inducer.at. the.correct. time.would.prevent.the.deletion.of.transgenes.in.pollen.and.seeds..For.instance,.the.ethanol.induc-ible.ALCR/alcA.(alc).two-component.system123.could.be.used.to.control.RNAi-FLP.gene.expression..With. the.ALCR/alcA (alc). system,.application.of. ethanol.during.pollen/seed.development.would.activate.the.RNAi-FLP.gene..RNAi-FLP.represses.the.pollen/seed-specific.expression.of.FLP.recombinase..As.a.result,.all.transgenes.will.remain.in.the.pollen.or.seeds.during.that.generation..However,.if.the.inducer.is.not.applied.in.a.subsequent.generation,.FLP.will.be.expressed..Consequently,.FLP.expression.will.lead.to.deletion.of.all.functional.transgenes.in.pollen.and.seeds.

The.gene.deletor.technology.may.be.extended.to.generate.non-transgenic.edible.parts. from. transgenic. plants. to. mitigate. consumer. concerns. over. the. presence. of.transgenes. in. food. products.. For. instance,. in. the. case. of. the. transgenic. process-ing.tomatoes.grown.in.California,.the.final.products,.tomato.paste,.ended.up.being.exported. to. many. countries. around. the. world.2. Approvals. prior. to. exporting. the.tomato.paste.to.these.countries.had.to.be.obtained.from.each.of.these.countries..If.the.processed.product.contains.viable.seeds,.environmental. studies.and.approvals.are.then.required.in.the.importing.country,.even.if.the.importation.is.intended.only.for.food.consumption..A.fruit-specific.gene.deletor.system.to.eliminate.all.transgenes.from.fruit.tissues.might.help.to.simplify.some.of.these.procedures.and.thus.reduce.associated.costs..With.desirable.organ-.and.tissue-specific.gene.promoters.or.devel-opmental.stage.specifically.or.conditionally.inducible.gene.promoters.to.control.the.FLP.gene.expression,.it.is.possible.to.obtain.non-transgenic.organs.from.transgenic.plants.or.to.revert.the.transgenic.plants.back.to.“non-transgenic”.plants.when.needed.

Volunteer.crops.are.plants.growing.from.seeds. that.are. left. in. the.field.from.a.previous.planting..Volunteer. transgenic.plants.are.a.source.of.perceived.or.actual.environmental.risks.and.have.resulted.in.lawsuits.from.neighboring.farms.or.seed.companies..Because. the.gene.deletor. technology.can. remove.all. functional. trans-genes. from.pollen.and.seeds,.volunteer.plants. in.subsequent. seasons.will. then.be.non-transgenic.

Deleting.all.transgenes.from.pollen,.seeds.or.other.organs.once.their.functions.are.completed.probably.is.one.of.the.best.transgene.containment.concepts,.but.efficiency.


of. the. gene. deletor. systems. under. field. environments. and. in. agricultural. crops.remains. to.be.demonstrated..While.the.gene.deletor. technology.should.be.readily.used.in.vegetatively.propagated.crops,.its.application.in.sexually.propagated.plants.requires.further.modifications.and.refinements.

PROSPECTIVES

Transgenic.technology.will.undoubtedly.play.an.important.role.in.genetic.improve-ment.of.horticultural.crops.in.the.future..As.discussed.earlier.even.though.transgenic.horticultural.crops.are.among.the.first.group.of.commercialized.transgenic.crops,.the.market.values.of.transgenic.horticultural.crops.is.insignificant.1,2.In.recent.years,.the. potential. utilities. of. many. trait. genes. have. been. well. demonstrated. in. plants.including.in.some.horticultural.crops.(see.other.chapters.of.this.book);.their.applica-tions.in.horticultural.plants.remain.uncertain.because.of.high.costs.associated.with.deregulation.approvals.and.environmental.and. food.safety.concerns.. If. the. safety.concerns.over.transgenic.horticultural.crops.can.be.reduced.and.if.the.costs.associ-ated.with.the.regulatory.approval.for.deregulation.of.a.transgenic.horticultural.crop.can.also.be.reduced,.commercialization.of.transgenic.horticultural.crops.should.be.accelerated..Thus,.it.is.crucial.to.develop.and.use.highly.reliable.gene-based.technol-ogies.to.help.minimize.transgene.flow.or.food.safety.concerns.over.transgenic.horti-cultural.crops.and.to.help.reduce.costs.associated.with.their.deregulation.approvals.

Finally,.we.would.like.to.end.this.chapter.with.quotation.from.a.report.by.The.National.Research.Council124.on.biological.confinement.of.genetically.engineered.organisms:.“Making.biosafety.a.primary.goal.from.the.start.of.any.project.will.be.a.more.effective.and.efficient.way.to.prevent.safety.failures.and.it.will.increase.com-mercial. investment. ratings. and. reduce. financial. risks. posed. by. possible. liability.claims. and. loss. of. consumer. confidence.”. “Each. (gene. containment). method. has.its.strengths.and.weaknesses,.and.all.vary.in.efficacy.depending.on.circumstances..No.one.method.will.achieve.100%.confinement.in.the.real.world.”.“Therefore,.it.is.sometimes.necessary.to.employ.more.than.a.single.method..In.many.technological.applications,. the. principle. of. redundancy. reduces. the. occurrence. of. predictable.hazards.while.achieving.the.benefits.of.technological.application.”

ACKNOWLEDGMENTS

The.work.described. in. the.Yi.Li.Laboratory.at. the.University.of.Connecticut.has.been.supported.by.grants.from.USDA,.DOE,.New.England.Invasive.Plant.Center,.and.the.Storrs-Connecticut.Agriculture.Experiment.Station..The.authors.would.like.to.thank.K..S..Cao.for.his.help.in.editing.references.cited.in.this.chapter.

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305

15 Prospects for the Commercialization of Transgenic Ornamentals

Michael S. Dobres

INTRODUCTION

Over. the.past.20.years,. there.has.been. an. explosion. in. the. range. of. technologies.available.to.breeders.of.commodity.crops..This.has.included.the.use.of.molecular.markers1.and. transgenic. technologies2. to.bring.about. significant. increases. in.crop.yields..Despite.this.availability.and.a.significant.amount.of.academic.and.industrial.experimentation,.these.technologies.have.not.been.effectively.applied.to.the.develop-ment.and.commercialization.of.ornamental.plants..This.essay.seeks.to.explain.the.commercial.and.business.reasons.behind.this.apparent.missed.opportunity.

In.the.United.States,.the.entire.wholesale.value.of.the.ornamental.market.exceeds.$8.billion.annually.3.The.market.can.be.divided.into.several.major.product.sectors.including.cut.flowers,. potted.plants,. as.well.as.all. categories.of.garden.and. land-scape.plants.(trees,.shrubs.and.herbaceous.annuals,.and.perennials)..In.the.nursery.and.landscape.industry,.there.is.significant.consumer.and.grower.demand.for.low-maintenance.plants..This.includes.plants.with.improved.disease.resistance,.drought.tolerance,.and.cold.and.heat.tolerance.as.well.as.plants.with.an.extended.seasonal.flowering.period..Disease.and.insect.resistance.traits.have.the.potential.to.reduce.the.environmental.and.human.health.impact.of.gardening.and.landscaping.in.terms.of.reduced.pesticide.and.chemical.input,.as.has.been.reported.for.the.use.of.transgenic.commodity. crops.4,5. Traits. such. as. compact. habit. and. improved. branching. could.reduce.the.time.and.labor.needed.to.maintain.landscaped.areas.

Compared.to.the.achievements.of.conventional.breeders,.genetic.engineers.have.a.tough.act.to.follow..For.centuries,.breeders.of.ornamental.plants.have.pursued.genet-ics.that.confer.improved.disease.resistance,.improved.habit,.more.flowers,.extended.

CONTENTS

Introduction.............................................................................................................305Patents,.Plants,.and.Costs........................................................................................307Field.Testing.and.Deregulation...............................................................................309Global.Distribution.and.Deregulation..................................................................... 311Consumer.Acceptance............................................................................................. 313References............................................................................................................... 314


flowering.time.range,.increased.branching,.and.better.shelf.life..However,.once.created,.these.improvements.are.almost.always.restricted.to.the.genus,.sometimes.restricted.to.the.species,.and.occasionally.restricted.to.the.variety.in.which.they.are.bred.

Commercial.examples.of.shrubs.and.herbaceous.perennials.displaying.some.of.the.above.listed.traits.have.been.and.continue.to.be.developed.by.conventional.breed-ing..These.include.The.Knock.Out®.Rose.(U.S..patent.PP11836),.Endless.Summer.Hydrangea.(U.S..plant.patent.15298),.and.Salvia.‘May.Night.’.The.Knock.Out.Rose.is.highly.resistant.to.black.spot,.is.drought.tolerant,.and.flowers.from.early.summer.to. fall..Endless.Summer.Hydrangea.blooms. throughout. the.summer. into. late. fall,.and.Salvia.‘May.Night’.is.repeat.blooming.and.highly.drought.tolerant..The.market.success.of.these.examples.demonstrates.that.there.is.indeed.a.strong.demand.market.for.these.traits..At.the.same.time,.it.emphasizes.that.the.technology.used.to.develop.new.varieties.needs.to.be.comparable.in.cost.to.conventional.approaches.or.needs.to.confer.traits.unobtainable.by.conventional.breeding.

Transgenic.technology.is.unique.among.other.breeding.tools.in.its.ability.to.pre-cisely.move. single. traits. over. large.genetic. distances.. Although.crossing. the. spe-cies.and.the.genus.barrier.has.been.achieved.by.conventional.breeding.on.numerous.occasions,6,7.genetic.engineering.differs.in.its.ability.to.move.specific.genes.across.greater. distances:. between. families,. between. divisions,. and. between. kingdoms..Transferring.genes.for.color,.fragrance,.and.other.desirable.traits.to.species.devoid.of.such.traits.represents.a.prime.target.for.genetic.engineering.8

To. date,. only. Suntory. has. commercialized. a. transgenic. ornamental. in. the. form.of.cut.carnations.in.the.United.States,.Europe,.and.Japan9.and.more.recently.lavender-colored. “blue”. cut. roses. in. Japan.10.Given. the.dearth.of. commercial. exploitation. in.ornamentals,.it.is.ironic.that.one.of.the.first.published.examples.of.a.transgenic.plant.involved.petunia.11.Since.then,.there.has.been.considerable.excitement.about.the.pros-pect. for. improving. ornamental. crops. and. other. specialty. food. and. nonfood. crops.through.genetic.engineering..Several.companies.were.founded.either.partially.or.solely.on.the.concept.of.developing.and.commercializing.genetically.engineered.ornamen-tal. plants.. Companies. included. NovaFlora. Inc.,. Sanford. Scientific,. Calgene. Pacific,.and.Florigene.NV..Many.established.companies.formed.their.own.genetic.engineering.groups..These.included.Scotts,.Ball.Seeds,.Syngenta,.Suntory,.and.Kirin..Corporate.and.university-based.activity.in.genetic.engineering.can.be.tracked.by.examining.the.online.United.States.Department.of.Agriculture-Animal.and.Plant.Health.Inspection.Service-Biotechnology.Regulatory.Service.(USDA-APHIS-BRS).movement.and.release.permit.database.(www.aphis.usda.gov/biotechnology)..More. than.91.movement.permits.and.73.release.permits.(field.trials).for.ornamental.genera.have.been.issued..Permits.were.issued.equally. to.both.corporations.and.university.and.government.institutions..The.targets.cover.more.than.10.genera.of.bedding.plants,.foliage.plants,.and.shrubs.

In.the.history.of.technology,.this.spurt.of.corporate.and.entrepreneurial.interest.in.a.new.technology.is.fairly.common..Similar.entrepreneurial.bursts.have.been.seen.in.the.advent.of.electrical,.automotive,.photographic,.computer,.and.medical.technology.industries..As.with.other.new.technologies,.the.growth.of.commercial.interest.was.matched.with.skepticism.and.sometimes.fear..This.has.been.true.for.the.automotive.industry,. the. computer. industry. as.well. as.biotechnology.and.genetic. engineering..Some.of.the.above-mentioned.“ornamental.biotech”.companies.are.still.in.existence..

307Prospects for the Commercialization of Transgenic Ornamentals

Others.have.long.since.gone.out.of.business.or.have.been.absorbed.into.larger.corpora-tions.or.have.curtailed.or.terminated.their.in-house.programs..The.main.reason.were.cost.of.patents.and. increased.regulatory.oversight.12.Although. there.are.significant.costs.in.the.development.and.introduction.of.conventionally.bred.ornamentals,.these.do.not.carry.with.them.the.same.administrative.and.legalistic.burden.associated.with.the.testing.and.government.approval.of.transgenic.ornamental.crops..These.issues.are.described.in.more.detail.in.the.following.paragraphs..Despite.the.decrease.in.corpo-rate.expenditures,.university.and.government.funds.are.still.used.at.a.significant.level.for.research.centered.around.the.development.of.genetically.engineered.ornamentals..This.includes.research.on.fragrance,.flower.form,.and.flower.color.13,14

PATENTS, PLANTS, AND COSTS

For.genetic.engineering,.like.any.new.and.innovative.technology,.its.success.and.per-sistence. in. the.marketplace.centers.around. the.patent.protection.of.new. inventions.and.discoveries..The.ornamental.industry.is.no.stranger.to.the.legal.protection.of.new.inventions..Since.1930,.new.varieties.of.trees,.shrubs,.and.herbaceous.plants.generated.as.spontaneous.or.induced.mutations,.or.by.cross-hybridization.have.been.protected.by.Plant.Patents.15.The.Plant.Patent.Act.provides.patent.protection.for.such.clonally.prop-agated.varieties..This.includes.varieties.propagated.by.grafting.of.scions.onto.root-stock,.the.direct.rooting.of.cuttings,.or.the.division.of.plants.or.bulbs..For.recalcitrant.varieties.that.are.difficult.or.slow.to.propagate,.this.can.also.be..performed.by.tissue.culture.micropropagation..Others.can.breed.with.these.varieties.but.cannot.propagate.them.without.a.license.from.the.patent.owner..For.the.past.90.years,.this.method.of.protection.has.provided.a.simple,.affordable,.and.enforceable.means.of.protecting.the.inventive.endeavors.of.hobby.breeders,.university.researchers,.and.corporations.alike..Ornamental.varieties.that.are.produced.and/or.propagated.by.seed.can.potentially.be.protected.under.the.Plant.Variety.Protection.(PVP).Act.16.In.practice,.however,.novel.ornamental.F1.hybrids.are.not.protected.by.their.inventors..Instead.the.F1.parents.are.held.by.the.inventors.as.a.trade.secret..Since.a.novel.F1.variety.will.not.breed.true,.the.breeder.has.little.to.fear.from.a.competitor.or.consumer.who.attempts.to.produce.the.new.variety.through.seed..Most.of.the.varieties.protected.as.PVPs.are.pure.breeding.open.pollinated.varieties.of.grasses.and.vegetables.(http://www.ars-grin.gov/cgi-bin/npgs/html/pvplist.pl)..The.developer.of.F1.hybrid.seed.avoids.the.cost.of.patent.pro-tection,.but.in.turn.invests.more.in.the.development.of.the.genetics.required.to.develop.true.breeding.parent.lines.needed.for.large.scale.F1.seed.production..Production.of.hybrid.seed.is.also.more.technically.demanding.and.less.predictable.than.asexually.propagated.varieties..In.general,.the.ornamental.industry.has.shifted.away.from.F1.hybrid.seed.production.and.more.and.more.towards.vegetative.propagation.

Outside. of. the. United. States,. new. plant. varieties. can. be. protected. under. the.International.Convention.for.the.Protection.of.New.Varieties.of.Plants.(UPOV).in.any.country.that.is.a.signatory.of.the.1991.convention.17.There.are.currently.67.mem-ber.states,. including.countries. in.Europe,.Asia,.Africa,.and. the.Americas..UPOV.provides.a.codified.system,.whereby.a.developer.can.seek.protection.in.parallel.in.multiple.member.states..This.greatly.simplifies.the.protection.for.a.developer.seek-ing.to.produce,.distribute,.and.market.new.varieties.in.multiple.member.states.


At.first.sight.transgenic.technology.represents.a.rapid.and.simple.way.of.modifying.plant.traits.for.the.development.of.novel.patentable.varieties..At.its.simplest.level,.it.allows.the.modification.of.traits.by.transfer.of.specific.trait.genes.to.a.targeted.vari-ety..If.the.gene.brings.about.a.change.in.a.protected.variety,.for.example.petal.color,.a.plant.patent.in.the.United.States.could.potentially.protect.that.variety..However,.in.UPOV.countries,.the.new.variety.would.be.viewed.as.essentially.the.same.as.the.par-ent.variety,.or.“essentially.derived.”.The.developer.of.the.transgenic.variety.would.require.a.license.from.the.owner.of.the.original.variety.before.commercialization.

In. practice,. however,. transgenic. technology. has. proven. to. be. both. technically.demanding.and.expensive,.especially.when. the. full. range.of. intellectual.property.issues.are.considered..Unlike.other.breeding.technologies,.the.transgenic.approach.requires.use.of.multiple.novel. technologies,.genes,.and.vectors.each.of.which.can.be.covered.by.a.utility.patent.restricting.the.use.of.that.technology.in.one.or.more.territories..These.patent-related. issues.have.been.discussed. in.detail. in.a.previous.review.12.In.short,.the.breadth.and.strength.of.a.patent.can.vary.greatly..For.exam-ple,.engineering.novel.petal.color.might. involve. the.use.of.a.coding.sequence.for.an.enzyme.involved.in.the.formation.of.a.novel.pigment..In.a.hypothetical.case,.a.broad.patent.covering.all.uses.of.the.coding.sequence.to.modify.pigment.formation.may.cover.this.coding.sequence..In.other.cases.the.breadth.of.the.patent.might.be.restricted.to.a.narrower.subset.of.uses..For.example,.claims.may.only.be.granted.for.use.of.the.coding.sequence.for.its.use.in.color.modification.of.petals.of.a.specific.species..The.developer.and.his/her.patent.counsel.need.to.carefully.read.the.patent,.its.claims,.and.the.prosecution.history.to.fully.understand.the.breadth.of.a.patent..If. the. developer’s. use. of. the. coding. sequence. falls. outside. the. granted. claims. of.the.patent,. the.developer’s. legal.counsel.may.determine.that.no.license.is.needed..Conversely,.the.patent.may.be.sufficiently.broad.to.make.commercialization.without.a. license.from.the.patent.holder. impossible..In.such.cases,. the.developer.needs.to.negotiate.with.the.patent.holder.for.a.license..Licenses.may.also.be.required.for.the.use.of.promoter.and.other.regulatory.sequences,.as.well.as.for.selectable.marker.cas-settes.and.vectors.used.in.the.plant.transformation.process.

In. addition. to. those. covering. trait. gene. technology,.patents. exist. for. all. of. the.commonly. used. plant. transformation. methods.. Although. many. of. the. major. pat-ents.have.recently.expired,.or.are.close.to.expiring,.the.existence.of.even.a.few.patents.for.which.licenses.are.required.can.add.significantly.to.the.cost.of.developing.and.commercializing.a.transgenic.ornamental..Even.if.the.dominant.patent.for.a.given.transformation. method. has. expired,. there. may.be. genus. specific. transformation.patents.covering.the.use.of.a.specific.transformation.method.in.a.particular.genus..The.situation.is.further.complicated.by.complexities.of.patent.law.and.the.complex.way.in.which.a.patent.is. issued.and.defined.or.claimed..In.some.cases.a.patent. is.so.narrowly.claimed.that.it.is.possible.to.easily.circumvent.the.patent..However,.to.do.this.often.requires.engaging.services.of.a.skilled.patent.lawyer.familiar.with.the.technology.field.in.hand..A.full.legal.opinion.can.add.tens.of.thousands.of.dollars.to.the.cost.of.product.development..Future.prospects.are,.however,.not.quite.so.gloomy..Thus,.within.the.next.decade.or.so,.most.of.the.key.enabling.patents.should.expire.and.fall.into.the.public.domain.and.the.prospect.for.developing.transgenic.orna-mentals. should. improve.. In.addition,.university.and.non-profit.efforts.such.as. the.


Public.Sector.Intellectual.Property.Resource.(PIPRA).(www.pipra.org).and.Cambia.(www.cambia.org).should.help.facilitate.access.to.the.technology.needed.to.develop.transgenic.ornamentals.and.other.specialty.crops.

FIELD TESTING AND DEREGULATION

In. addition. to. the. complex. intellectual. property. issues. discussed. above,. an. orna-mental.breeder.needs.to.consider.the.increased.burden.associated.with.field-testing.a. transgenic. variety.. For. conventionally. bred. varieties,. the. focus. during. field-testing.involves.evaluation.of.such.characteristics.as:.phenotypic.stability,.hardiness,.flowering.time.and.length,.diseases.resistance,.and.in.some.cases.invasiveness..The.breeder.needs.to.ensure.that.the.variety.will.perform.as.expected.in.the.market.place..Ornamentals.released.in.the.United.States.have.to.perform.well.in.a.wide.variety.of.conditions.that.range.from.the.hot.humid.summers.and.cold.damp.winters.of.the.East.Coast.to.the.year-round.arid.conditions.of.the.Southwest..For.this.reason,.developers.often.test.in.multiple.locations..In.some.cases,.this.is.organized.through.grower.orga-nizations.such.as. the.All.America.Rose.Selections.(AARS,.http://www.rose.org/)..The.AARS.organizes.multi-site.testing.for.new.varieties.developed.by.its.members..New.varieties.are.ranked.based.on.disease.resistance,.ease.of.care,.and.beauty..The.best.varieties.are.awarded.the.prestigious.AARS.winners.certificate..The.developer.uses.the.award.to.promote.the.variety.to.the.trade.and.consumers..Before.entering.an.AARS.trial,.developers.perform.their.own.evaluations.for.several.years..Such.field.tests.for.conventionally.bred.ornamentals.are.often.conducted.under.tight.security.to.ensure.the.public.and.competitors.cannot.gain.access.to.commercially.valuable.varieties.before.they.are.released.or.undergo.industry.trials..Transgenic.landscape.plants.would,.in.addition.to.the.private.and.public.field.evaluations,.need.to.undergo.studies.on.trans-gene.flow.and.impact.on.insect.populations..These.studies.would.significantly. add. to. the. size.and.complexity.of. the.field.evaluations..Such.studies.are.aimed.at.understanding. the.ecological.benefits.and/or.risks.of. introducing.the.transgenic.crop..For.large.acreage.commodity.crops,.a.great.deal.of.scientific.effort.and.literature.has.been.devoted.to.the.subject.of.gene.flow.and.impact.on.nontarget.organisms.18.Such.data.is.used.in.the.compilation.and.review.of.regulatory.packages..The.garden.and.landscape.industry.represents.a.special.case..Plants.are.produced.on.a.much.smaller.scale.than.commodity.crops..For.a.popular.perennial.or.shrub,.this.could.be.in.the.range.of.100,000.to.a.million.plants.covering.0.5–10.ha..The.final.end.use.in.the.landscape.might.range.from.a.single.specimen.plant.to.a.few.hundreds.in.a.large.municipal.landscape.planting..Hybridization.and.gene.flow.between.native.species. is. a.naturally.occurring. event..Spontaneous. interspecific.hybrids. are.well.documented.in.numerous.ornamental.genera.19.The.exact.level.and.frequency.of.out-crossing.from.a.cultivated.variety.(whether.transgenic.or.not).will,.however,.likely.vary.from.species.to.species,.and.variety.to.variety..A.2005.study.in.Germany.docu-mented.the.level.of.outcrossing.from.non-transgenic.hybrid.roses.to.wild.roses.20.The.study.made.use.of.microsatellite.markers.to.follow.gene.flow..The.authors.concluded.that.the.frequency.of.outcrossing.from.cultivated.to.wild.roses.was.rare..A.develop-ing.body.of.similar.literature.is.helping.to.define.the.level.of.regulatory.oversight.needed.for.transgenic.ornamentals.21


Studies.on.gene.flow.and. impact.on.flora.and.fauna. for.a.new. transgenic.crop.are.compiled.and.submitted.as.a.petition. for.deregulation.by. the.USDA.. In.addi-tion. to. the. detailed. molecular. and. ecological. studies. required. by. the. USDA,. the.Environmental.Protection.Agency.(EPA).requires.registration.of.what.it.defines.as.Plant.Incorporated.Protectants.(PIP).12.A.PIP.is.a.“pesticidal.substance.that.is.pro-duced.in.a.living.plant.and.the.genetic.material.necessary.for.the.production.of.the.substance,.where.the.substance.is.intended.for.use.in.the.living.plant.”.This.definition.does.not.include.genes.moved.into.a.variety.through.conventional.breeding.or.cell.culture.techniques,.but.only.covers.genes.transferred.to.a.variety.by.genetic.engi-neering..It.also.does.not.cover.pesticidal.genes.transferred.from.closely.related.spe-cies..To.conduct.the.necessary.field.experiment.prior.to.filing.for.EPA.approval.and.registration.of.a.PIP.containing.variety,.a.developer.must.obtain.an.Experimental.Use.Permit.(EUP).in.addition.to.the.USDA-APHIS.permit..Final.registration.requires.a.commitment.to.post-registration.monitoring.and.stewardship..For.commodity.crops.registered.to-date,.this.has.included.preparation.of.an.Insect.Resistance.Management.Plan.(IRM).including.the.use.of.non-transgenic.refuges.to.reduce.the.potential.for.insects.resistant.to.the.PIP..There.are.currently.no.examples.of.EPA.approved.PIPs.in.ornamental.crops,.and.it.is.unclear.what.refuge.and.stewardship.requirements.would.be.required.for.transgenic.ornamentals..Natural.refuges.would.exist.in.all.other.non-transgenic.plants.in.the.surrounding.landscape..Furthermore,.EPA.PIP.registration.for.commodity.crops.is.valid.for.about.3–5.years..This.would.not.be.practical.for.plants.used.in.a.garden.or.landscape.environment..Would.the.consumer.be.expected.to.discard.his/her.aphid-resistant.rose.bush.after.5.years?

Perhaps.one.of.the.most.significant.differences.in.design.of.transgenic.and.non-transgenic.field.tests.is.that.conventional.field.tests.and.trials.are.conducted.in.par-allel. on. hundreds. of. candidate. varieties.. Commercial. field-tests. typically. contain.several.hundred.distinct.varieties..Each.variety.may.differ.in.one.or.more.traits..It.is.fairly.common.for.a.developer.to.include.lines.selected.from.a.broad.array.of.parents.that.exhibit.wide.variation. in.color. range,.habit,. and.disease.resistance. traits. in.a.single.field-test.location..In.contrast,.for.transgenic.varieties,.gene.flow.studies.and.insect. impact.population.studies.are.typically.conducted.on.one.variety.at.a. time..In.some.cases,.multiple.lines.each.carrying.a.distinct.gene.insertion.event.may.be.characterized.in.parallel..This.greatly.increases.the.cost.per.variety.for.transgenic.varieties.compared.to.non-transgenic.varieties.

Many. of. the. genes. proposed. for. use. in. ornamentals. are. derived. from. plants..These.include.genes.involved.in.pigment.biosynthesis.and.genes.conferring.altered.plant.habit.8.The.consequences.of.gene.flow.between.related.species.of.ornamentals.will. probably. depend. on. the. exact. transgene. in. question.. In. a. hypothetical. case,.would.a.gene.derived. from.Arabidopsis. and.conferring.dwarf.phenotype22.confer.any.selective.advantage.if.transferred.from.a.transgenic.hybrid.rose.to.a.multiflora.rose?. Would. it. make. a. difference. if. the. gene. were. derived. from. rose. instead. of.Arabidopsis?. Indeed.in.2007,. the.USDA.proposed.a. tiered.regulatory.structure. in.which. certain. low-risk. transgenics. are. regulated. less. stringently. than. higher. risk.transgenics.23.In. this.context,. technical.refinements.may.help.lower.the.regulatory.cost.and.burden..The.development.of.native.selectable.markers,24.positive.selection.systems,25,26.marker. removal. systems,27. and.marker-free. systems. is.one. approach..


The.development.of.cisgenic.technology,.in.which.all.genes.used.in.the.gene.transfer.process.are.derived.from.the.same.genus.as.the.target.genus,.is.another.approach.28,29

Other.approaches.that.could.be.adopted.by.a.breeder.to.mitigate.risk.and.reduce.costs.include.the.careful.selection.of.parental.breeding.lines..One.approach.would.be.to.introduce.the.transgene.into.male.and/or.female.sterile.lines..Many.varieties.or.ornamentals.have.been.bred.by.conventional.means. to.have.full.flowers.with.multiple.whorls.of.petals.but.no.male.or.female.reproductive.organs..Commercial.examples.include.Impatiens.‘Cameo.Pink.Surprise’.(U.S..patent.13,.308),.Petunia.‘Condowhite’.(U.S..patent.14,.509),.and.Begonia.‘Double.White’.(U.S..patent.12,.674).. Such. transgenic. versions. of. these. sterile. ornamentals. would. be. unable. to.hybridize.with.wild.or.other.related.cultivated.species,.and.would.be.unable.to spread.by.seed.dispersal..This.approach,.however,.might.be.expensive.from.a.regulatory.approach,.since.every.new.variety.would.need.to.be.separately.deregulated..Thus,.the.U.S..system.regulates.transformation.“events.”.By.first.introducing.the.trans-gene.into.a.good.parent.or.breeding.line,.a.high-expressing.stable.transgenic.line.can. be. characterized. and. deregulated. and. used. as. a. parent. line. for. subsequent.breeding.and.development.of.new.varieties.in.accordance.with.conventional.breed-ing.and.evaluation.practices..The.two.approaches.could.be.potentially.combined.if.the.final.variety.created.by.the.breeder.were.rendered.sterile.through.use.of.male.sterile.lines.or.parents.of.different.ploidy.levels..Even.if.issues.related.to.gene-flow.could.be.addressed,.there.would.still.be.costs.related.to.monitoring.and.post-dereg-ulation.stewardship.that.could.add.significant.costs.to.field.trials.and.commercial.release.of.transgenic.varieties.

GLOBAL DISTRIBUTION AND DEREGULATION

Difficulties.also.arise.in.the.way.that.different.regions.and.countries.regulate.trans-genic.crops..Thus,.although.many.countries.are.signatories.of.the.Cartagena.Protocol.on.Biosafety,30.significant.differences.exist.in.the.exact.way.in.which.regulatory.pol-icy.is.practiced.and.enforced..For.example,.in.the.European.Economic.Community.(EEC),.transgenic.crops.are.not.regulated.based.on.the.insertion.events,.but.based.on. the. variety.31,32. Thus,. the. EEC. must. separately. approve. two. or. more. varieties.derived.from.a.single.transformation.event..Such.differences.in.regional.and.national.regulatory.practices.add.a.further.level.of.cost.and.complexity.to.the.development.and.commercialization.of.transgenic.ornamentals.and.can.impact.the.prospects.for.international.distribution..This.is.discussed.in.more.detail.below.

Varieties.of.bedding.plants,.perennials,.shrubs,.and.cut.flowers.are.produced,.dis-tributed,.and.sold.to.consumers.in.many.different.regions.and.countries..The.supply.chain.for.a.new.bedding.plant.variety.typically.starts.in.a.low-cost.production.region.such.as.Africa.or.Central.America..From.there,.cuttings.are.shipped.to.rooting.sta-tions.in.Europe,.North.America,.or.Asia..These.rooting.stations,.in.turn,.sell.plant.plugs.to.growers.within.that.country.or.region..The.grower.then.sells.finished.plants.to.garden.centers.and.large.chain.retailers..Similarly,.the.supply.chain.for.cut.flowers.starts. in.South.America.or.North.Africa. for. the.United.States.or.Europe,. respec-tively..Cut.flowers.are.harvested.and.shipped.by.overnight.air-freight.to.major.distri-bution.centers.in.the.United.States.and.Europe..At.this.point.commercial.brokers.are.


instrumental.in.distributing.products.to.large.regional.wholesalers..The.wholesalers.distribute.products.to.local.florists.and.supermarkets..There.are.some.exceptions.to.these.general.patterns..For.example,.a.few.internet-direct. importers.of.cut.flowers.allow.florists.to.ship.directly.from.farms.in.South.America.to.destinations.in.Europe.and.the.United.States..Some.of.the.large.chain.retailers.control.their.own.distribu-tion.chain.directly.from.Africa.or.South.America.to.the.United.States.or.Europe..Certain. locations,. such.as.California.or.Holland,.have.either. favorable. climate.or.favorable.infrastructure.that.allow.for.the.production.of.cut.flowers.in.the.Northern.Hemisphere.

For. international. trade.of. conventionally.bred.varieties,. growers. routinely.deal.with.the.phytosanitary.regulations.required.for.the.export.and.import.of.plant.mate-rial..These.are,.for.the.most.part,.fairly.similar.from.country.to.country..They.are.mainly.written.and.enforced.to.ensure.that.plant.material.is.pest.and.disease.free.as.it.travels.from.one.country.to.another..For.a.transgenic.variety,.however,.a.developer.would.need.to.apply.for.government.approval.and.permission.to.import.and.distribute.in.each.member.state.or.region.of.the.supply.chain..A.considerable.amount.of.time.and.effort.would.be.required.to.identify.the.appropriate.government.agency,.under-stand.the.authorization.process,.liaise.with.the.appropriate.government.officials,.and.prepare.the.appropriate.application.documents.required.for.government.approval.

For.example,.in.a.hypothetical.case,.starting.in.Costa.Rica,.a.developer.of.a.trans-genic.petunia.would.need. to.gain.approval. for. import.and.growth.of. stock.plants.from. the. Costa. Rican. government.. Costa. Rica. is. one. of. the. largest. producers. of.un-rooted.cuttings.for.the.North.American.bedding.plant. industry..In.Costa.Rica,.the. National. Technical. Biosafety. Commission. (NTBC). regulates. the. production.of.transgenic.crops.33.The.NTBC.has.approved.the.production.of.transgenic.cotton.and.soybean.seeds.and.would.presumably.be.amenable.to.the.production.of.trans-genic.cuttings.of.ornamentals..Imports.of.such.cuttings.into.the.United.States.would.require.that.the.developer.files.a.petition.for.deregulation.with.USDA-APHIS-BRS.and.the.EPA,.if.the.variety.contained.a.PIP.

If.the.developer.wanted.to.ship.transgenic.petunia.cuttings.to.Europe,.he.would.be.required.to.file.and.obtain.approval.for.release.of.the.variety.throughout.the.EEC.31,32.In.theory,.a.developer.can.file.for.approval.in.a.single.member.state..If.the.variety.is.approved.in.that.member.state.(e.g.,.Holland).and.no.other.member.state.objects,.the.variety.can.be.released.and.commercialized.in.all.member.states..Even.if.one.member.state.objects,.an.opinion.is.sought.from.the.European.Food.Safety.Authority.(EFSA).and.used.by.the.European.Commission.to.approve.or.reject.the.application.

For. cut. flower. production. and. import. into. the. United. States,. Columbia. and.Ecuador.are.the.two.main.sites.of.production..The.regulatory.system.in.Columbia.is. more. accommodating. for. the. production. of. transgenic. crops. than. Ecuador’s.34.A.2006.law.in.Ecuador.prohibits. the. trade,.use,.and.handling.of. transgenic.prod-ucts. for. human. consumption.. Though. limited. quantities. of. transgenic. carnations.have.been.produced.in.Ecuador,. transgenic.cotton. is.widely.planted. in.Columbia,.and.the.government.and.agricultural.industry.appear.to.be.generally.supportive.of.biotechnology.35

Similar.technology.driven.import.barriers.exist.for.countries.such.as.Japan36.and.China.37.Thus.import.of.cutting.material.for.growth.and.sale.in.Japan.would.require.


approval. by. the. Plant. Safety. Division. of. the. Food. Safety. and. Consumer. Affairs.Bureau.of. the.Japanese.Ministry.of.Agriculture,.Forestry,.and.Fisheries.(MAFF)..Due.to.consumer.and.industry.resistance,.no.transgenic.crops.have.been.commer-cially.cultivated.in.Japan..This.is.despite.the.approval.for.import.of.more.than.90.transgenic.crop-derived.products.. In.China,. the.application.process.appears. to.be.more.complicated.than.in.most.other.countries..There.are.only.two.application.dead-lines.per.year..Before.applying.for.approval. in.China,. the.transgenic.variety.must.first. be. approved. in. the. country. of. origin.. Thereafter,. it. must. be. retested. for. the.Chinese.application.

Overall.a.company.seeking.to.commercialize.a.transgenic.crop.in.one.or.more.of.the.major.markets.of.the.developed.world.has.to.deal.with.the.regulatory.agencies.not.only.in.the.country.where.the.crop.will.be.sold.but.also.in.any.country.in.which.the.crop.is.produced..This.presents.a.significant.challenge.in.terms.of.the.resources.needed. to.compile. the. regulatory.packages.and.communicate.with. the. regulatory.agencies.in.each.of.these.countries.

In.some.respects,.the.regulatory.process.is.just.as.much.about.managing.the.per-ception.of.risk.and.the.perception.of.benefits.as.it.is.about.managing.the.risks.and.benefits. themselves.. In. this. regard.effective.communication.between. industry,. the.regulatory.agencies,.and.the.public.is.important..Furthermore,.the.effectiveness.of.the.regulatory.process.will.be.aided.by.the.growing.body.of.literature.on.the.ecologi-cal.consequences.and.extent.of.gene.flow.for.transgenic.and.non-transgenic.crops..Additional.funding.to.support.basic.research,.and.the.dispersal.of.this.research.to.industry.and.the.public.could.substantially.increase.the.prospects.for.the.develop-ment.of.transgenic.ornamentals.for.garden.and.landscape.use.

In.the.attempt.to.reduce.the.impact.of.the.regulatory.process.on.the.development.of.ornamentals.and.other.specialty.crops.the.Specialty.Crop.Regulatory.Assistance.consortium. (SCRA).was. formed. in.2005..SCRA. is. a.public–private. effort. that. is.modeled.somewhat.on.the.Federal.Drug.Administration’s.successful.Orphan.Drug.Program..It.plans.to.work.alongside.the.existing.regulatory.agencies.and.their.exist-ing.regulatory.framework.to.facilitate.passage.of.transgenic.specialty.crops.through.the.regulatory.process..This. is.expected. to. include.clear.delineation.of. regulatory.requirements.for.specific.crop-trait.combinations,.assistance.in.compiling.regulatory.packages,.and.general.guidance.for.navigating.the.regulatory.process.(http://www.specialtycropassistance.org).. The. effort. is. currently. seeking. industry. and. govern-ment. funding. to. run.and.maintain. the.necessary.support.programs.. If. successful,.this.effort.could.significantly.increase.the.prospects.for.the.development.and.com-mercialization.of.transgenic.ornamentals.and.other.specialty.crops.

CONSUMER ACCEPTANCE

Genetically.engineered.carnations,.sold.as.cut.flowers,.have.been.marketed.in.Europe,.the.United.States,.and.Japan.for.many.years.9.There.has.been.little.adverse.public.reaction.to.these.varieties..This.bodes.well.for.the.marketing.of.transgenic.landscape.plants..Indeed,.a.2004.survey.of.602.Master.Gardeners.in.Tennessee.revealed.that.73%.of. respondents. reported.an. interest. in.buying. transgenic.ornamentals,. if. and.when.they.become.available.38.Overall.respondents.perceived.slight.environmental.


and. human. health. benefits. due. to. transgenic. ornamentals.. Respondents. were.particularly.interested.in.modifications.that.confer.increased.disease.resistance,.pest.resistance,.plant.longevity,.and.increased.flowering.time..When.asked,.respondents.indicated.a.strong.preference.for.the.labeling.of.transgenic.ornamentals..The.authors.of.the.study.suggest.that.the.results.indicate.that.developers.should.emphasize.the.benefits.gained,.not.the.technology.used.to.develop.new.varieties.

Consumer. and. grower. acceptance. will. also. be. influenced. by. any. added. cost.related. to. the. regulatory. and.patent. hurdles. discussed. above.. Will. there.be. suffi-cient.added.value.to.justify.the.increased.cost.and.can.the.developer.pass.the.cost.on.to.the.grower.and/or.the.consumer?.If.so,.how.will.this.be.done?.In.commodity.crops,.such.as.herbicide-tolerant.soybeans,.this.has.been.successfully.accomplished.by.use.of. a. technology. fee..The. farmer.buys. the. seed. and.pays.an.additional. fee.in.consideration.of.the.additional.cost.and.benefits.of.the.herbicide.tolerance.gene.technology.contained.within.the.seed.39.It.is.unlikely.that.such.a.fee-based.system.would.be.necessary.or.be.practical.for.transgenic.landscape.plants..However,.if.the.value.added.can.be.clearly.demonstrated.to.the.grower.and.consumer,.it.is.possible.that.transgenic.landscape.plants.could.be.marketed.at.a.premium.relative.to.existing.varieties..Indeed,. in.the.ornamental.sector,. it. is.not.uncommon.for.new.improved.varieties.(developed.by.conventional.breeding).to.carry.a.higher.wholesale,.royalty,.and.retail.price.than.older.established.varieties.

The.changing.structure.of.the.industry.itself.may.eventually.favor.the.development.of.transgenic.landscape.plants..The.past.few.years.have.seen.tremendous.consolida-tion.in.the.ornamental.industry.40.As.a.result.of.these.consolidations,.the.number.of.breeding.companies.has.decreased.while.their.size.has.increased..This.does.not.nec-essarily.change.the.likelihood.that.the.remaining.large.companies.will.adopt.trans-genic.technologies..Most.of.the.major.ornamental.companies.have.experimented.with.genetic.engineering.and.most.have.shelved.their.efforts.in.transgenic.ornamentals..Only. Suntory. has. commercialized. a. transgenic. product. in. the. form. of. cut. carna-tions. in. the.United.States,.Europe,.and.Japan9.and.more.recently. lavender-colored.“blue”.cut.roses.in.Japan.10.In.other.words,.the.size.of.a.company.does.not.necessarily.change.the.fundamental.economics.of.commercializing.transgenic.ornamentals.

Currently,. the. main. factors. influencing. the. prospects. for. the. development. of.transgenic.ornamentals.are.those.related.to.regulatory.oversight.and.those.related.to.freedom.to.operate..Additional.streamlining.of.the.regulatory.process.and.additional.research.aimed.at.a.further.assessment.of.the.risks.and.benefits.associated.with.trans-genic.ornamentals.will.help.facilitate.the.commercialization.and.public.acceptance.of.transgenic.ornamentals.21,41.At.the.same.time,.the.expiration.of.key.enabling.patents.and.the.activities.of.organizations.such.as.Cambia.and.PIPRA.should.help.facilitate.access.by.breeders.to.the.necessary.technology.

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317

16 Genetic Engineering of Grapevine and Progress toward Commercial Deployment

Dennis J. Gray, Sadanand A. Dhekney, Zhijian T. Li, and John M. Cordts

CONTENTS

Introduction............................................................................................................. 318Grape.Genetic.Transformation................................................................................ 318

Culture.Systems................................................................................................. 319Methods.of.Gene.Insertion................................................................................. 319Marker.Genes..................................................................................................... 319Functional.Genes................................................................................................ 319Field.Tests.......................................................................................................... 320

Issues.Related.to.Commercialization.of.Transgenic.Grapevines............................ 322Native.Species.and.Gene.Flow........................................................................... 322

Grape.Speciation.and.Range......................................................................... 322Potential.for.Environmental.Risk.................................................................. 322

Potential.Impacts.of.Transgene.Flow.in.Grapevine........................................... 323Potential.for.Transgene.Pollen.Flow.into.Non-GE.Vineyards....................... 323Potential.for.Contamination.of.Commercial.Vineyards.via.Seed.................. 324Potential.for.Transgene.Movement.into.Native.Vitis.Populations................. 324

Mitigating.Environmental.Risks.via.Cisgenics.Engineering............................. 324Endogenous.Genes........................................................................................ 324Marker-Free.Plants........................................................................................ 326Rootstocks..................................................................................................... 327

Conclusion.............................................................................................................. 327References............................................................................................................... 327


INTRODUCTION

Grapevine.is.the.world’s.most.valuable.fruit.crop.due.to.its.multipurpose.uses.for.fresh. fruit,.wine,.and.other.processed.products..While.many.species.of.grape-vine. exist,. as. described. below,. the. predominant. species. used. in. commerce. is.Vitis vinifera.L..This.species.originated.in.the.Middle.East.and.is.adapted.to.a.Mediterranean. environment.. It. has. diversified. into. hundreds. of. varieties. since.antiquity.. Major. production. areas. in. the. world. tend. to. be. those. possessing.Mediterranean. climatic. conditions.. Despite. widespread. consumption. of. grape.products,.attempts.to.grow.V. vinifera.in.climates.that.are.too.cold.or,.especially,.too.humid.have.met.with. failure.. It.would.be.advantageous. to.have.V. vinifera.varieties.with.more.resistance.to.diseases.caused.by.bacteria,.fungi,.and.viruses.in. order. to. foster. more. widespread. production. and. thus. address. local. market.needs.and.opportunities.

While.V. vinifera.has.been.hybridized.with.native.species.to.create.land.races.for.suboptimal.regions,.this.approach.has.met.with.limited.success.due.to.the.consum-er’s.strict.preference.for.the.exact.phenotypic.characteristics.of.well-known.varieties..This.is.because.grapevine.is.genetically.self-incompatible,.making.it.impossible.to.create.inbred.lines.needed.to.introgress.desirable.traits.from.resistant.native.species.into.existing.elite.varieties..Although.clonal.selection.of.randomly.occurring.muta-tions.has.been.successful. in.creating.new.varieties,. it. is.a.haphazard.process.and.cannot.produce.grapevines.with.desirable.resistance.traits.1

Genetic.transformation.offers.an.alternate.method.for.adding.specific.traits.into.otherwise.desirable.varieties..Presumably,.varieties.altered.only.by.the.addition.of.genes. for. resistance. (or.other. traits).would. retain. their. elite. characteristics.2.Only.recently.has.genetic.transformation.technology.matured.for.grapevine,.such.that.it.is.now.commonplace.in.certain.laboratories.to.insert.genes.and.produce.transgenic.ver-sions.of.popular.varieties.3–6.It.now.remains.to.study.grapevines.containing.various.transgenes.for.their.performance,.which.includes.expression.of.new.desirable.traits.as.well.as.whether.the.high.level.of.clonal.fidelity.demanded.of.grape.is.maintained.in.transgenic.vines.

This. chapter. will. review. the. progress. in. genetic. transformation. of. grapevine,.including. discussion. of. the. most. recent. methods. and. procedures. that. have. led. to.successful.transformation..Status.of.field.tests.and.issues.related.to.environmental.release.of.transgenic.grapevines.will.be.presented.

GRAPE GENETIC TRANSFORMATION

Significant. progress. in. grapevine. transformation. has. been. made.1–3,5,6. Genetic.transformation. has. become. relatively. routine. for. ‘Chardonnay’,. ‘Merlot’,.‘Superior.Seedless’,. and. ‘Thompson.Seedless’. (=‘Sultana’,. ‘Sultanina’),. among.others..However,.several.fundamental. technologies.were.required.before.trans-formation.of.grapevine.could.be.studied..These. included.regenerative.cell.cul-ture.systems,.methods.for.gene.delivery,.and.then.advances.in.molecular.genetic.techniques.to.identify.useful.genes.and.place.them.into.functional.DNA.cassettes.that.could.be.tested.

319Genetic Engineering of Grapevine

culture SyStemS

Somatic.embryogenesis.is.well.documented.for.grapevine.7–9.Most.successful.exam-ples. of. transformation. have. utilized. such. embryogenic. culture. systems. because.embryogenic.cells.are.capable.of.being.genetically.transformed.and.can.be.induced.to.regenerate.into.plants.1,2.Thus,.once.a.cell.is.stably.transformed,.a.transgenic.plant.can.be.produced..A.drawback.to.use.of.embryogenic.cultures.is.that.they.have.been.difficult. to.obtain.for.many.varieties..However,. this.obstacle.is.slowly.being.over-come.through.experimentation.and.refinement.of.culture.techniques.7,10

Cells. of. the. shoot. apical. meristem. obtained. through. in. vitro. micropropagation.were.considered.as.a.potential.alternative. to.embryogenic.cells. for. transformation..The.ease.of.producing.and.maintaining. in.vitro.micropropagation. cultures. from.a.large.number.of.cultivars11,12.makes.development.of.a.shoot.tip-based.transformation.system.attractive..However,.use.of.shoot.apical.meristems.for.transformation.has.met.with.limited.success.in.grapevine.13.Problems.that.inhibit.their.use.likely.include.a.rel-atively.low.number.of.totipotent.cells.in.the.shoot.apex.to.be.transformed,.when.com-pared.to.embryogenic.cultures,.and.the.propensity.for.chimeric.plants.to.be.recovered.

methodS of Gene InSertIon

As.reviewed.by.Gray.et.al.,1.the.two.commonly.used.methods.of.gene.insertion,.biolistic-mediated.and.Agrobacterium-mediated.transformation,.have.been.successfully.utilized.for.grape..Biolistic.bombardment.resulted.in.intense.transient.beta-glucuronidase.(GUS).expression.in.somatic.embryos.of.V. vinifera.‘Thompson.Seedless’14.and.has.been.used.to.produce.transgenic.plants.from.embryogenic.suspension.cultures.of.‘Chardonnay’,.‘Merlot’,.and.‘Chancellor’.15,16.However,.Agrobacterium-mediated.transformation.has.been.utilized.more.frequently.and.currently.appears.to.be.the.transformation.system.of.choice.for.grape.(see.Dhekney.et.al.3–5.and.Li.et.al.6.for.recent.examples).

marker GeneS

Development.of.transformation.protocols.for.grape.has.been.greatly.facilitated.by.refinement.of.genetic.vectors.that.efficiently.express.various.reporter.genes..Visible.reporter.genes. inserted. in.grape.include.GUS,14.green.fluorescent.protein.(GFP),17.and. luciferase.18.The. selectable.marker. genes,. phosphinothricin. acetyl. transferase.(PAT,.or.BAR.for.Bialaphos. resistance).gene,19. hygromycin.B.phosphotransferase.(HPH).gene,19. and.neomycin.phosphotransferase. (NPTII).gene,17. have.been.used,.with.the.latter.being.most.common.

functIonal GeneS

Establishment. of. reproducible. transformation. protocols. has. enabled. the. insertion. of.transgenes.for.quality.trait.improvement.including.fungal,.bacterial,.and.viral.resistance.in. elite. grapevine.varieties..Genes. encoding. pathogenesis-related. (PR). proteins. have.been.widely.tested.to.enhance.grapevine.fungal.resistance..‘Thompson.Seedless’.grape-vines.transformed.with.an.endogenous.thaumatin.like.protein.(VVTL-1).gene.exhibited.significant.resistance.to.powdery.mildew.(Uncinula necator).and.black.rot.(Guignardia bidwellii).in.greenhouse.and.field.tests.20,21.Berries.of.‘Shiraz’.grapevines.transformed.


with.an.endogenous.proprietary.Vitis.gene.(EG2).exhibited.significantly.lower.devel-opment.of.powdery.mildew.(Uncinula necator).compared. to.control.grapevines.22–24.Resistance. to. fungal.diseases. in.grapevines. transformed. with. other. PR.proteins. has.also.been.reported.25,26.Low.molecular.weight.chain.antimicrobial.peptides,.also.known.as.lytic.peptides,.have.been.widely.tested.to.impart.bacterial.resistance.in.grapevines..Transgenic. ‘Chardonnay’,.carrying.an.antimicrobial.peptide.magainin,.exhibited.sig-nificant.reduction.in.crown.gall.symptoms.caused.by.Agrobacterium vitis.27.‘Merlot’,.‘Shiraz’,. and. ‘Thompson. Seedless’. grapevines. transformed. with. proprietary. hybrid.lytic.peptides,.LIMA-A.and.LIMA-B,.exhibited.enhanced. resistance. to.Pierce’s.dis-ease.(Xylella fastidiosa).in.greenhouse.tests.28.Field.tests.are.currently.underway.to.test.resistance.of.a.number.of.transgenic.Vitis.varieties.and.rootstocks.to.Pierce’s.disease.29,30.Virus. resistant. transgenic. grapevines. have. also. been. developed. using. virus-derived.genetic.elements..Grapevine.varieties.and.rootstocks.carrying.the.coat.protein.for.grape.fan.leaf.virus.(GFLV).and.grape.leaf.roll.virus.(GLRV).have.been.produced31–34.and.tests.are.under.way.to.study.the.response.of.vines.to.virus.infection.under.field.conditions.

fIeld teStS

Field.trials.of.GE.grapevines.(Vitis vinifera.and.other.Vitis.species).have.been.con-ducted.worldwide.since.the.mid-1990s.(Table.16.1)..Early.trials.in.Colmar,.France,.consisted. of. gene. constructs. for. resistance. to. GFLV.. Other. trials. in. the. United.States.(several.locations),.Canada,.and.Germany.were.designed.to.evaluate.resis-tance. to. fungal.diseases. (powdery.mildew.and.botrytis,.primarily)..Those. trials.included. a. number.of.different.genes. including.chitinases/endochitinases,. glu-canases,.a. ribosome.inactivating.protein,.a.polygalacturonase. inhibiting.protein,.lytic. peptides,. antimicrobial. peptides,. a. lignin. biosynthesis. protein,. and. endog-enous.grape.genes..Field.trials.that.have.ended.include.work.in.Canada.by.Chateau.des. Charmes. to. develop. both. fungal. resistance. and. improved. cold. tolerance. in.vines. (Cordts,. personal. communication);. work. by. AgriVitis/GenApps. in. the.United.States.to.develop.resistance.to.GFLV,.GLRV,.and.crown.gall.(http://www.isb.vt.edu/cfdocs/fieldtests1.cfm);. research. in. Italy. to. improve.berry.number. and.size. (http://www.gmo-compass.org/eng/database/plants/73.grape_vine.html)35;.research.by.SUNY/Geneseo.to.develop.mildew.resistant.V. labrusca.(http://www.isb.vt.edu/cfdocs/fieldtests1.cfm);. and. trials. by. the. University. of. California. and.Anton.Caratan.&.Son.for.fungal.resistance.and.improved.product.quality.(http://www.isb.vt.edu/cfdocs/fieldtests1.cfm).

Current.field. trials.of. Vitis. species. worldwide. are. being.conducted. by. Cornell.University,. the. University. of. Florida,. and. by. the. Commonwealth. Scientific. and.Industrial.Research.Organisation.(CSIRO).(http://www.isb.vt.edu/cfdocs/fieldtests1.cfm,. http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/ir-1).. The. Cornell.vines. have. been. developed. to. evaluate. resistance. to. GFLV,. GLRV,. crown. gall.disease,. fungal. pathogens,. and. Xylella fastidiosa. (http://www.isb.vt.edu/cfdocs/fieldtests1.cfm)..The.University.of.Florida.vines.are.being.evaluated.for.resistance.to.Xylella.and.fungal.diseases..CSIRO.is.evaluating.vines.for.a.number.of.traits.includ-ing.alterations. to. tannins,.anthocyanins,.sugars,.browning,.and.various.flowering/fruiting.parameters.(Table.16.1).


TABLE 16.1Field Tests of Transgenic Grapevine

Country Species Traits Tested

Australia

CSIRO.(1999–2009),.http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/ir-1

V. vinifera Expression.of.modified.color,.sugar.composition,.flowering.and.fruit.development/improved.product.quality.(ppo,.sh4,.ufgt,.dfr,.inv,.ARMGs,.GFP,.GUS).browning,.flowers,.anthocyanins,.tannins,.sugars

Canada

Chateau.des.Charmes,.Ontario,.Canada (ended.2001)

V. vinifera Fungal.tolerance,.cold.tolerance

France

INRA,.Colmar,.France.(1994,.1999,.2004).(DeFrancesco36;.http://www.gmo-compass.org/eng/database/plants/73.grape_vine.html)

V. vinifera GFLV.resistance.using.viral.coat.proteins.(VCPs)

Germany

Bundesanstalt.fur.Zuchtungsforschung,.Germany.(2000).(DeFrancesco36;.http://www.gmo-compass.org/eng/database/plants/73.grape_vine.html)

V. vinifera Improved.product.quality.(fungal.resistance—chitinase,.glucanase,.ribosome.inactivating.protein)

Italy

Universita.degli.Studenti.Ancona,.Italy.(1999).(DeFrancesco36;.http://www.gmo-compass.org/eng/database/plants/73.grape_vine.html)

V. vinifera Fecundity.(Increased.auxin,.increased.tryptophan-2-mono-oxygenase).Def.H9-iaaM.designed.to.increase.fruit.size,.number35

United States of America

Cornell.(2007–2009) Vitis.rootstock.species

GFLV,.GVLR.resistance.based.on.coat.protein.gene.insertions

University.of.Florida.(2006–2009) V. vinifera Xylella.resistant.and.powdery.mildew.resistant.(lytic.peptides.and.endogenous.grape.genes)

Cornell.(2000–2009) V. vinifera Crown.gall.resistant,.Xylella.resistant,.Botrytis.resistant,.powdery.mildew.resistant.(antimicrobial.peptides,.chitinases,.endochitinases)

SUNY/Geneseo.(1999–2007) V. labrusca Powdery.mildew.resistant.(lignin.biosynthesis.protein,.glucanase)

. University.of.California/Davis.(2004+) V. vinifera Fungal./.bacterial.resistance.(polygalacturonase.inhibiting.protein)

(continued)


No.submissions.for.regulatory.approvals.for.unconfined.release.of.GE.grapevines.have.been.noted.from.current.sources.

ISSUES RELATED TO COMMERCIALIZATION OF TRANSGENIC GRAPEVINES

natIve SpecIeS and Gene flow

Because.several.species.of.grapevine.are.endemic.to.temperate.and.tropical.areas.of.the.world,.genetic.modification.to.adapt.V. vinifera. into.such.regions.should.be.studied.for.environmental.impact.on.native.flora.

Grape Speciation and RangeVitis. is.divided.into. two.subgenera,.Euvitis.Planch,. the.bunch.grape.species. that.all.contain.38.somatic.chromosomes,.and.Muscadinia.Planch,.the.muscadine.grapes.with.40.somatic.chromosomes.37,38.Species.in.each.subgenus.are.interfertile.but.are.only.par-tially.fertile.between.subgenera..Estimates.of.the.number.of.Euvitis.species.range.from.28.to.43..These.are.separated.into.an.American.group.of.18–28.species,.an.Asian.group.of.10–15.species,.and.a.European.or.central.Asian.group.of.one.species.(V. vinifera).37.Muscadinia.contains.three.species;.V. munsoniana.and.V. rotundifolia.are.native.to.the.southeastern.United.States.and.V. popenoeii.Fennell.is.native.to.Central.America..With.the.exception.of.muscadine.cultivars,.most.cultivated.grapes.are.either.pure.strains.or.hybrids.of.V. vinifera..Two.wild.subspecies.of.V. vinifera,.ssp..sylvestris.Gmel..and.ssp..caucasis.Vav.,.and.one.cultivated.subspecies,.ssp..sativa.D.C.,.are.recognized.37

Potential for Environmental RiskTransgenic.grapevines.that.contain.genes.to.make.them.resistant.to.diseases,.much.like.native.species,.could.pose.the.possibility.of.environmental.risk.if.they.became.invasive..It.is.important.to.consider.whether.there.is.potential.for.adverse.environ-mental. impacts. from. field. release. of. genetically. modified. disease-resistant. grape-vines..Research.should.be.accomplished.to.determine.the.characteristics,.rates,.and.methods.of.gene.transfer.that.may.occur.between.genetically.engineered.non-native.grapevines.and.cross-fertile.wild.and.agricultural.species.currently.in.the.environ-ment.to.determine.if.the.GE.vines.or.progeny.produced.by.cross-fertilization.could.

TABLE 16.1 (continued)Field Tests of Transgenic Grapevine

Country Species Traits Tested

AgriVitis/GenApps.(1997–2000) Rootstocks GLRV,.GFLV,.crown.gall.resistant.(based.on.VCPs.and.other)

Anton.Caratan.&.Son.(2000+) V. vinifera Improved.product.quality

South Africa

University.of.Stellenbosch,.South Africa

V. vinifera Traits.to.be.tested.unclear


become.invasive.pests..Because.transgenes.are.dispersed.from.genetically.engineered.vines.by.pollination.and/or.seed.production,.certain.aspects.of.grape.reproductive.biology.should.be.evaluated..Molecular.methods.using.marker.genes.for.detection.of.transgenes.and.transproteins.in.grape.seed/fruit.tissues.to.monitor.movement.of.such.genes.through.sexually.produced.progeny.are.readily.available..In.addition.to.gene.movement,. the. relative.vigor.and.adaptation,. including.survivability.of. transgenic.progeny,.must.be.determined.to.assess.their.potential.to.become.invasive.pests.

Gene Flow via PollenIn.varieties.with.perfect.flowers,.the.calyptrate.flower.structure.and.arrangement.of.many.flowers.on.a.panicle.favor.self-pollination.37.The.extent.to.which.natural.cross-pollination.occurs. in.grape. is.controversial..Natural.hybridization.among.native. species.has.been.detected.39.Grape.has.been.considered.to.be.wind-pollinated,.with.pollen.flow.occurring.no.further.than.20.ft..from.the.source.40.However,.cross-pollination.has.been.suggested.to.be.enhanced.by.insect.pollinators.in.certain.instances..For.example,.in.muscadine.grape.vineyards.composed.of.female.(self-sterile).vines.that.require.pollinator.vines,.fruit.set.has.been.enhanced.by.incorporation.of.honeybee.hives.(reviewed.by.McGregor41)..However,.in.a.controlled.study,.vines.isolated.from.wind-blown.pollen.by.cages.did.not.produce.appreciatively.more.fruit.when.bee.hives.were.added.42.Airborne.pollen.yields.have.been.accurately.measured.in.order.to.predict.seasonal.fruit.yield.43,44.The.latter.studies.described.airborne.pollen.loads,.but.did.not.address.hybridization/gene.flow..Thus,.the.maximum.distance.that.viable.pollen.moves.from.one.grapevine.to.another.and/or.the.frequency.of.hybridization.resulting.from.such.movement.have.not.been.adequately.documented..This.issue.has.taken.on.new.importance.with.the.advent.of.genetically.GE.grapevine.

Gene Flow via SeedThe.possibility.for.gene.flow.via.seed.dispersal.from.transgenic.vines.would.be.low.if.seeds.result.from.selfs,.as.mentioned.above,.and.because.grape.has.a.high.level.of.inbreeding.depression.45.Therefore,.the.resulting.selfed.plants.lack.vigor.and.tend.not.to.survive..For.example,.of.plants.grown.from.over.one.thousand.selfed.seeds,.none.were.able.to.survive.to.flowering.when.planted.in.a.research.vineyard.at.Leesburg,.Florida.(Gray,.personal.observation)..Further,.when.seedless.varieties.are.used,.there.is.no.opportunity.for.seed.dispersal..Thus,.frequency.of.natural.self.versus.cross-fertilization.should.be.investigated.

potentIal ImpactS of tranSGene flow In GrapevIne

Potential for Transgene Pollen Flow into Non-GE VineyardsTransgene. movement. into. non-GE. vineyards. as. a. consequence. of. pollen. flow.should.have.minimal. impact..This. is.because.only. the. embryo.and.endosperm,.which.are.encased.in.a.non-transgenic.hard,.durable.seed.coat,.can.become.trans-formed. by. pollen. from. a. transgenic. vine.. Our. current. research. using. the. GFP.marker.system.confirms.this..Grape.seed.is.discarded.whether.fruit.is.used.fresh.or. for. processed. products,. with. the. exception. of. use. for. oil. extraction.46. Thus,.if. seed. is. properly. disposed,. there. is. little-to-no. risk. of. transgene. movement..


Furthermore,.seedless.varieties.used.for.fruit,.raisins,.or.other.processing.contain.abortive.ovules.without.embryos.47,48

Potential for Contamination of Commercial Vineyards via SeedSeeds.could.be.distributed.from.transgenic.vines.into.non-transgenic.vineyards.via.animals.or.unintentionally.by.workers..However,.as.discussed.above,.the.selfed.seeds.that.typically.would.result.lack.vigor.and.plants.from.them.do.not.mature.to.flower-ing..Regardless,.even.if.selfed.or.hybrid.seeds.capable.of.flowering.were.produced,.the.plants.would.tend.to.be.out.of.place.and.discarded.in.the.normal.practices.of.a.commercial.vineyard.

Potential for Transgene Movement into Native Vitis PopulationsThere.are.native.species.and.feral.vines.of.Euvitis.grape.that.are.cross.fertile.with.transgenic. varieties.. The. possibility. that. transgenes. may. be. transferred. to. native.populations.of.Vitis.centers.both.on.the.frequency.of.selfing.versus.out.crossing.that.naturally.occurs.and.the.dynamics.of.pollen.flow..If.only.selfed.seeds.are.produced,.inbred. progeny. transferred. from. transgenic. plants. into. the. wild. will. tend. to. lack.sufficient.vigor.to.survive..How.frequently.hybridization.occurs,.how.far.viable.pol-len.moves,.and.whether.hybrid.progeny.containing.transgenes.for.disease.resistance.have.a.competitive.advantage.in.the.ecosystem.are.issues.that.should.be.considered..For.example,.if.out.crossing.occurs.to.produce.hybrid.seed.among.transgenic.vines,.or.if.transgenes.are.detected.in.seeds.of.outlying.vines,.or.progeny.are.more.vigorous.in.nature.than.their.parents,.more.stringent.management.schemes.might.be.required.than.if.transgene.flow.is.found.to.be.restricted.or.its.consequences.benign.

mItIGatInG envIronmental rISkS vIa cISGenIcS enGIneerInG

The.advancements.in.molecular.genetics.and.its.application.now.allow.a.genetic.engi-neering. approach. that. utilizes. only. grape-derived. genes. (cisgenes). and.genetic. ele-ments.23.Such.a.cisgenic.approach.to.genetic.engineering.solves.issues.related.to.the.release.of.foreign.genes.into.the.environment.and.should.overcome.reluctance.to.GM.foods,.which.normally.contain.foreign.genes.and.gene.products.36.There.are.a.number.of.grape-derived.(endogenous).genes.already.available. for. testing..Furthermore,. the.common.use.of.grafting.in.viticulture.may.allow.transgenic.rootstocks.to.be.utilized.to.which.non-transgenic.scions.could.be.grafted.

Endogenous GenesCertain. concerns. about. implementation. of. GE. technology. might. be. allayed. if.endogenous. genes. from. the. grape. genome. itself. were. used. for. genetic. improve-ment.through.overexpression.strategies..Recent.advances.in.plant.molecular.biology.including. whole. genome. sequencing,. rapid. cloning,. and. efficient. gene. expression.techniques.have.greatly.facilitated.efforts.to.exploit.plant.genes.associated.with.agro-nomic.traits.based.on.manipulation.of.native.endogenous.genes.already.existing.in.a.plant’s.genome.49.Manipulation.of.endogenous.genes.includes.removal.of.introns.and.codon.optimization.to.increase.efficiency..Endogenous.genes.may.be.placed.in.a.construct.with.a.promoter.chosen. to.modify.expression.characteristics.and. then.


reinserted.into.the.plant..Such.genes.often.modify.metabolism.in.a.manner.similar.to.natural.or.induced.mutations.without.causing.genetic.contamination.49.The.improve-ments.achieved.via.use.of.such.endogenous.genes.should.be.qualitatively.similar.to.those.obtained.via.traditional.breeding.approaches,.but.significantly.more.precise,.due.to.the.ability.to.overexpress.desired.proteins,.both.temporally.and.spatially..In.addition,.endogenous/homologous.transgenes.can.be.defined.and.regulated.differen-tially.as.compared.to.use.of.foreign.transgenes.for.crop.genetic.engineering.50.It.is.possible.that.transgenic.modification.of.native.genes.tends.to.inherently.balance.the.selection.process.and.maintain.natural.variance.49

PR ProteinsA.number.of.genes,.commonly.known.as.PR.(pathogenesis.related).protein.genes,.have. been. cloned. from.and. expressed. in. the. plant. species. from. which. they. were.cloned..They.have.been.grouped.(PR.1–PR.5).based.on. their.structure.and.mode.of.action.51.A.number.of.PR.proteins.exhibit.antifungal.properties,.which.variously.cause.inhibition.of.fungal.cell.wall.synthesis.and/or.a.disruption.in.cell.wall.struc-ture.leading.to.cell.lysis.52.PR.proteins.are.classified.into.different.groups.including.cysteine.rich.antimicrobial.peptides,53–57.glucanases.and.chitinases,52.chitin.binding.proteins53,58.and.thaumatin.like.(TL).proteins.52

The.PR.5,.or.TL.proteins,.share.significant.amino.acid.homology.to.thaumatin.52.TL.proteins.inhibit.a.wide.range.of.plant.pathogens.in.vitro52.and.have.been.cloned.from.several.plant.species.59–62.TL.proteins.are.known.to.be.differentially.expressed.in. reproductive. tissues. such. as. pistils. and. ripening. fruits.63–66. The. University. of.Florida/Institute.of.Food.and.Agricultural.Sciences.(UF/IFAS).grape.biotechnology.laboratory.cloned.V. vinifera.thaumatin-like.protein.(VVTL-1).gene.from.grapevines.derived.from.embryogenic.cultures.that.were.subjected.to.in.vitro.selection.with.the.culture. filtrate. of. Elsinoe ampelina,. the. causal. agent. of. grapevine. anthracnose.67.Protein.produced.from.VVTL-1.significantly.inhibited.E. ampelina.spore.germina-tion.and.hyphal.growth.in.vitro..Plants.regenerated.from.in.vitro-selected.cultures.similarly.inhibited.fungal.growth.in.leaf.assays..Similar.results.have.been.obtained.with.VVTL-2.cloned.from.grape,.which.was.expressed.in.leaves.and.ripening.berries.in.response.to.powdery.mildew.infection68,69.and.exhibited.antifungal.properties.70

Seed ProteinsSeeds.contain.a.wide.array.of.antimicrobial.compounds.that.can.be.expressed.in.a.constitutive.or.inducible.manner.71.Although.2S.albumins.are.generally.considered.as. storage. proteins,. they. also. inhibit. growth. of. pathogenic. fungi. in. part. by. per-meabilization.of.hyphal.plasmalemma.72,73.2S.albumin.genes.have.been.cloned.from.sunflower,74.Brazil.nut,75.grape,76.and.other.crop.plants..In.grape,.we.have.cloned.the.entire.2S.albumin.gene.along.with.the.coding.sequence.and.promoter.elements76.and.we.are.evaluating.its.expression.(Li.et.al.,.unpublished.data).

Stilbene SynthasePhytoalexins. are. low.molecular.weight. antimicrobial.compounds. that. are. synthe-sized.by.and.accumulate.in.plants.in.response.to.biotic.and.abiotic.stresses..Stilbenes.and.their.derivatives.have.been.regarded.as.phytoalexins.that.contribute.to.defense.


against.fungal.infection.77.Stilbene.synthase.is.the.key.enzyme.that.catalyzes.the.formation.of.resveratrol.via.phenolic.substrates.in.the.shikimic.acid.pathway..Stilbene.biosynthesis.only.specifically.requires.the.presence.of.stilbene.synthase..The.family.of.stilbene.synthase.genes.have.been.cloned.and.characterized.from.grape.78.Studies.with.transgenic.apple,79.papaya,80.rice,81.tobacco,77.wheat,.and.barley82.overexpress-ing.the.STS.1.gene.from.grape.reported.enhanced.resistance.to.various.fungal.dis-eases.like.blast,.fruit,.root,.and.stem.rot.

Polyphenol OxidasePolyphenol. oxidases. (PPOs). are. nuclear-encoded. enzymes. of. almost. ubiquitous.distribution. in.plants.and.are.known. to.catalyze. the.oxygen.dependent.oxidation.of. phenols. to. quinones.83. Systemic. induction. of. PPO. in. response. to. wounding.and. pathogen. attack. may. protect. plants. against. further. attack. by. pathogens. and.insects.84.PPOs.have.been.cloned.from.a.number.of.plant.species.including.grape,85.potato,86.tomato,87.and.Vicia.species.88.Transgenic.plants.overexpressing.PPO.were.found.to.exhibit.enhanced.resistance.to.bacterial.diseases.caused.by.Pseudomonas synringae.89,90.Additionally,.an.antisense.downregulation.of.polyphenol.oxidase.in.transgenic.tomato.plants.resulted.in.increased.disease.susceptibility.as.compared.to.non-transgenic.controls.90.Although.only.bacterial.resistance.has.been.specifically.demonstrated,.PPOs.are.considered.to.have.broad.antimicrobial.activity.

Marker-Free PlantsGenetic.transformation.requires.a.method.to.separate.transformed.cells.from.non-transformed.cells.and.typically.is.achieved.by.expression.of.a.marker.gene.that.pro-vides.a.growth.advantage.to.transformed.cells.in.a.selection.medium.(reviewed.by.Dutt.et.al.91)..In.the.absence.of.a.selectable.marker.gene,.transformed.cells.tend.to.be.at.a.competitive.disadvantage.compared.to.nontransformed.cells.and.die.out..The.marker.gene.is.needed.only.for.selection.of.transgenic.cells.and.typically.is.linked.to.a.gene.of. interest..Thus,.selection.for.ability. to.proliferate. in. the.presence.of.a.selective.agent. results. in. isolation.of. transgenic. cells.containing.both. the.gene.of.interest.and.the.marker.gene..However,.once.a.desired.plant.is.selected.from.trans-genic. cells,. the. marker. gene. is. no. longer. needed.. The. presence. of. marker. genes.may. complicate. future. commercialization. due. to. concerns. regarding. their. effects.on.ecosystems.and/or.human.health.91.For.example,.one.concern. is. that.selectable.marker.genes.could.become.transferred.to.other.organisms,.leading.to.the.creation.of.antibiotic.resistant.bacterial.strains.(from.antibiotic.resistance.markers).or.new,.aggressive.weedy.plant.species.(from.herbicide.resistance.markers)..Development.of.transgenic.plants.without.marker.genes.alleviates.such.concerns..Dutt.et.al.91.used.a.cotransformation.treatment.to.demonstrate.production.of.transgenic.grapevines.free.of.the.selectable.marker.gene(s).that.are.typically.required.only.for.initial.identifica-tion.and.selection.of.transgenic.cells..Transgenic.V. vinifera.‘Thompson.Seedless’.plants.containing.only.a.single.stably.integrated.gene.of.interest.and.not.a.selective.marker.gene.were.produced..The.system.provided.the.possibility.of.producing.plants.that.contain.only.a.gene.of.practical.use.while.preventing.the.formation.of.genetic.chimeras.that.might.occur.through.meristem.transformation..The.technique.can.be.


used.readily.to.produce.grapevines.with.a.range.of.other.nonselectable.genes.and.may.be.adapted.for.use.in.other.vegetatively.propagated.crops.as.well.

RootstocksGrafting.of.scion.to.a.rootstock.is.commonly.used.for.grapevine.in.most.production.areas..The.possibility.of.using.a.transgenic.rootstock.that.resists.soil.stresses.and/or.transmits. a. useful. substance. into. the. scion. is. intriguing. for. several. reasons.. For.example,.only.the.non-transgenic.scion.would.reproduce.pollen.or.seed,.so.that.there.would.be.no.possibility.for.transgene.flow.to.occur,.and.virtually.any.scion.could.be.grafted.onto.a.transgenic.rootstock,.thus.reducing.the.number.of.transgenic.variet-ies.to.be.developed.and.tested..Rootstocks.that.produce.lytic.peptides.to.inhibit.the.xylem-limited.bacterium.that.caused.Pierce’s.disease.of.grapevine.are.in.field.tests.92.The.direct.vascular.connection.of.rootstock.to.scion.allows.lytic.peptide.produced.and.accumulated.in.rootstock.xylem.to.be.transported.into.the.scion.93

CONCLUSION

Genetic.transformation.of.grape.is.no.longer.an.obstacle.to.progress.in.development.of.improved.transgenic.grapevines..However,.it.is.difficult.to.predict.when.a.trans-genic.grape.product.will.become.commercially.available..While.it.is.now.possible.to.insert.virtually.any.gene.of.interest.into.an.increasingly.wide.range.of.elite.grape.varieties,.field. testing.has.been. insufficient. to.prove. the.expected.efficacy.of.new.traits;.one.problem.being.the.time.required.to.evaluate.such.a.perennial.fruit.crop..Also,.needs.to.conduct.risk.assessment.studies.and.develop.consumer.acceptance.of.any.transgenic.crop.have.proven.to.be.costly.and.difficult..One.alternate.approach.to.reducing.the.concerns.associated.with.transgenic.plants.is.to.adopt.the.aforemen-tioned.cisgenics.approach.to.crop.development.wherein.only.genes.and.other.genetic.elements. taken.directly. from.grapevine. are.used..Such.a.green.approach. is.more.akin.to.“precision.breeding”.inasmuch.as.it.disrupts.the.plant.genome.even.less,.and.is.more.predictable,. than.conventional.breeding..Similarly,. the. implementation.of.transgenic.rootstocks.to.combat.diseases.in.the.scion.could.have.a.similar.impact..We.believe.that.grapevines.developed.in.this.manner.to.have.improved.disease.resistance.and.quality.attributes.will.be.desirable.to.growers.and.be.accepted.by.consumers.

REFERENCES

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crops

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Agriculture

As the world debates the risks and benefits of plant biotechnology, the proportion of the global area of transgenic field crops has increased every year, and the safety and value continue to be demonstrated. Yet, despite the success of transgenic field crops, the commercialization of transgenic horticultural crops (vegetables, fruits, nuts, and ornamentals) has lagged far behind. Transgenic Horticultural crops: challenges and opportunities examines the challenges for the creation and commercialization of horticultural biotechnology and identifies opportunities, strategies, and priorities for future progress.

A “must read” for anyone working in the fields of genetic engineering or plant breeding, for policy makers, educators, students, and anyone interested in the issues of genetic engineering of fruits, vegetables and ornamentals, this book covers:

• Past achievements, newest developments, and current challenges in transgenic fruit, nut, vegetable, ornamental, and pharmaceutical crops • Reviews transgenic horticultural crops in the U.S., Europe, Africa, and Asia • Hurdles to the commercialization of transgenic technology in economics and the marketplace, consumer acceptance, intellectual property rights protection, public–private partnership, and regulation • Critical evaluation of the benefits and risks of genetically engineered horticultural crops, including risk assessment and transgene containment • Presents case studies and an industry perspective on transgenic horticultural crops

The production and commercialization of transgenic horticultural crops are enormous tasks—their progress and realization require an informed research community, horticultural industry, government, and body of consumers. To aid in this effort, this book provides facts, analyses and insights by leading experts in this field to inform a wide audience of students, agricultural and genetic professionals, and the interested public. Part of the global conversation on the pros and cons of transgenic foods, Transgenic Horticultural crops aims to stimulate more interest and discussion on the subject and to promote the development of safe and sustainable genetically modified horticultural crop varieties.

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