Plant Biotechnology and Agriculture Prospects for the 21st Century

E d i t e d b y

Arie Altman Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture Hebrew University of Jerusalem Rehovot, Israel

Paul Michael Hasegawa Bruno C. Moser Distinguished Professor Horticulture and Landscape Architecture Department Purdue University West Lafayette, Indiana, USA

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Contents

Contributors xxi

Foreword xxv Roger N. Beachy

Preface xxvii Me Altman and Paul Michael Hasegawa

Introduction to plant biotechnology 2011: Basic aspects and agricultural implications xxix Arie Altman and Paul Michael Hasegawa

Section A Introduction to basic procedures in plant biotechnology 1 1 Genetics and genomics of crop domestication 3

J.S. (Pat) Heslop-Harrison, Trude Schwarzacher Plants and Domestication 3

Scope 3 Domesticated crops 3 Weeds 4 Invasive species 4 Model species and crop sciences 5

Understanding Domestication Processes 5 Evidence of relatives and processes of early domestication 5 Genes of domestication 6 Genetic variation and domestication 6 Genetic control related to diversity and speciation 6 Domestication of maize 7 Domestication of legumes 7 Yield traits 8

Hybrid Species and New Polyploids in Domestication 8 Post-Domestication Selection 8

Modifications in crop characteristics 8 New Domestication 9

Domesticated species 9 Lost crops 9 Trees and biofuels 9 Genetics and breeding for new uses: Ecosystem services 10

Features of Domesticated Genomes 11 Superdomestication 14 Acknowledgments 16

2 The scope of things to come: New paradigms in biotechnology 19 Maheshi Dassanayake, Dong-Ha Oh, Dae-Jin Yun, Ray A. Bressan, John M. Cheeseman, J. Hans Bohnert Introduction 19

2 Contents

Progress Enabled by Next-Generation DNA Sequencing 20 Mapping of comprehensive, genome-wide, treatment-specific transcript profiles 23 Current next-gen sequencing 23 Behold the third generation 23

The Elephant in the Laboratory: Data Handling 24 From Sequences to Comparative Genomics 24

Transcriptome profiling 25 Broadening the Genomics Toolbox: Proteins and Metabolites 26

Proteomics advances 26 Metabolomics highlights 26

Genomics Unlimited: Getting Beyond Mere Genes 27 Into the Future: Genomics-Based Biotechnology and Agriculture 28

From models to crops, from labs to fields 28 Genetic resources from extremophile species 29 Exploring "unknown unknowns" 29 The importance of stress "tolerance" engineering 29

Acknowledgments 30

3 Protein targeting: strategic planning for optimizing protein products through plant biotechnology 35 Elizabeth Hood, Carole Cramer, Giuliana Medrano, Jianfeng Xu Introduction: Strategic Decisions about How to Express an Output Trait 35 Approaches 37

Routing proteins to the endomembrane system 37 Accumulating proteins in the ER 38 Accumulating proteins in ER-derived protein bodies 39 Accumulating proteins in the vacuole or vacuolar protein bodies 39 Accumulating proteins in the apoplast 40 Accumulating proteins in the chloroplast 40 Accumulating proteins on the surface of oil bodies 41

Seed-Based Expression Systems 41 Leaf Systems 44

Stable versus transient leaf expression systems 44 Protein bodies in leaves 47

Hairy Root Cultures 47 Advantages of the hairy root culture system 48 Recombinant proteins expressed with hairy root cultures 48 Hairy root cultures in bioreactors and scale-up 48

Summary and Conclusions 50

4 Proteomics and its application in plant biotechnology 55 Sylvain Bischof, Jonas Grossmann, Wilhelm Gruissem Introduction 55 Mass Spectrometry-Based Proteomics 56

Sample preparation prior to mass spectrometry 56 Mass spectrometry 57

- Spectra assignment for peptide and protein identification 58 Quantitative proteomics 58 Post-translational modifications 58

Proteomics in Plant Biotechnology 59 What has been achieved so far in crop proteomics? 59 Arabidopsis thaliana as plant model organism 59 Crops and other economically relevant plant species 60 Future applications and perspectives 61

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5 Plant metabolomics: Applications and opportunities for agricultural biotechnology 67 Diane M. Beckles, Ute Roessner Introduction 67 Metabolite Networks: The Basics 68 Metabolomics: Technologies for Analyses 69

Analytical platforms 70 Data analysis and interpretation 71

Metabolomics: Applications in Agricultural Biotechnology 73 Metabolite profiling to test substantial equivalence 73 Phytochemical diversity, phenotyping, and classification.... 74 Postharvest quality of horticultural crops 74 Stress responses 74 Functional genomics 75 Breeding and metabolite quantitative trait loci 75

Metabolomics: Challenges and Future Perspectives 76 From model organisms to crop plants 76 Compartmentation of plant metabolism 76 High-resolution sampling 76 Primary and secondary metabolism pose different challenges 76 Identifying the metabolome 77 Measurements of metabolic flux 77

Outlook 78 Acknowledgments 78

6 Plant genome sequencing: Models for developing synteny maps and association mapping.... 83 Delphine Fleury, Ute Baumann, Peter Langridge Introduction 83 Genome Sequencing 84

Strategies for plant genome sequencing 84 High-throughput sequencing technologies 86 Single molecule and real-time sequencing 86 Assembly and alignment programs 86 Genome browsers 87

Models for Developing Syntenic Maps 88 Definitions 88 Intraspecies comparison 88 Cytogenetics for interspecies comparison 89 Sequence comparison 89 Macro- versus micro-synteny 89 Nature of the differences 89 Applications of syntenic maps 91 Tools and limitations 91

Association Mapping 91 Definitions 91 Population size and structure 92 Markers and marker density .( 93

Implications 94

7 Agrobacterium-rr\ed\a\ed, plant genetic transformation 99 Yoel Shiboleth, Tzvi Tzfira Introduction 99 The Genetic Transformation Process 99 Agrobacterium as a Tool for Plant Transformation 104 Novel and Specialized Vectors for Plant Transformation 106 Manipulating the Plant Genome to Improve and Control Transformation 108

1 Contents

Using Novel Selection Methods and Restriction Enzymes to Control T-DNA Integration 109 Conclusions and Future Prospects 110 Acknowledgments 111

8 Biolistic and other non-Agrobacterium technologies of plant transformation 117 Trade K. Matsumoto, Dennis Gonsalves Introduction 117 Other Non-Agrobacterium Transformation 117

Electrophoretic transfection 117 Electroporation 118 Bioactive-beads-mediated gene transfer 118 Microinjection 118 Pollen-tube pathway 119 Silica carbide whisker-mediated transformation 119

Biolistic Transformation 120 The invention 120 Electric discharge particle acceleration 120 Current status of the "invention" hardware 121

Advantages of Biolistic Transformation 121 Implications of Biolistics in Agricultural Biotechnology 122

Application of biolistics in agriculture crops 122 Papaya: A case study of biolistic transformation 122

9 Plant tissue culture for biotechnology 131 Prakash P. Kumar, Chiang Shiong Loh Introduction 131 Plant Tissue Culture Technology 131

The basic laboratory setup 131 Preparation of tissue for culturing 132 Nutrient media 132 Types of culture 133 Environmental aspects of tissue culture 133 Modes of regeneration 134

Implications for Agricultural Biotechnology 134 Haploid tissue culture 135 Somatic embryogenesis 135 Artificial seeds 135 In vitro flowering 136

Future Perspectives 136 Acknowledgments 136

Section B Breeding biotechnologies 139 10 Somatic (asexual) procedures (haploids, protoplasts, cell selection) and their

applications 141 Tanya Tapingkae, Zul Zulkarnain, Masayo Kawaguchi, Takashi Ikeda, Acram Taji General Introduction 141 Somatic Embryogenesis 141

Introduction 141 Patterns of somatic embryogenesis 142 Factors affecting somatic embryo induction 142 Plant maturation 143 Plant regeneration 144

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Gene expression during somatic embryogenesis 144 Mass propagation and somaclonal variation 144

Haploid Technology 144 Introduction 144 Cytological basis underlying haploids plants induction 145 Factors affecting the induction of microspore embryos 146 Haploid induction via ovary and ovule cultures 147

Protoplast and Somatic Hybridization 148 Introduction 148 Types of somatic hybrids 148 Protoplast fusion methods 148 Selection of somatic hybrids 150 Identification of somatic hybrids 150 Factors affecting regeneration of hybrid plants 151

Screening and Development of Stress-Resistant Plants Using in vitro Selection Techniques 151 Introduction 151 General methods of screening and breeding using in vitro selection techniques 151 Biotic stress resistance 152 Abiotic stress tolerance 152 Future perspective of screening and breeding using in vitro selection techniques 155

Conclusions and Future Directions 155 Acknowledgments 155

11 Marker-assisted selection in plant breeding 163 Giora Ben-Ari, Uri Lavi Background 163

The concept of marker-assisted selection 163 Historical review 164

Plant Traits, DNA Markers, Technologies, and Applications 164 Genes controlling important traits 164 DNA markers 165 Modern genotyping technologies 168 Identification of genes controlling commercially important traits 170 Application of DNA markers to breeding 173 MAS in breeding programs 174

Discussion 176 Bottlenecks and difficulties in the application of MAS 176 Future prospects of application of genetic variations to breeding 177

Acknowledgment 178

12 Male sterility and hybrid seed production 185 Sally Mackenzie Introduction 185 Male Gametogenesis 185

Pollen mitosis 1 185 Pollen mitosis II 186

Male Sterility Mutants Elucidate Anther Development 187 Hormonal Influences on Male Reproduction in Plants 187

Gibberellic acid 187 GA regulates jasmonic acid biosynthesis 188 Brassinosteroids 188 Auxins 189

Cytoplasmic Male Sterility Systems in Agriculture 189 Plant mitochondrial mutations 189 Fertility restoration 189 Stability of the CMS trait 190

Contents V

Male Sterility: Metabolic and Evolutionary Implications 190 CMS is a naturally found condition 190 Organelle metabolism influences pollen development 190

Genetic Engineering of Male Sterility 191 Implementation of Male Sterility in Agricultural Systems 191

13 Advances in identifying and exploiting natural genetic variation 195 Christian S. Hardtke, Kaisa Nieminen Natural Genetic Variation in Crop Breeding: From Prehistory to the Green Revolution 195 The Genetic Limits of Evolving Domesticated Crops 196

; Tapping the natural genetic variation present in wild ancestors 196 Natural Genetic Variation in Arabidopsis 197 QTL Analyses in Arabidopsis 197

Novel Arabidopsis genes isolated through the natural variation approach 198 What to Expect: Intraspecific Variation in Gene Structure and Content 198

| Structural genome variation: Higher than expected? 198 QTL Analysis and Sequence Variation in Crops 199

Domestication genes of maize 199 Examples from rice 199 Examples from other cereals 200

Toward Prediction of Variation in Molecular Function: Why Model Organisms are here to Stay 200 Crucial support from model organism candidate genes 200 Model systems as references to characterize allele activities 201

Beyond Simple Traits: Epigenetics, Heterosis, Genetic Incompatibility, and Trade-offs 201 I Incompatibility between natural accessions 201 \ Trade-offs between different beneficial traits 202

Extending the Toolbox: Genome-wide Association Mapping 202 The Route to Effectively Exploit Natural Variation for Plant Biotechnology 202

14 From epigenetics to epigenomics and their implications in plant breeding 207 I Athanasios Tsaftaris, Aliki Kapazoglou, Nikos Darzentas

Mechanisms of Epigenetic Inheritance and their Interactions 207 Introduction 207 Epigenetic mechanisms and their interactions 208

From Epigenetics to Epigenomics 212 Deciphering epigenomes: A matter of scale and complexity 212 Epigenomic methods and the type of data collected 212 Epigenomic resources 213 Transposable elements on the emerging epigenomic landscape(s) 216 An illustrative and practical example of data and resources integration 217

Epigenetic Phenomena and their Implications in Plant Breeding 217 Epigenetic controls during vegetative development and the role of the environment 217 Epigenetic control of flowering 219 Endosperm development and parental imprinting 220

Conclusions and Prospects 222 Acknowledgments 222 Abbreviations 222

Section C Plant germplasm 227 15 An engineering view to micropropagation and generation of true to type and

pathogen-free plants 229 Eli Khayat Preface 229

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Shoot Multiplication Through Meristem Culture 229 Stage 0: disinfection and start of axenic culture 230 Stage I: Initiation of culture 230 Stage II: Multiplication 230 Stage III: Elongation and promotion of shoots and roots development 231 Stage IV: Acclimatization and hardening 231

Automation 231 Energy and Lights 232 Photoautotrophic Cultures 232 Micropropagation in Liquid Media 233 Plant—Microbe Interaction During in vitro and ex vitro Stages of Micropropagation 233 Inoculation with Beneficial Microorganisms 234 Elimination of Viruses by in vitro Techniques 238 Concluding Remarks 238 Acknowledgments 238

16 Regulation of apomixis 243 Peggy Ozias-Akins, Joann A. Conner Introduction 243 Overview of Ovule Development During Sexual Reproduction 244 Overview of Ovule Development During Apomictic Reproduction 244 Germline Specification 244 Apomeiosis 246 Megagametogenesis 247 Gamete Specification 247 Parthenogenesis 248 Endosperm Development 250 Chromatin Modification and Epigenetic Regulation 251 Conclusions and Future Prospects for Apomixis in Crops 251

17 Germplasm collection, storage, and conservation 255 Florent Engelmann Introduction 255

Strategies for conserving plant biodiversity 255 Ex situ conservation technologies 256

Applications of Biotechnologies for Conservation 257 In vitro collecting 257 Slow growth storage 258 Cryopreservation 259

Conclusions 264

Section D Controlling plant response to the environment: Abiotic and biotic stress 269

18 Integrating genomics and genetics to accelerate development of drought and salinity tolerant crops 271 Zvi Peleg, Harkamat Walia, Eduardo Blumwald Impact of Abiotic Stresses on Crop Plant Productivity 271 Water Deficit: A Major Abiotic Stress Factor 272 Salinity 272 Plant Responses to Abiotic Stress 272 Breeding for Drought and Salinity Tolerance: "The Conventional Approach" 273

Germplasm resources for drought and salinity tolerance 274 Genetic dissection of plant responses to abiotic stress 274 Introducing new technologies for abiotic stress breeding 275

Contents

Engineering-Tolerant Crop Plants: The Transgenic Approach 275 Genes for osmoregulation 275 Dehydration-responsive element 278 NAC proteins 279 Genes for ionic balance 279 Genes for redox regulation 279 Other transcription factors 280

Hormones and Abiotic Stress 280 Challenges and Prospects 280 Acknowledgments 281

19 Molecular responses to extreme temperatures 287 Rafael Cataia, Aurora Diaz, Julio Salinas Introduction I. 287 Plant Response to Low Temperature 287

Low temperature perception 288 Transducing the low-temperature signal 289

Cross-talk between Plant Responses to Extreme Temperatures 297 The membrane as a node in the perception of temperature oscillations 298 Transducing the signals initiated by temperature variations 298

Conclusions 300 Acknowledgments 301

20 Biotechnological approaches for phytoremediation 309 Om Parkash Dhankher, Elizabeth A. H. Piion-Smits, Richard B. Meagher, Sharon Doty Introduction 309

Overview of results from biotechnological approaches for different pollutants 311 Organic pollutants 317

Future Prospects 323 Acknowledgments 323

21 Biotechnological strategies for engineering plants with durable resistance to fungal and bacterial pathogens 329 Dor Salomon, Guido Sessa Introduction 329 Choosing the Target gene for Transgenic Expression 330

Plant immune receptors mediating pathogen recognition 330 Elicitors of plant immunity 331 Plant genes involved in signaling networks of plant immunity 332 Antimicrobial genes 334 Genes targeting pathogen virulence determinants 335

How Many Transgenes Should be Expressed in a Single Plant for Efficient Disease Control? 335 When and Where Should the Transgene(s) be Expressed? 336

Pathogen-responsive and tissue-specific promoters 337 Pathogen-responsive elements and synthetic promoters 338

Conclusions and Prospects 339 Acknowledgments 339

22 Controlling plant response to the environment: Viral diseases 343 Munir Mawassi, Abed Gera Introduction 343 Phytosanitation and Quarantine Regulation 344 Transmission of Plant Viruses 344

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Cultural Strategies of Virus Control 344 Management of soil-borne viruses 344 Management of airborne viruses 345

Resistance to Virus Transmission by Insects 345 Pathogen-derived resistance 345 RNA-mediated resistance 346

Application of the PDR Concept for Developing Transgenic Virus Resistance to Horticultural Crops 346 RNA silencing-based applications for developing virus resistant plants 347 PDR stability and suppression of RNA silencing 348

Assessment of Risks Associated with Transgenic Virus Resistance in Plants 348 Conclusion 349

23 Insects, nematodes, and other pests 353 Philip R. Watkins, Joseph E. Huesing, Venu Margam, Larry L. Murdock, T.J. V. Higgins Introduction — Genetically Modified Crops for Insect Resistance 353

History of B. thuringiensis ; 353 Cry proteins 354

Commercially Available Insect Protected Crops 354 Bt maize 354 Bt cotton 356 Discontinued Bt crops 357

Bt Crops Under Development 357 Bt brinjal 357 Bt rice 358 Other Bt crops 358

Impact of Bt 359 Benefits of Bt crops 359 Concerns about Bt crops 359 Improving Bt 360

Cowpea Trypsin Inhibitor 360 Novel Insecticidal Protection 361

VIP genes 361 Microorganism-derived toxins 361 Plant-derived toxins 361 Secondary metabolites 362 Other toxins 363 RNAi 363

Nematode-Resistant Crops 364 Recombinant Insecticides 364 Conclusion 364

Section E Biotechnology for improvement of yield and quality traits 371

24 Growth control of root architecture 373 Christopher N. Topp, Philip N. Benfey Introduction to Root System Architecture 373 Genetic and Developmental Aspects of Root Growth 373

Stereotypical organization of root tissues 374 Architectural possibilities 374 Signaling 375 Systems biology concept of cell identity 376

Plant-Environment Interactions 376 Environmental sensing and root exudation 376 Microbial interactions 377 Architectural responses to nutrient availability 378

Contents

Crop Root Systems 379 Types of root systems 379 Embryonic and post-embryonic root systems 379 Evolutionary strategies and trade-offs 380

Approaches to Study Root Architecture 380 Quantitative analysis 380 High-throughput sequencing 381 Phenomics 381

Concluding Remarks 382

25 Control of flowering 387 Alon Samach Introduction 387

A plant's perspective 387 A farmer's perspective 387

Proteins Controlling Flowering Time 388 Florigen and FLOWERING LOCUS T (FT) 388 Transcription factors regulating FT 389 Proteins parallel or downstream of FT. 390

Processes Affecting Flowering Time Proteins 391 Histone modifications 391 Gibberellin 392 MicroRNAs 393 The circadian clock 394 Regulated proteolysis 394 Sugars 394

Developmental Decisions on Timing of Flowering 395 Juvenility 395 Seasonality 395 Reproductive cycles and alternate bearing 397

Summary 398 Acknowledgment 398

26 Fruit development and ripening: A molecular perspective 405 Avtar K. Handa, Martfn-Ernesto Tiznado-Hernandez, Autar K. Mattoo Fruit Classification 405 Fruit Development 406

Fruit shape, size, and mass 406 Fruit Ripening 409

Ripening mutations 409 Nutritional mutations 411 Shelf life mutations 412

Ethylene and Fruit Ripening 413 Ethylene biosynthesis 413 Ethylene perception and signal transduction 414 Genetic intervention in ethylene biosynthesis and perception 416

Fruit Texture 417 Cell wall depolymerizing enzymes 417 Expansins 418 Protein glycosylation 418

Future Perspectives 418

27 Potential application of biotechnology to maintain fresh produce postharvest quality and reduce losses during storage 425 Amnon Lers Introduction 425

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Ethylene Biosynthesis or Perception and Its Relation to Postharvest Quality of Fresh Produce 426 Senescence in Postharvest of Leafy Vegetables and Flowers 427

Background 427 Senescence regulatory genes . 427 Senescence-associated hormone biosynthesis or perception 428 Oxidative stress involvement in senescence 429 Chlorophyll degradation 429

Abscission of Fruits, Flowers, and Leaves During Postharvest 429 Background 429 Development of the dedicated AZ tissue 430 Regulatory genes involved in abscission control or mediating hormonal signal transduction 430 Genes involved in actual execution of cell separation in the later stage of abscission 431 Ethylene and abscission 431 Regulated manipulation of abscission 431

Reducing Postharvest Chilling Sensitivity 431 Background ., 431 Membrane structure and chilling sensitivity 432 Oxidative stress and chilling sensitivity or tolerance 433 Regulation of low-temperature responses 433 Molecules with protective functions during cold stress 434

Affecting Postharvest Texture and Appearance Qualities 435 Background 435 Softening and cell wall hydrolysis 435 Softening and turgor i. 435 Tissue lignifications 435

Implications for Plant and Agricultural Biotechnology 436

28 Engineering the biosynthesis of low molecular weight metabolites for quality traits (essential nutrients, health-promoting phytochemicals, volatiles, and aroma compounds) 443 Fumihiko Sato, Kenji Matsui General Introduction 443 Lessons from Essential Nutrients 444

Essential amino acids : 444 Fatty acids 446 Vitamins 446 Improvement of the bioavailability of minerals through metabolic engineering 449 Multigene transfer for improved food quality 449

General Strategy for the Engineering of Secondary Metabolites with Nutritional Value 449 Identification of biosynthetic genes 449 Identification of transcription factors and engineering through integrated "omics" 450 Modulation of organelle development 450

Quality Improvement of Plants as Functional or Medicinal Food 451 Resveratrol 451 Anthocyanins and flavonoids 452 Catechins and proanthocyanidins 452 Sesamins 452

Beloved Metabolites: Plant Volatiles 452 Biochemistry of plant volatile secondary metabolites 453 Flavor compounds in fruits 454 Scent/aroma of flowers 455 Volatile organic chemicals in vegetative organs of plants 455

Perspectives 456 Conclusion 458 Acknowledgments 458

Section F Plants as factories for industrial products, pharmaceuticals, biomaterials, and bioenergy 463

29 Vaccines, antibodies, and pharmaceutical proteins 465 Yuri Y. Gleba, Anatoli Giritch Introduction 465 Expression Technologies: Nuclear Transformation 466 Expression Technologies: Plastid Transformation 469 Expression Technologies: Transient Expression Systems 469

"Full virus" vectors 470 Magnifection 470 Derisking the new manufacturing process 471

Plant-Made Pharmaceuticals: A Unique Selling Proposition? 471 Plant-Based Manufacturing, Post-Translational Modifications, and Plant-Specific Sugars 472 Plant-Based Manufacturing and Downstream Issues 473 Plant-Based Expression Systems: Advantages and Limitations 474

Nuclear transformation 475 Plastid transformation 475 Transient expression 476

Conclusions and Outlook 476 Acknowledgments 476

30 Plants as factories for bioplastics and other novel biomaterials 481 Jan B. van Beilen, Yves Poirier Introduction 481 Major Natural Plant Biopolymers 482

Starch 482 Cellulose 482 Rubber 483 Proteins 485

Novel Polymers Produced in Transgenic Plants 485 A role for transgenic crops in the production of biopolymers? 485 Which biopolymers should be targeted for production in transgenic crops? 486 Which crops should be targeted? 487 Fibrous proteins 487 Cyanophycin 488 Polyhydroxyalkanoate 489

Conclusion and Prospects 491

31 Bioenergy from plants and plant residues 495 Blake A. Simmons Introduction 495 Biochemical Conversion 497

Comminution 498 Pre-treatment 499 Saccharification 500 Fuel synthesis 500

Thermochemical Conversion 501 Pyrolysis 501 Gasification 502

Concluding Remarks 503 Acknowledgment 503

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Section G Commercial, legal, sociological, and public aspects of agricultural plant biotechnologies 507

32 Containing and mitigating transgene flow from crops to weeds, to wild species, and to crops 509 Jonathan Gressel Introduction: Does Transgene Flow Matter? 509

Transgene flow: To what ecosystem? 510 Thresholds matter 511 Gene containment and/or mitigation is often necessary 511

Methods of Containment 511 Containment by targeting genes to a cytoplasmic genome 512 Male sterility 512 Rendering crops asexual 513 Genetic use restriction technologies alias "terminator" 513 Chemically induced promoters for containment 513 Recoverable block of function 514 Repressible seed-lethal technologies 514 Trans-splicing to prevent movement. 514 A genetic chaperon to prevent promiscuous transgene flow from wheat to its wild and weedy relatives 515 Transiently transgenic crops 515

Mitigating Transgene Flow 516 Demonstration of transgenic mitigation 516 Will transgenic mitigation traits adversely affect wild relatives of the crop? Models that suggests that mitigation is deleterious 517

Traits that can be Used in Tandem Transgenic Mitigation Constructs 518 Morphological traits and genes for mitigation 518 Chemical mitigation of transgene flow 519 Special cases where transgenic mitigation is needed 520

Concluding Remarks 521

33 Intellectual property rights of biotechnologically improved plants 525 Antoine Harfouche, Richard Meilan, Kannan Grant, Vincent K. Shier Introduction: Capitalizing on Research and Development in Agricultural Biotechnology with Intellectual Property Protection 525 Intellectual Property Protection of Biotechnologically Improved Plants 526

International intellectual property protection agreements 526 Types of intellectual property protection in plant biotechnology 528

Freedom-to-Operate in Agricultural Biotechnology: The Road from a Research Idea to Commercialization of a Biotechnologically Improved Plant Product 532 Technology Transfer as a Means to Facilitate the Development of Biotechnology-Based Agriculture 534 Conclusion and Future Needs 536 Acknowledgments 537

34 Regulatory issues of biotechnologically improved plants 541 Elizabeth £ Hood, Deborah Vicuna Requesens, Kellye A. Eversole Introduction 541 Commercializing an Agricultural Biotechnology Product 542 The Regulatory Framework 543

The U.S. Coordinated Framework. 545 Perspectives 547

Specialty crops regulatory assistance: A new paradigm 547

Contents

Standardization 548 Conclusions 548

35 Prospects for increased food production and poverty alleviation: What plant biotechnology can practically deliver and what it cannot 551 Martina Newell McGloughlin Introduction 551 Progress to Date 552 The Next Generation 554 Barriers to Introduction 557

36 Crop biotechnology in developing countries 563 Hugo De Groote Introduction 563 Agriculture and Food in Developing Countries: The Needs 564

Feeding a growing world population 564 Undernutrition and poverty 564 Technology 565

Current State of GM Crops 565 Geographic distribution 565 Crops, traits, and farmers 565 Future and trends 565

Economic Impact of Transgenic Crops in Developing Countries 567 Main effects of current GM crops 567 Empirical evidence of farm level benefits 567 Effect of GM crops on poverty and inequality 568 Combined effects on farmer income 568 Macro level impacts 569

Health Impact 569 Safety concerns 569 Nutritional benefits of biofortification 570 Nutritional impact of GM biofortification 570 Reduced exposure to toxins, pesticides, and anti-nutrients 571

The Environment 571 Consumer Acceptance of GM Food 572

Regional differences 572 Factors influencing acceptance 572

Regulatory Systems 573 Importance of regulatory systems 573 Regional differences 573 Economics of regulation 573 The way forward. 574

Conclusions 574 Acknowledgments 574

Index 577

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