beckles - biotechnology and postharvest quality · 2015-06-19 · advantages disadvantages 2....

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Diane M. Beckles

Department of Plant Sciences

University of [email protected]

http://www.plantsciences.ucdavis.edu/plantsciences_faculty/beckles/index.htm

BIOTECHNOLOGY AND POSTHARVEST QUALITY

Biotechnology

The application of molecular biology and novel analytical tools to:

Identify ‘key’ genes underscoring traits

Modify these genes toenhance selected traits

What Determines Traits ?

DNA

mRNA

Metabolite

Gene Gene

ProteinTraits

(Postharvest)

Sweetness

Texture

Aroma

Shelf-life

Low microbialadherence

Postharvest Quality

• “The factors that ensure maximum income for producers and meet the nutritional and aesthetic needs of the consumer after horticultural crops are harvested.” (Kader, 2002).

Texture

Flavour

AromaSweetness

Acidity

Color

Appearance

Nutrition

Shelf-life

Chilling-tolerance

ReducedMicrobes

Reduced Browning

FirmnessDiseaseresistance

CONSUMERS

It is often difficult to define quality and the causal genes

PRODUCERS

Traits are determined by the genetic make-up (genotype) of the organism and the environment

Ripeningmutant

25°C

25°C

NormaltomatoRipe

yDifferent Genotype Different Environment

Normaltomato

All crop plants have been genetically modified

These are all the products of human intervention. These plants have modified genomes that make them unfit for surviving in the wild.

Beckles, Diane "Biotechnology and Postharvest Quality" Postharvest Technology of Horticultural Crops Short Course 2015 (c) Postharvest Technology Center, UC Regents

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Biotechnological sophistication

Scientific Breeding

1903

Transgenic Technology

1994

Mutagenesis

1965

GenomeEditing

2012

Marker-Assisted Breeding

2009

*These timelines are approximate only

T.I.L.L.I.N.G

2005

Domestication

8-10,000YA

Natural mutations creates diversity

The effects of natural genetic mutations in carrots

Wieczorek, A. M. & Wright, M. G. (2012) History of Agricultural Biotechnology: How Crop Development has Evolved. Nature Education Knowledge 3(10):9

1. Domestication of Plants

5-10 000 years ago, natural mutations, changes in DNA, occurred in wild plants creating desirable traits. These ‘mutants’ were propagated by early farmers.

Wild banana

with seeds

Cultivated banana- sterile

Extreme in size in

cultivated tomato vs. wild fruit

2. Selective Breeding

Solanumlycopersicum

Solanumperuvianum

Percent of “wild” genes 50%

25%

12.5%Using the wild tomato species as a source of novel genes.6.25%

3.125%

1.5%

0.75% Kent Bradford, Department of Plant Sciences, UC Davis

Donor ParentRecurrent Parent

• Limited to sexually compatible crosses. Therefore the amount of variation that can be introduced is limited.

• Slow. Cumbersome and inefficient.

• Very successful.

• No regulatory hurdles.

• Technology accessible and relatively cheap.

• Potential may be untapped.

ADVANTAGES DISADVANTAGES

2. Selective BreedingPostharvest traits developed through selective breeding

Uniform Ripening1 Longer Shelf-life2

1) A mutant ‘uniform ripening’ gene was bred into most tomatoes in 1920s (US). 2) This made selection of the ‘correct’ maturation stage at harvesting was easier. 3) However the mutation also reduced sugars (10-15%) and flavor compounds. 4) Most heirloom tomatoes were not altered in this way.

-Rin is a naturalRipening mutant

-Poor organolepticquality but long

shelf-life

Normal tomatowith short shelf-lifeand good quality

Tomato withlonger shelf-life

good quality

X

1Powell et al (2012) Science 336 no. 6089 1711-17152.Kedear (1989) Long shelf life heterozygous tomato plant US 4843186 B1

Conventional Heirloom


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3. Marker Assisted Selection (MAS)

By applying genomics i.e. using DNA markers for selection of desirable progenyinstead of the phenotype, the breeding time can be cut in half or more.

• Limited to sexually compatible crosses. Therefore the amount of variation that can be introduced is limited.

• Cost of markers can be very high.

• Markers are not always reliable when plants are grown in different environments

• No regulatory hurdles.

• Marker development is becoming more accessible and cheaper.

• Enhances the efficiency of conventional breeding.

ADVANTAGESDISADVANTAGES

3. Marker Assisted Selection

Several Products Have Been Bred by Marker Assisted Selection – Example of One With Enhanced Postharvest Trait - Beneforte

Broccoli were bred with 2-3 times the amount of glucoraphanin, a compound that stimulates the body’s antioxidant (defense) system.

http://www.superbroccoli.info/why-beneforte

Whole Genomesequencing projects ofof several Horticultural

crops are complete(this list is already

outdated)

Now, 1000 bp of DNA sequence cost less than 5 cents to produce. Genotyping of all crops will soon be feasible and will enable efficient MAS.

4. MutagenesisSeeds* are exposed to a chemical or physical agent that causes a high frequency of changes in the DNA sequence of that organism. A mutation in a gene that determines an important trait will create a line with a new, desirable phenotype.

Mutagenesis has led to the production of 3088 varieties from 170 different plant species including some with improved postharvest traits.

http://www-naweb.iaea.org/nafa/pbg/ *Other plant parts may be mutagenized as well

• Inefficient: relies on random events.

• Screening for new phenotype is slow and expensive.

• High failure rate if the aim is to select for a particular phenotype.

• Can introduce genetic change quickly


• Little knowledge of the genome of the target plant needed.


4. Mutagenesis


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5. Targeted Induced Local Lesions In Genomes (TILLING).

Colbert T et al. Plant Physiol. 2001;126:480-484

Applying genomics to mutagenesis. Treated plants are first screened for genes with altered sequences i.e. gene mutations not for an altered phenotypes.

Mutant Melons

Ripening Firmness Shape Brix Time

ACO MutantControl

Dahmani-Mardas et al (2010) PLoS One vol 5 (12) 15776http://www.arcadiabio.com/extendedshelflife

Mutant ACC Oxidase melons found by TILLING have a longer shelf-life

Arcadia Biosciences(Davis, CA):

Long shelf-life:-Strawberry-Lettuce-Tomato

• Inefficient: relies on random events.

• Moderate-high failure rate.

• Limited in the types of changes that can be introduced i.e. mainly loss-of-function.

• Gene target must be known.

• Can introduce genetic change quickly.


• No DNA is added to the plant.


2. T.I.L.L.I.N.G

4. Transgenic manipulation

Gene Construct

Splice gene intoPlasmid vector

Gene of interest

Plasmid(Vector)

Plasmids are circular molecules of DNA naturally found in bacterial cells.

The gene to be modified Is usually introduced intothe plant using bacterialplasmid as vectors.

Step 1

A soil bacterium that naturally inserts its plasmid DNA into the plant genome.

DNA construct physically introduced into tissue

Transgenesis: Introducing the gene into the plant

Direct transfer Agrobacterium tumefaciens

Step 2.

The gene construct is integrated into a cell from which a whole plant may be regenerated

Only cells with recombinant DNA

survive and divide in culture

DNA inserted in plant chromosome.

Cells regenerate into transgenic plant p

Plantlet grow into plants with new traits

Step 3.

drawing by Celeste Rusconi, © Regents of the Univ. California


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Which method creates more genetic changes in a plant?

A. Mutagenesis.

B. Transgenic manipulation.

C. Conventional Breeding.

Ricroch et al (2011). Plant Physiology vol. 155 no. 4 1752-1761Herman & Price. (2013) Journal Agriculture Food Chemistry 61 (48) 11695–11701

Summary of how plants are genetically modified

Marker-Assisted SelectionTransgenic modification

Mutagenesis

http://www.nationalgeographic.com/foodfeatures/green-revolution/

Conventional Breeding

• Regulatory process for approving transgenic products for sale is very expensive and slow.

• Traditionally relied on using bacterial (foreign) genes.

• Many important traits in plants are complex and modifying these by knocking out single or multiple genes have not been successful.

• Distrust of GMOs by the public.

• Novel phenotypic variation can be introduced rapidly.

• Minimal alteration to the genome compared to other processes.



2. Transgenesis

* Moving away from this

Problems: Transgenic manipulation of plants offers many possibilities but has created a lot of angst among consumers

Chrispeels and Sadava, 2002 Plant, Genes and Crop Biotechnology; ASPB

Please note: Plants with animal genes have not been marketed.

Problem: GMOs create angst among the public and has driven the desire for labeling products as non-GMO.

A. Two

B. Three

C. Four

D. Five

TRUTH: Few fruits and vegetables are transgenic i.e. ‘GMO’

How many inaccuracies can you spot in this product description?

One of first plant GMOs: Flavr Savr tomatoes was altered in a postharvest trait. Extended shelf-life. Discontinued in 1999

Chrispeels & Sadava, 2002 Plant, Genes and Crop Biotechnology.

Transgenic Normal


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Lab GMOs: enhanced shelf-life In transgenic apples: ethylene biosynthesis suppressed

Control

Dandekar et al (2004) Transgenic Res 13 : 373-384

Transgenic

After 3 monthsat room

temperature

At harvest

*ACO = 1-aminocyclopropane-1-carboxylic acid oxidase

Normal

Lab GMOs: transgenic tomato expressing an anthocyanin gene.

http://www.norfolkplantsciences.com/projects/ripening-tomatoes/

Normal Red Tomato

Transgenic Purple Tomato

Transgenic papaya resistant to Papaya Ringspot Virus

California Agriculture vol 58 #2; http://CaliforniaAgriculture.ucop.edu

Transgenic papaya accounts for 90% of all grown in Hawaii.

Cultivated since 1999.

Normal Transgenic

Other commercialized transgenic horticultural crops

Ear Worm Resistant Sweet Corn

Florigene Moonshadow carnations

Normal Transgenic Normal Transgenic

Roundup Ready Sugarbeet

Virus Resistant Squash

Problem: Steady increases in the proportion of biotech crops grown but few are horticultural crops

A100-fold increase in area cultivated with GMOs since commercialization began in 1996. Littletranslation of technology from lab-to-table

http://isaaa.org/resources/publications/pocketk/16/default.asp

• Between 2003-2008:

• More than 77 specialty type crops transformed with 206 traits altered.

• Still only 5 transgenic lines currently on market for consumption: sweet corn, papaya, zucchini squash, sugar beet and Innate potato.

Miller & Bradford (2010) Nature Biotech 28: 1012-1214

Why there are few GM Horticultural Crops are on the market?• Getting GM crops to market is expensive.

• Estimates of regulatory costs:

• US$15 M per transgenic line; Time to market: 10 - 12 years.

• Fruits and vegetables are niche crops, no economies of scale.

• Public resistance to GMOs.

• More intimate ‘association’ with fruit and vegetables. Not so with processed maize, soybean.

• ‘Fear’ of the technology.

• Difficult to obtain transformative robust genetic changes in keys traits that would make GM produce economical

Miller & Bradford (2010) Nature Biotech 28: 1012-1214


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What are the legitimate problems with GMOs?

• Gene transfer to non-GMO crops (evidence in maize).

• SOLUTION: Better ‘Buffer’ growth zones

• Use of antibiotic markers, bacterial plasmid (foreign DNA).

• SOLUTION: Marker-free systems*, cisgenics and intragenics*

• Positional effects due to non-specific insertion of plasmid

• SOLUTION: Genome editing*

• ‘Monopolization’ of currently used GMOs by seed companies.

• SOLUTION: Greater public investment in traits with humanitarian goals.

• Molecular analysis of GMOs produced over 20 years and modifying 100s traits show them to be substantially equivalent to conventionally produced crops.

• Further, while consumption of GMOs is a personal decision, there is no credible evidence that current products are harmful to human or animal health.

• Herman (2013) J. Agriculture food Chemistry (61): 11695

• http://www.geneticliteracyproject.org/2015/01/29/pewaaas-study-scientific-consensus-on-gmo-safety-stronger-than-for-

global-warming/

Solution: Transgrafting

A. Yes

B. No

C. Not Sure

Do you consider this plant to be GMO?

Escobar et al (2001) Proc Natl Acad Sci USA. 98:42; Haroldsen et al (2012) California Agriculture 66 (2) 62-69.

Use transgenic rootstock – harvest fruit from non-transgenic scion. Limited to traits altered by mobile systemic proteins.

Cisgenics & Intragenics

Holme et al 2013. Plant Biotech J 11: 395

A segment of DNA from the same (intragenic e.g. RNAi) or sexually compatible plant (cisgenic) is introduced into the genome. No DNA from a foreign species is transferred.

Intragenenics: gene silencing by RNAi (RNA interference)

BLOCKGENE---------ENEGKCOLB

BLOCKGENEBLOCKGENE

Design a palindromic DNA sequence so that the RNA produced forms a hairpin

Short Sequence forming a Hairpin Loop

Introducing a DNA sequence in a hairpin conformation into the cell causes the destruction of all RNA with that identical sequence. We say that the gene is silenced.

Transformation can be done without a plasmid vector construct.

‘Intragenic’ GMO melon: Plant ACO gene silenced by RNAishowed enhanced shelf-life.

Hao et al (2011) Biotechnol Lett 2011 33:55-61

Melon transformed with melon ACO RNAi hairpin sequence. No plasmid DNA, antibiotic selectable gene, and no tissue culture used. The process worked but was inefficient.

12 days at room temperature

Normal Transgenic


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Postharvest GMO products close to market: reduced browning in transgenic apples (NA)* and potatoes (A)*

Genes suppressed by RNAi technology

Haroldsen et al (2012) California Agriculture 66(2): 62-69

• NA = Not approved by FDA• *A = Approved by FDA

Genome Editing: The exact region of the genome to be modified can be defined using methods such as CRISPR-CAS*

Pennisi E (2013) The CRISPR craze. Science 341: 833–836 *Clustered Regulatory Interspaced Short Palindromic Repeats

The CAS9 protein cuts at the region of the genome desired by the Researcher. The resulting mutation can alter gene function, or, a fragment of new DNA can be introduced at that specific region. CRISPR is fast, easy, accurate and cheap.

CAS 9)

Kyndt et al (2015) Proceedings National Academy Sciences 112 (18):5844-5849

A need to rethink GMOs. Gene transfer among species may be more common than we originally thought

Summary• Crop improvement techniques have moved away from a phenotype-based approach for

selection to a DNA or genotyping approach. Marker Assisted Selection and TILLING use genotyping to improve the efficiency of traditional breeding and mutagenesis respectively.

• Transgenic manipulation is a successful way to introduce new traits into commercial lines but has not gained much traction. Investments in improving PH quality of specialty crops may not be profitable in the short term.

• Recent genome editing discoveries, plus the use of intragenics and cisgenics will make gene manipulation more accurate with fewer unintended consequences.

• It still remains difficult to define quality and hence genes that underscore “ ‘postharvest quality’ in specialty crops, which hampers genetic improvement efforts.

Online Resources

• http://sbc.ucdavis.edu/Outreach/Biotechnology_Tutorials_Online.htm

• http://www.agbioworld.org/

• http://californiaagriculture.ucop.edu/0402AMJ/toc.html

• http://californiaagriculture.ucanr.org/collectionview.cfm?collection=13786

• http://www.pacificbiosciences.com/

• http://www.nanoporetech.com

• http://www.gmo-compass.org/eng/home/

• http://tilling.ucdavis.edu/index.php/Main_Page

• http://solgenomics.net/

• http://www.newscientist.com/blogs/culturelab/2009/11/creationist-bananas.html

• http://www.washingtonpost.com/local/scientists-breed-a-better-seed-trait-by-trait/2014/04/16/ec8ce8c8-9a4b-11e3-80ac-63a8ba7f7942_story.html

• http://mvgs.iaea.org/PDF/Mutation%20Induction%20for%20Breeding%20101.pdf

References• Chrispeels and Sadava, 2002 Plant, Genes and Crop Biotechnology; ASPB.

• Bradford KJ & Alston, J. (2004) California Agriculture vol 58(2):84-85 http://CaliforniaAgriculture.ucop.edu

• Bradford et al (2004) California Agriculture 58(2):68-71

• http://CaliforniaAgriculture.ucop.edu

• Clark et al (2004) California Agriculture 58(2): 89-98

• http://CaliforniaAgriculture.ucop.edu

• Lemaux, P. (1998-2010) Biotechnology presentations?

• http://ucbiotech.org/resources/presentations/presentations.html retrieved 6/11/12

• “What’s for dinner – Genetic engineering from the lab to your plate.

• http://mail.wecdsb.on.ca/~a_altenhof/FOV1-000460CD/S0595122D.3/geneticapplications.pdf retrieved 6/11/12

• Prakash C.S. : Agricultural Biotechnology”

• www.agbioworld.org retrieved 6/11/12

• US Regulatory Agencies Unified Biotechnology website

• http://usbiotechreg.nbii.gov/lawsregsguidance.asp (No longer active)

• Wieczorek, A. M. & Wright, M. G. (2012) History of Agricultural Biotechnology: How Crop Development

• has Evolved. Nature Education Knowledge 3(10):9.


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References (cont’d)• Griffiths et al (2010) Introduction Genetic Analysis 10th Edition. WH Freeman Press.

• Kader (2002) HortScience 37 (3) 467-468

• Dandekar et al (2004) Transgenic Res 13 : 373-384• Escobar et al (2001) Proc Natl Acad Sci USA. 98:42

• Lynch et al (1999) J. Econ Entomol; 92 (1): 226.

• Tricoli et al. (1995). Nat Biotech. 13: 1458.

• Chandler SF (2003) J. Plant Biotech 5: 69-77

• Collard et al 2005 Euphytica vol 142: 169

• Dahmani-Mardas et al (2010) PLoS One vol 5 (12) 15776• Hao et al (2011) Biotechnol Lett 2011 33:55-61

• Bruening & Lyons (2000) California Agriculture 54(4):6-7

• Miller & Bradford (2010) Nature Biotech 28: 1012-1014

• Bradford et al., (2005) Nature Biotechnology 23: 439-444

• Colbert et al 2001 Plant Physiol. 126: 480-484

• Haroldsen et al (2012) California Agriculture 66(2): 62-69.• Kyndt et al (2015) Proceedings National Academy Sciences 112 (18):5844-5849

• Pennisi E (2013) The CRISPR craze. Science 341: 833–836

• Hou et al (2014) Frontiers in Plant Science 5 (389): 1-5

• Herman & Price (2013) J. Agriculture food Chemistry (61): 11695

Commonly asked questions regarding GMOs

• Is it necessary to label food containing GMOs?http://www.ext.colostate.edu/pubs/foodnut/09371.pdfGMO Labeling: How to end the fight!Is Labeling GMOs a good idea?

• Do GMOs contribute to a loss of biodiversityhttp://www.gmo-safety.eu/pdf/biosafenet/Gepts.pdfGMOs lead to loss of Biodiversity

• Are we properly exploiting natural variation in wild species for crop improvement?Wild species are untapped source of genetic diversity

• Wasn’t there a study that showed GMOs caused cancer in rats?That GMO Rat Study!

• Did GMOs cause Indian Farmer Suicide?• http://www.theguardian.com/global-development/gallery/2014/may/05/india-cotton-

suicides-farmer-deaths-gm-seeds• http://www.economist.com/blogs/feastandfamine/2014/03/gm-crops-indian-farmers-

and-suicide• http://www.theguardian.com/environment/2008/nov/05/gmcrops-india• http://www.bbc.com/news/magazine-21077458\\

• Does Monsanto Sells Suicide Seeds?• http://www.nature.com/news/seed-patent-case-in-supreme-court-1.12445• http://www.monsanto.com/newsviews/pages/terminator-seeds.aspx

• Do GMOs cause allergies?• Twenty Year Study of GMOs

• Are Heirlooms substantially different and more ‘natural’ than conventional tomatoes?• http://www.scientificamerican.com/article/case-against-heirloom-tomatoes/

Commonly asked questions regarding GMOs


beckles - biotechnology and postharvest quality · 2015-06-19 · advantages disadvantages 2....

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