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Diane M. Beckles

Department of Plant Sciences

University of California-Davisdmbeckles@ucdavis.edu

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 the ‘key’ genes that underscore important traits in living organisms.

• Modify these genes to enhance selected traits.

Postharvest Quality (PHQ)

• “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

Producers and Consumers sometimes have opposing wants

PRODUCERS

Traits Are Determined By The Genetic Make-Up (Genotype) Of The Organism And The Environment

Normal

Ripeningmutant

25°C

25°C

Normaltomato

Ripe

Different Genotype Different Environment

Normaltomato

Biotechnology

NewKnowledge

New Products

Chilling Injury In Tomato(example)

NovelPostharvest

Attributes

What Determines Traits ?

DNA

mRNA

Metabolite

Gene Gene

ProteinTraits

(Postharvest)

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Biotechnology Provides The Tools To Monitor These Processes

Postharvest Chilling Injury (CI) In Tomato Fruit

-Temperature below 12.5°C will lead to CI. -Function of temperature and time.-Symptoms only visible after rewarming.-Temperature pre-treatments can reduce the occurrence of CI in fruits

What Are The Molecular Differences In These Fruits? (Environment)

12.5 °C 2.5 °C 2.5 °C StorageTemperature

Chilling Injury

12.5 °C

45°C45°C PretreatmentTemperature

LessChillingInjury

Normal Normal

Metabolomics Of Chilled Tomato Fruit

LuengwilaI, K., Saltveit, MS, Beckles, DM (2012) PBT 63: 116-122

Found metabolites associated with chilling and the protective treatment. Goal: Identity metabolite biomarkers for chilling stress

Identifying Tomato Species With Resistance To Postharvest CI (Genotypes)

Wild tomatoes grow in the Andes at 3000 m above sea level. What about the postharvest changes in wild fruits?

Monitoring the spatio-temporal occurrence of chilling injury symptoms in fruits with greater specificity

Visualizing Tomato Noninvasively during Chilling by MRI

Data of: F. Tao; L. Zhang; Saltveit, MS; McCarthy, M.; Beckles, DM

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Genetic modification of Plants

Using Biotechnology to Create Novel Postharvest Traits

All Crop Plants Have Been Genetically Modified

Selective Breeding For Improved Traits

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 ParentTransgenic Manipulation Can Create More Novel Traits than Selective Breeding

• Allows the transfer of genes between different organisms.

• More commonly used to transform an organism using a native gene sequence.

• The action of that gene, normally present in the plant, may be suppressed, overexpressed or modified.

• Crops so produced are often described as Genetically Modified Organisms (GMOs).

The Gene to be Modified Is Introduced into the Plant Using Bacterial Plasmid as Vectors

Gene Construct

Splice Gene intoPlasmid vector.

Gene of interest

Plasmid(Vector)

Plasmids are circular molecules of DNA naturally found in bacterial cells. drawing by Celeste Rusconi, © Regents of the Univ. California

The Construct Is Integrated Into A Cell From Which A Whole Plant May Be Regenerated

Only cells containing recombinant DNA

survive and divide in culture

DNA inserted in plant chromosome.

Cells regenerate into transgenic plant

Plantlet grow into plants with new traits

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Transgenic Manipulation Of Plants Offers Many Possibilities

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

Plants with animal genes have not been marketed.

Transgenic Papaya Resistant To Papaya RingspotVirus

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

Other examples: Flavr Savr tomatoes – Extended Shelf-life

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

Transgenic Normal Polygalacturonase a ‘ fruit softening’ gene was suppressed.

Transgenic fruits could be harvested ripe but withstand shipping and handling.

Transgenic Plums Resistant To Plum Pox Virus

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

Normal Transgenic

Enhanced Shelf-life In Transgenic Apples : Ethylene Biosynthetic Gene ACO* Was 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

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Reduced Browning In Transgenic Apples With Low Levels Of PPO*

Normal Transgenic PPO was suppressed which

reduced browning in cut or wounded fruits.

Artic apple™ by Okanagan Specialty Fruits Canada.

Haroldsen et al (2012) California Agriculture 66(2): 62-69*Polyphenol oxidase (PPO)

Database Of Plant GMOs Produced Worldwide

Transgenic Fruits & Vegetables – Where Are We?

• Between 2003-2008: • 313 publications on transgenic research of produce.

• Research in diverse countries: USA, Europe, India, Japan, Brazil, South Korea, Israel, Tunisia among others.

• 77 specialty type crops transformed with 206 traits altered.

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

• Little transition from lab-to-table.

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

Limitations To Marketing GM Horticultural Crops

• Expensive.• Estimates of regulatory costs:

• US$15 M per transgenic line.

• Time to market: 10 - 17 years.

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

• Public resistance. • More intimate ‘association’ with fruit and vegetables. Not so with processed

maize, soybean.

• ‘Fear’ of the technology.

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

While some argue that transgenic plants are a cure-all

© Drawing by Nicholas Eattock, Dept Plant Sciences UC Davis

Public opinion has been less than enthusiastic

Unresolved Issues• Gene transfer to non-GMO crops.

• Disturbing evidence that native maize landraces in Mexico pollinated by GMO crops*

• Use of markers, bacterial plasmid; tissue culture.

• Not naturally found in plants; somaclonal variation.

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

• Perception : Profits over humanity and the environment

*http://www.plantsciences.ucdavis.edu/gepts/mec_3993_LOW.pdf **Gepts, P. (2002) Crop Science 42:1780-1790

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Solution: Transgrafting. Use Transgenic Rootstock –Harvest Fruit From Non-Transgenic Scion

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

Solution: Engineer Plants With No Plasmid, No Antibiotic Selectable Gene And No Tissue Culture.

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

Melon with the ACO gene silenced showed enhanced shelf-life. Process worked but was inefficient.

12 days at room temperature

Normal Transgenic

What Lessons Can We Learn From Nature?

Still Alternative Approaches Are Necessary!

Natural Mutations That Occurred In Wild Plants Created Desirable Traits

Wild banana with seeds Cultivated banana- sterile

We can accelerate this natural process by subjecting seeds to mutagens.

Induced Mutations In Genes Can Also Lead to Novel Traits

CreCre DNA

mRNA

Metabolite

Gene Gene

Protein

Trait

Mutation

NewTrait

Targeted Induced Local Lesions InGenomes (TILLING): Creating Mutations In Plants

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

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Mutant Melons

Ripening Firmness Shape Brix Flesh RindTime Color Color

ACO MutantControl

Dahmani-Mardas et al (2010) PLoS One vol 5 (12) 15776

Mutant ACC Oxidase Melons Found By TILLING Have A Longer Shelf-life

Kornneef and Stam (2001) Plant Physiology vol 125, 156-159

Great Genetic Diversity Exists In Nature – Can We Exploit It?

Finding DNA Markers Among Diverse Genotypes To Serve As ‘Tags’ for a Trait

Using DNA Markers In Plant Breeding: Marker Assisted Selection (MAS)

Potential Efficiency Of MAS

Collard et al 2005 Euphytica vol 142: 169

Finding DNA Markers Is Feasible Because Genome Sequencing Is Relatively Cheap

1 Million bp or units of DNA sequence cost $5000 in 2001 now it costs just 6 cents

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Whole Genomesequencing projects ofof several Horticultural

crops are complete(this list will soon to be

outdated)

Summary• A repertoire of sophisticated tools have been developed

that can be used to identify key ‘Postharvest’ genes and to alter the genetic makeup of crop plants.

• Transgenic manipulation is a successful way to introduce new traits but has not gained much traction.

• Marker Assisted Selection and TILLING may be viable alternatives.

• Genomics of horticultural crops has been revolutionized by cheap sequencing. This will help to select fruits, vegetables and nuts with improved postharvest traits.

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.454.com/

• 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

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.pdfretrieved 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)

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

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

• Luengwilai & Beckles (2010) JSPPR 1:1

• 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.