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Biotechnology and Postharvest Quality

Diane M. BecklesDiane M. Beckles

Department of Plant Sciences

University of California-Davisdmbeckles@ucdavis.edu

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

Overview of Topics

Biotechnology.

Postharvest Quality (PHQ).

Transgenic manipulation Transgenic manipulation.

Targeted Induced Local Lesions INGenomes (TILLING).

Marker Assisted Selection.

Biotechnology

A set of tools used to modify the genetic makeup of an organism. Produces new productProduces new product

Product perform new function(s)

Postharvest Quality The factors that ensure maximum income for producers as well

as meeting the nutritional and aesthetic needs of the consumer after horticultural crops are harvested.

Producers and consumers often have opposing needs

Texture

Flavour

Aroma

Sweetness

Acidity

Color

Appearance

Nutrition

Shelf-life

Chilling-tolerance

MicrobialcontaminationBrowning

Firmness

Diseaseresistance

Postharvest traits are due to the interaction of the environment and genotype

25°CRipened fruit

25°

Identical genotype Different environment

Different genotype Identical environment

Normal tomato

rin: ripening mutant Chilling injuredFruit

C

5°C

Normal tomato

Luengwilia & Beckles (2010) JSPPR

Selective breeding for improved traits

Solanum lycopersicum

Solanumperuvianum

Wild species has resistance to nematodes

Donor ParentRecurrent Parent

Percent of “wild” genes 50%

25%

12.5%

Using the wild tomato species as a source of genes for nematode resistance

Six or more generations of backcrosses to the cultivated parent, selecting for resistance at each generation

6.25%

3.125%

1.5%

0.75%Kent Bradford, Depart Plant Sciences, UC Davis

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All crop plants have been genetically modified Transgenic manipulation

Allows the transfer of genes between different organisms.

Crops so produced are said to be Genetically Modified Organisms (GMOs)Modified Organisms (GMOs).

Native gene may be suppressed, overexpressed or modified.

Transgenes are expressed in the plant using bacterial plasmid as vectors.

Gene to be suppressed or overexpressed

Vector Construct

Plasmids are circular molecules of DNA found in bacterial cells. They confer selective advantage e.g. survival on Antibiotics, fertility etc to bacteria

Different methods of introducing the vector construct into the plant

2. Agrobacterium tumefaciens(more precise).

1. Particle bombardment (random insertion of genes).

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

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

Transgenic manipulation of plants offers many possibilities

Can cross species barrier.

NOTE:The famous “fishberry” was ynever pursued.

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

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Transgenic papaya resistant to papaya ringspot virus

Transgenic papaya accounts for 90% of all grown in Hawaii

C lti t d i

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

Non-transgenic Transgenic

Cultivated since 1999.

Other commercialized transgenic horticultural crops

Sweet corn resistance to earworm

Freedom II squash

Florigene Moonshadow carnations

Examples: Flavr Savr tomatoes –extended shelf-life

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

Transgenic plums resistant to plum pox virus

Non-transgenic fruit Transgenic fruit

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

Delayed softening/ripening in ACC Oxidase (ACO) silenced transgenic apples

Control

ACO silenced

At harvest

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

Control

ACO silenced After 3 months at

room temperature

Database of GMO produced worldwide:

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Transgenic fruit & vegetables – where are we? From 2003-2008: 313 publications on transgenic research of

produce.

USA, Europe, India, Japan, Brazil, South Korea, Israel, Tunisia among others.

77 specialty type crops.

206 traits.

Still only 4 transgenic lines currently on market: sweet corn, papaya, zucchini squash, carnations

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

Limits to marketing GMO Horticultural crops

Several postharvest traits are complex Multigenic; strongly affected by the environment. Difficult to

engineer.

Public resistance to the idea. More intimate ‘association’ with fruit and vegetables than maize or More intimate association with fruit and vegetables than maize or

soybean products

Economies of scale. Expensive to apply to niche crops and cultivars

Estimates of cost: US$15 M per transgenic line

Technologies not sufficiently efficient for some crops. Poor transformation efficiencies; long generation times - not

tractable.

© Drawing by Nicholas Eattock, Dept Plant Sciences UC Davis

While some argue that transgenicplants are a cure-all

Public opinion has been less than enthusiastic

Gene transfer to non-GMO crops.

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

Positional Effects. ⦁ Disruption of native genes at the site where the

t t i i t d**

Unresolved Issues

construct is inserted**

Use of markers, bacterial plasmid; tissue culture. Not naturally found in plants; somaclonal variation.

Monopolization/Concentration power by seed companies They determine the traits worthy of investment.

Humanitarian interests may not be prioritized.*http://www.plantsciences.ucdavis.edu/gepts/mec_3993_LOW.pdf**Gepts, P. (2002) Crop Science 42:1780-1790

Grafting: Use transgenic rootstock –harvest fruit from control scion

Limitations. Trait must be determined by: Activity in root Activity in root.

A transgene that moves systemically through the plant.

But is the fruit really non-GMO?

Escobar et al (2001) Proc Natl Acad Sci USA. 98:42

Engineered melons: ACC Oxidase (ACO) knockdown – no plasmid, no markers and no tissue culture used.

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

Control ACO1 knock down

Stored at room temperature for 12 days

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Still Alternative approaches necessary!

What lessons can weWhat lessons can we learn from nature?

Spontaneous mutations in wild have created novel/useful traits

Wild banana with seeds Cultivated banana- sterile

⦁ We can accelerate this natural process by subjecting seeds to mutagens. The mutant seeds are grown and the plants screened for useful traits.

⦁ DNA may also be extracted from mutants and screened for defects in gene of interest (TILLING)

Targeted Induced Local Lesions In Genomes (TILLING)

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

Mutant population TILLED for lines with ACC Oxidase defective gene

ACO MutantControl

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

Ripening Firmness Shape Brix Flesh RindTime Color ColorMutant Melons

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

Great genetic diversity exists - can we exploit it?

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

Breeding using Genomics Part of the variation in the population is due to differences in

gene sequence.

Ideally – identify the gene sequence that is the basis of the trait.

Alternately – identify a marker sequence – one that is associated with, and can act as a proxy for the trait., p y

Chances of finding good markers increases if the whole genome of a species is sequenced.

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Cost of sequencing now cheap : Whole genomeWhole genome sequencing ofplants feasible.

Genome Sequencing Projects of Horticultural CropsHorticultural Crops: Genome Sequencing Projects

Crop Status Website

Papaya Complete http://asgpb.mhpcc.hawaii.edu/papaya/

Solanaceae

Tomato Complete http://solgenomics.net/

Potato Complete http://www.potatogenome.net/

Rosaceae

Apple Complete http://www.rosaceae.org/species/apple

P h C l h // / h/Peach Complete http://www.rosaceae.org/peach/genome

Strawberry Complete http://www.rosaceae.org/projects/strawberry_genome

Cherry In Progress

Lettuce Transcriptome http://compgenomics.ucdavis.edu/

Grape (Pinot) Complete http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/

Citrus

Mandarin Complete http://www.citrusgenomedb.org/

Sweet orange Complete http://www.citrusgenomedb.org/

Cucurbita

Cucumber Complete http://www.icugi.org/

Rose In Progress http://www.rosaceae.org/species/rose

Banana In Progress http://www.musagenomics.org/

Finding molecular markers for a trait occurs by trial and error

Plants bearing Plants bearing Chilli i t t

Extract DNA fromplants with contrasting traits.

Collard et al 2005 Euphytica vol 142: 169

Chilling-susceptible fruit

Chilling-resistant fruit

Find molecular markersassociated with each trait

Chilling-susceptiblefruit

Chilling-resistant fruit

Genetic Markerx

Marker-Assisted Selection (MAS)

Select these seedlings for advancement through breeding program

x x x x x x x

Thousands of plants

Adapted from Collard et al 2005 Euphytica vol 142: 169

Potential Efficiency of MAS

Collard et al 2005 Euphytica vol 142: 169

Summary

A repertoire of sophisticated tools have been developed to alter the genetic makeup of crop plants.

Transgenic manipulation has proven to be very successful but has not gained much tractionsuccessful but has not gained much traction.

Alternate approaches such as Marker Assisted Selection and TILLING may be viable alternatives.

Genomics of horticultural crops will be revolutionized by Next-Gen sequencing.

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Online Resources http://sbc.ucdavis.edu/Outreach/Biotechnology_Tutorials

_Online.htm

http://www.agbioworld.org/

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

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/

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) What is biotechnology?

http://ucbiotech.org/resources/biotech/slides/biotech.html retrieved 5/20/11

“What’s for dinner – Genetic engineering from the lab to your platehttp://www.foodsafetynetwork.ca/biotechres/newpdfs/pg9-18.pdf retrieved 5/20/11

Prakash C.S. : Agricultural Biotechnology and Food Security”www.agbioworld.org retrieved 5/20/11

US Regulatory Agencies Unified Biotechnology website http://usbiotechreg.nbii.gov/lawsregsguidance.asp

References Griffiths et al (2009) Introduction Genetic Analysis 9th Edition. WH

Freeman Press.

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

C ll d t l 2005 E h ti l 142 169 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