beckles- biotechnology and postharvest (6-18-13) 215 pm (2 ...ucce.ucdavis.edu › files ›...
TRANSCRIPT
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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 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.