biotechnology and overview of topics postharvest...
TRANSCRIPT
1
Biotechnology and Postharvest Quality
Diane M. BecklesDiane M. Beckles
Department of Plant Sciences
University of [email protected]
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
2
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
3
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:
4
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
5
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.
6
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.
7
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