Biotechnology and Postharvest Quality

Transcription

1 Biotechnology and Postharvest Quality Diane M. Beckles Department of Plant Sciences University of California-Davis Overview of Major Topics Biotechnology. Postharvest Quality (PHQ). manipulation. Targeted Induced Local Lesions IN Genomes (TILLING). Marker Assisted Selection. Summary. Biotechnology A set of tools used to modify the genetic makeup of an organism so that: It produces a new product. A native product performs a new function(s). Postharvest Quality (PHQ) The factors that ensure maximum income for producers and meet the nutritional and aesthetic needs of the consumer after horticultural are harvested. Kader, 2002 Sweetness Aroma Texture Acidity CONSUMERS Nutrition Color Flavour Appearance Reduced Browning Firmness Shelf-life PRODUCERS Reduced Microbes Chillingtolerance Disease resistance Producers and Consumers sometimes have opposing wants Postharvest traits (like most plant traits) are determined by the interaction of the plant genotype and the environment Selective breeding for improved traits Recurrent Parent Donor Parent GENOTYPE ENVIRONMENT Solanum lycopersicum Solanum peruvianum 25 C 25 C tomato tomato rin: ning mutant 25 C Partially Different genotype Identical environment 5 C tomato Partially Identical genotype Different environment Percent of wild genes 50% Using the wild tomato species 25% as a source of novel genes. 12.5% 6.25% Six or more generations of backcrosses to the cultivated 3.125% parent, selecting for desired 1.5% trait at each generation 0.75% Kent Bradford, Department of Plant Sciences, UC Davis 1

2 All crop plants have been genetically modified manipulation 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). Basic steps needed to genetically engineer a plant. Genes are expressed in the plant using bacterial plasmid as vectors. Select Gene Make Gene Construct Transform Plant Select cells Regenerate Plant Plasmid (Vector) Gene of interest Splice gene into Plasmid vector. Gene Construct What trait do you wish to modify? Splice gene into plasmid. Gene now competent for expression in plant Physically introduce the gene construct into plant tissue. Grow cells on select media. Only cells with construct survive. Make a whole plant from transgenic cells using special media -Test Gene Stability. -Evaluate Traits. Verify Plants are Plasmids are circular molecules of DNA naturally found in bacterial cells. Different methods of introducing the gene construct into the plant The gene is integrated into a cell from which a whole plant may be regenerated Particle bombardment. Agrobacterium tumefaciens DNA inserted in plant chromosome Gold particles coated with DNA construct shot into tissue A Soil bacterium that naturally inserts its plasmid DNA into plant genome. Only cells containing recombinant DNA survive and divide in culture Cells regenerate into transgenic plant drawing by Celeste Rusconi, Regents of the Univ. California Plantlet grow into plants with new traits 2

3 manipulation of plants offers many possibilities papaya resistant to papaya ringspot virus DNA has the same chemical makeup in different species. DNA from one species can sometimes be stably expressed in another. NOTE: The famous fishberry was never pursued. papaya accounts for 90% of all grown in Hawaii. Cultivated since Chrispeels and Sadava, 2002 Plant, Genes and Crop Biotechnology; ASPB California Agriculture vol 58 #2; Other commercialized transgenic horticultural Other examples: Flavr Savr tomatoes extended shelf-life Sweet corn resistant to earworm Florigene Moonshadow carnations Freedom II Squash virus resistant Bt corn: Lynch et al (1999) J. Econ Entomol; 92 (1): Squash: Tricoli, D. M., et al. (1995). Nat Biotech. 13: Carnations: Chandler SF (2003) J. Plant Biotech 5: Polygalacturonase increases as fruit ripens and leads to fruit softening. This gene was suppressed. fruits could be harvested ripe but withstand shipping and handling. Chrispeels & Sadava, 2002 Plant, Genes and Crop Biotechnology. plums resistant to plum pox virus Delayed softening/ripening in transgenic apples ACO suppressed Control Ethylene biosynthetic gene AC Oxidase* was suppressed. ning was significantly delayed increasing shelflife. At harvest After 3 months at room temperature *ACO = 1-aminocyclopropane- 1-carboxylic acid oxidase California Agriculture vol 58 #2; Dandekar et al (2004) Res 13 :

4 Reduced browning in transgenic apples with low levels of PPO* Database of plant GMOs produced worldwide High activity of *Polyphenol oxidase (PPO) promotes browning in cut or wounded fruits. apple with suppressed PPO gene shows reduced browning. Artic apple by Okanagan Specialty Fruits Canada. Haroldsen et al (2012) California Agriculture 66(2): fruit & vegetables where are we? From : 313 publications on transgenic research of produce. Research in diverse contries: USA, Europe, India, Japan, Brazil, South Korea, Israel, Tunisia among others. 77 specialty type transformed. 206 traits altered. Still only 4 transgenic lines currently on market: sweet corn, papaya, zucchini squash, carnations Little transition from lab-to-table. Limits to marketing GMO Horticultural Economies of scale. Expensive to apply to niche and cultivars such as fruits and vegetables. Estimates of regulatory costs: US$15 M per transgenic line Time to market: years. Public resistance to the idea. More intimate association with fruit and vegetables. Not so with processed maize, soybean. Fear of the technology. Miller & Bradford (2010) Nature Biotech 28: Miller & Bradford (2010) Nature Biotech 28: Limits to marketing GMO Horticultural Several postharvest traits are complex Multigenic. Genes for traits unknown. Traits strongly affected by the environment. Technologies not sufficiently efficient for some. Poor transformation efficiencies; Long generation times. Not tractable. While some argue that transgenic plants are a cure-all Public opinion has been less than enthusiastic Drawing by Nicholas Eattock, Dept Plant Sciences UC Davis 4

5 Unresolved Issues Gene transfer to non-gmo. Disturbing evidence that native maize landraces in Mexico pollinated by GMO * Positional Effects. Disruption of native genes at the site where the construct is inserted** Disruption of native genes at the site where the construct is inserted** Use of markers, bacterial plasmid; tissue culture. Not naturally found in plants; somaclonal variation. Monopolization by seed companies Perception : Profits over humanity and the environment * **Gepts, P. (2002) Crop Science 42: Solution? Grafting: Use transgenic rootstock harvest fruit from control scion Limitation - trait must be determined by a transgene or product that moves systemically through the plant. proteins conferring virus or bacterial resistance. But is the fruit really non- GMO? Escobar et al (2001) Proc Natl Acad Sci USA. 98:42 Haroldsen et al (2012) California Agriculture 66 (2) Solution? Engineer plants with no plasmid and no tissue culture. Still alternative approaches necessary! 12 days at room temperature Melon transformed with ACO gene showed enhanced shelf-life. Construct introduced via pollen tube. Gene construct did not contain bacterial plasmid DNA. No selectable marker included. No tissue culture needed. What lessons can we learn from nature? Hao et al (2011) Biotechnol Lett :55-61 Spontaneous mutations in the wild have created desirable traits Targeted Induced Local Lesions In Genomes (TILLING) 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). Colbert T et al. Plant Physiol. 2001;126:

6 Mutant ACC Oxidase melons found by TILLING have longer shelf-life Great genetic diversity exists in nature can we exploit it? Control ACO Mutant Mutant Melons ning Firmness Shape Brix Flesh Rind Time Color Color Dahmani-Mardas et al (2010) PLoS One vol 5 (12) Kornneef and Stam (2001) Plant Physiology vol 125, Marker Assisted Breeding using Genomics Variation within species due to differences in DNA sequence. Identify DNA sequences that are associated with trait of interest. Those sequences can act as a proxy or marker for that trait. Hachiya Resistant Fuyu Susceptible 1. Find DNA sequences unique to resistant lines and sequences unique to the susceptible lines 2. Use these distinctive DNAs as markers for the trait. Finding molecular markers for a trait occurs by trial and error Extract DNA from several Chilling-resistant lines Extract DNA from plants with contrasting traits. Extract DNA from several Chilling-sensitive lines Find molecular markers associated with each trait. Collard et al 2005 Euphytica vol 142: 169 Marker-Assisted Selection (MAS) Potential Efficiency of MAS Chilling-susceptible fruit Chilling-resistant fruit x Genetic Marker Thousands of plants x x x x x x x Select these seedlings for advancement through breeding program Adapted from Collard et al 2005 Euphytica vol 142: 169 Collard et al 2005 Euphytica vol 142: 169 6

7 Finding DNA markers helped by genome sequencing. Whole genome sequencing projects of horticultural Horticultural Crops: Genome Sequencing Projects Crop Status Website Papaya Complete Solanaceae Cost of DNA sequencing now cheap. Whole genome sequencing of plants feasible. Facilitates marker identification. Tomato Complete Potato Complete Rosaceae Apple Complete Peach Complete Strawberry Complete Cherry In Progress Lettuce Transcriptome Grape (Pinot) Complete Citrus Mandarin Complete Sweet orange Complete Cucurbita Cucumber Complete Rose In Progress Banana In Progress Beckles & Kader (2011) Unpublished Summary A repertoire of sophisticated tools have been developed to alter the genetic makeup of crop plants. manipulation has proven to be very successful but has not gained much traction. Alternate approaches such as Marker Assisted Selection and TILLING may be viable alternatives. Genomics of horticultural will be revolutionized by Next-Gen sequencing. This will help to select fruits, vegetables and nuts with improved postharvest traits. Online Resources References Chrispeels and Sadava, 2002 Plant, Genes and Crop Biotechnology; ASPB. Bradford KJ & Alston, J. (2004) California Agriculture vol 58(2): Bradford et al (2004) California Agriculture 58(2): Clark et al (2004) California Agriculture 58(2): Lemaux, P. ( ) Biotechnology presentations? retrieved 6/11/12 What s for dinner Genetic engineering from the lab to your plate. retrieved 6/11/12 Prakash C.S. : Agricultural Biotechnology retrieved 6/11/12 US Regulatory Agencies Unified Biotechnology website (No longer active) References (cont d) Griffiths et al (2010) Introduction Genetic Analysis 10 th Edition. WH Freeman Press. Kader (2002) HortScience 37 (3) Luengwilai & Beckles (2010) JSPPR 1:1 Dandekar et al (2004) Res 13 : Escobar et al (2001) Proc Natl Acad Sci USA. 98:42 Collard et al 2005 Euphytica vol 142: 169 Dahmani-Mardas et al (2010) PLoS One vol 5 (12) Hao et al (2011) Biotechnol Lett :55-61 Bruening & Lyons (2000) California Agriculture 54(4):6-7 Miller & Bradford (2010) Nature Biotech 28: Bradford et al., (2005) Nature Biotechnology 23: Colbert et al 2001 Plant Physiol. 126: Haroldsen et al (2012) California Agriculture 66(2):