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D J Gray, Z T Li, S A Dhekney,M Dutt, D L Hopkins D J Gray, Z T Li, S A Dhekney, M Dutt, D L Hopkins Mid-Florida Research & Education Center University.

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Presentation on theme: "D J Gray, Z T Li, S A Dhekney,M Dutt, D L Hopkins D J Gray, Z T Li, S A Dhekney, M Dutt, D L Hopkins Mid-Florida Research & Education Center University."— Presentation transcript:

1 D J Gray, Z T Li, S A Dhekney,M Dutt, D L Hopkins D J Gray, Z T Li, S A Dhekney, M Dutt, D L Hopkins Mid-Florida Research & Education Center University of Florida/IFAS T W Zimmerman Biotechnology & Agroforestry University of the Virgin Islands Genetic Engineering of Grapevine and Field Testing for Bacterial & Fungal Disease Resistance

2 Field Testing Transgenic Grapevine for Bacterial and Fungal Disease Resistance Objectives To test GM grapevines in Florida and the US Virgin Islands under USDA/APHIS approved conditions Evaluate for Pierce’s disease & fungal disease resistance Evaluate for commercially-useful qualities Begin to assess environmental risks of GM grape Determine extent of gene flow via pollen Determine if weedy hybrids occur

3 Grapevine & Genetic Improvement World’s most valuable fruit cropWorld’s most valuable fruit crop 10 – 12 th most valuable agricultural crop in US10 – 12 th most valuable agricultural crop in US But - Grape is particularly difficult to improve geneticallyBut - Grape is particularly difficult to improve genetically –Genetic self-incompatibility makes breeding new varieties difficult –Long grape lifecycle – years to make recurrent crosses –Fine discrimination of wine type makes breeding a new “Cabernet” impossible These obstacles have led to little improvement of desirable varieties, such as adding disease resistance to established wine varietiesThese obstacles have led to little improvement of desirable varieties, such as adding disease resistance to established wine varieties –An exception has been for table grape breeding where improved fruit types have been created by laborious breeding The Opportunity:

4 Why Grape Research in the Subtropics? The Virgin Islands Grape is a high-value crop suited for small farm production A new source of agricultural income The Virgin Islands must import all grape products BUT - Disease-resistant cultivars are needed Therefore, like Florida, a large untapped local market exists

5 Why Grape Research in the Subtropics? Florida Conventional varieties are needed for wine & seedless fruit But - All such varieties will die from Pierce’s disease & various fungal diseases if grown in Florida Florida is the US’s # 2 consumer of wine & grape products Conventional breeding cannot be used to create resistant versions of desirable varieties Therefore a large untapped local market exists Existing muscadine-based industry satisfies less than 1% of market

6 Genetic Engineering of Grapevine Insertion of genes for disease resistance into otherwise desirable varieties might result in grapevines that can be grown in the Virgin Islands and Florida and address the existing market Agrobacterium-mediated genetic transformation of certain Vitis vinifera varieties and rootstocks is now routine & efficient Li et al In Vitro Cell. Dev. Biol. Plant 42: Dhekney et al ACTA Hort 738: Therefore all needed technology is in place to evaluate use of genetic transformation in grapevine improvement

7 Diseases of Grapevine Bacterial Disease Pierce’s Disease – Florida & California Pierce’s Disease – Florida & California Prohibits production of Vitis vinifera in Prohibits production of Vitis vinifera in the Southeastern US & is increasing in Ca the Southeastern US & is increasing in Ca Fungal Diseases – Florida, Virgin Islands & Worldwide Powdery Mildew – Most important disease of grape worldwide Powdery Mildew – Most important disease of grape worldwide Anthracnose Anthracnose Downy Mildew Downy Mildew Ripe Rot Ripe Rot Many others Many others Viral Diseases – Worldwide Grape fan leaf virus – most severe & widespread virus Grape fan leaf virus – most severe & widespread virus Leaf roll, corky bark, many others Leaf roll, corky bark, many others

8 Pierce’s Disease Endemic in Florida (the southern coastal plain) & California Endemic in Florida (the southern coastal plain) & California Caused by Xylella fastidiosa Caused by Xylella fastidiosa Xylem limited bacterium that infects a wide range of Xylem limited bacterium that infects a wide range of vascular plants vascular plants Transmitted by xylem-feeding insects Transmitted by xylem-feeding insects Primarily leaf hoppers & sharpshooters – Glassy Winged Sharpshooter new in CA Primarily leaf hoppers & sharpshooters – Glassy Winged Sharpshooter new in CA Lethal to all Vitis vinifera & other non-native varieties Lethal to all Vitis vinifera & other non-native varieties

9 Pierce’s Disease Total loss of vineyard results

10 Pierce’s Disease Tolerant Vines

11 Anthracnose of Grapevine

12 Powdery Mildew of Grapevine Powdery Mildew infects leaves, stems & fruit Typical chlorotic lesions Aerial hyphae

13 Genetic Engineering of Grape Direct insertion of genes (DNA)Direct insertion of genes (DNA) Allows integration of single traits without disturbing desirable characteristicsAllows integration of single traits without disturbing desirable characteristics –Important for grape where varieties are highly prized and change is resisted (e.g. wine varieties) Adds traits that normally are not found in grapeAdds traits that normally are not found in grape –Such as novel types of disease resistance

14 Genetic Engineering of Grape Modern techniques of molecular biology allow DNA to be analyzed and manipulatedModern techniques of molecular biology allow DNA to be analyzed and manipulated Functional DNA, including genes, literally can be cut and pasted together into new and useful combinationsFunctional DNA, including genes, literally can be cut and pasted together into new and useful combinations Potentially, unlimited possibilities resultPotentially, unlimited possibilities result

15 Understanding Genetic Control Elements Basic definitions:EnhancerElement Promoter Gene GeneTerminator RNA to Protein A segment of DNA that causes genes to become active. Promoters serve as a binding site for the enzyme RNA polymerase, which performs the first step of transcribing DNA in the adjacent gene. A segment of DNA that influences the activity of promoters by serving as a binding site for specific proteins. Enhancers may be physically separated from the promoter, but still influence it. Duplicating enhancers or modifying their DNA sequences can dramatically alter activity. A heritable sequence of DNA that determines a particular characteristic in an organism. A gene may code for a wide range of functions. Most commonly, proteins are produced. A segment of DNA that signals the end of a gene. Terminators serve to stop transcription by detaching RNA polymerase. Also known as “transcription” and “translation”. RNA polymerase binds at the promoter and moves along the DNA strand, transcribing the sequence into Messenger RNA. When the terminator is reached, mRNA detaches and is translated into a protein, the structure of which is dictated by the original DNA sequence.

16 A Conventional Genetic Control Element GeneTerminator EnhancerElementCorePromoterGeneRNAProtein* Located on Chromosome Chromosome (Double-Stranded DNA)

17 Bidirectional Dual Promoter Complex US Patent # 7,129, Duplicated Enhancer CorePromoter Transgene - 2 RNAProteinEnhancerElement CorePromoterGeneTerminatorRNAProteinEnhancerElement GeneTerminator Transgene - 1 The patented BDPC is a Genetic Control Elements. The BDPC provides more efficient control of genes and results in better protein production. The patented BDPC is a new way to arrange Genetic Control Elements. The BDPC provides more efficient control of genes and results in better protein production. Duplicated Elements in Divergent Orientation Conventional Arrangement of Gene Controlling Elements

18 Assembly of DNA for Insertion into Grape Source Genes Unique DNA Sequence for Genetic Engineering Functional Gene GFP Antibiotic Resistance Molecular Biology Antibiotic Resistance GFP Functional Gene (To select GE cells) (To see GE cells) (Improved Trait)

19 Bi-Functional Fusion Gene EGFPNPTII A bi-functional marker gene + Yang et al Burk et al ReporterSelectable

20 Functional Genes Tested in Grape at MREC Vitis vinifera thaumatin-like protein geneVitis vinifera thaumatin-like protein gene –Tested for fungal resistance Grape albumen protein geneGrape albumen protein gene –Tested for fungal resistance and seedlessness Lytic peptide genesLytic peptide genes –Tested for PD and fungal resistance Hybrid resistance genesHybrid resistance genes –Tested for PD and fungal resistance

21 Example of Transformation Vector: Modified Enhancer CorePromoterCorePromoterCsVMV-BDPC EGFP/NPTII Lyt Pep Term Term. Transgene - 1 Transgene - 2 A Lytic Peptide-Containing Transformation Vector Based on a Bidirectional Promoter Complex (BDPC)

22 Grape Transformation System Embryogenic culturesEmbryogenic cultures –Totipotent cells of somatic embryos are the target Agrobacterium mediated transformationAgrobacterium mediated transformation Kanamycin selection of transgenic cellsKanamycin selection of transgenic cells –Uses NPTII Gene

23 Selection of Transgenic Grape Transient GFP expressionTransient GFP expression –First visualized at 2 days and faded by 20 days Stable GFP expressionStable GFP expression –Apparent within 20 days GFP-positive embryogenic culturesGFP-positive embryogenic cultures –Isolated within 6 weeks Plants produced from embryos are acclimated to ex vitro conditionsPlants produced from embryos are acclimated to ex vitro conditions

24 CC AA E D BB A.Embryos B. Leaf/stem/tendril C. Flowers E. Anther/stigma A-C = V. vinifera ‘Thompson Seedless’ D-E = V. rotundifolia ‘Alachua’ Green Fluorescent Protein Expression in Vitis vinifera & Vitis rotundifolia

25 Preparing Transgenic Grape Lines for Greenhouse Testing Plants from tissue culture are propagated to produce multiple clones Plants are arranged into populations to study disease resistance

26 Screening for Powdery Mildew- Resistant Transgenic Grapevines Powdery Mildew Test Site in UF/IFAS Greenhouse Transgenic vines producing “Thaumatin- like” protein are grown with non-transgenic controls in an area of high powdery mildew incidence and without chemical control. Vines are rated 3x’s per week for disease development throughout the growing season. Experimental population of transgenic and control vines

27 Powdery Mildew-Resistant Transgenic Grapevines ( January 2005 ) Initial Powdery Mildew Test Site in Greenhouse Susceptible Control Vines Resistant Transgenic Vines Certain transgenic vines grown in an area of high powdery mildew pressure remained vigorous and show resistance to PM throughout the growing season.

28 Powdery Mildew Resistant Transgenic Grapevines Selected in Greenhouse at MREC Susceptible ‘Thompson Seedless’ Transgenic ‘Thompson Seedless’ Selected transgenic vines that express Vitis vinifera thaumatin-like protein (VVTL-1) exhibit an 8 day delay in visible lesion development compared to control vines VVTL-1 is an endogenous gene from grapevine 5 resistant lines have been selected for field trials

29 Testing Transgenic Plants for Resistance to Pierce’s Disease The Xylella bacterium, which causes Pierce’s disease, is injected directly into the xylem tissue of transgenic grape plants and control plants. The plants are then evaluated for resistance.

30 Testing Xylem Sap for Lytic Peptide Pure xylem sap is exuded from decapitated plants due to root pressure CK Purified Protein Test Samples The presence of lytic peptide is determined by ELISA Transgenic Vines

31 Tracking Bacterial Concentrations in Test Plants After stem inoculation, the xylem sap from leaf petioles is placed on Xylella-specific culture mediaAfter stem inoculation, the xylem sap from leaf petioles is placed on Xylella-specific culture media The presence and number of bacterial colonies provides an early estimate of plant resistanceThe presence and number of bacterial colonies provides an early estimate of plant resistance Periodic sampling provides information on internal spread of bacteria over timePeriodic sampling provides information on internal spread of bacteria over time Dead Dead Seasonal dormancy

32

33 PD Resistant Transgenic Grapevines Selected in Greenhouse at MREC These vines were inoculated in July 2004 Since even resistant controls developed symptoms, the PD test is considered to be stringent Lack of symptoms in transgenic plants that contain proprietary lytic peptide genes suggest high level of resistance Approximately 100 highly resistant lines have since been selected, 15 of which were propagated for field trials Susceptible Control Vines Transgenic Vines w Lytic Peptide Resistant Control ‘Tampa’ ‘Thompson Seedless’

34 Production of Transgenic Grapevines at MREC Over 2,200 unique transgenic plants have been producedOver 2,200 unique transgenic plants have been produced –90%+ have been Thompson Seedless (the most widely planted variety in the US) Nine different functional genes have been testedNine different functional genes have been tested –Most have been discarded due to poor response The hybrid LIMA gene appears to provide PD resistanceThe hybrid LIMA gene appears to provide PD resistance The native thaumatin gene appears to provide fungal resistanceThe native thaumatin gene appears to provide fungal resistance Field tests must now be establishedField tests must now be established

35 Selected Transgenic Plants Propagated For Field Trial

36 The Virgin Islands UVI Field Site Approved through USDA APHIS notification process in October 2006 A protected site used for evaluation of GM plants  5 lines containing VVTL-1, replicated 5-7 times  29 transgenic vines plus 9 controls planted 1/2007

37 The Virgin Islands Field Site University of the Virgin Islands, St. Croix Established January Two ‘Thompson Seedless’ lines containing VVTL-1 gene (June 2007)

38 The Florida Field Site Approved through USDA APHIS notification process in October 2006 Isolated from cross-fertile wild & cultivated vines  15 lines containing either of 2 experimental lytic peptide, replicated 4-5 times  5 lines containing VVTL-1, replicated 8 times Transgenic varieties used (180 plants = 60%)  30% ‘Thompson Seedless’, 10% ‘Merlot’, 10% ‘Seyval Blanc’  10% ‘Freedom’ rootstock Non-transgenic controls (120 plants = 40%)  Same scions and rootstocks as above (20%)  PD-resistant hybrids ‘Tampa’ and BN5-4 (20%)

39 The Florida Field Site UF/IFAS Mid-Florida Research & Education Center

40 Trellises constructed in March 2007 The Florida Field Site

41 Planting April 2007

42 The Florida Field Site ‘Thompson Seedless’, June 2007

43 The Florida Field Site July 6, 2007

44 What’s Next? Evaluation for disease resistance & clonal fidelity are ongoing Environmental risk assessment studies planned Greenhouse screening of endogenous genes and varieties will lead to new field planting in

45 Screening Endogenous Genes for use in Powdery Mildew Resistance ‘Syrah’ before veraision Non-transgenic Transgenic

46 Screening Endogenous Genes for use in Powdery Mildew Resistance ‘Syrah’ ripe Non-transgenic Transgenic

47 Acknowledgments Florida Department of Agriculture & Consumer Services Viticulture Trust Fund Long-term support of grape biotech research USDA Tropical, Sub-Tropical Agricultural Research Grants Program Support for endogenous gene discovery and field tests Florida Genetics LLC (www.flgenetics.net) Support for patent costs and commercialization efforts


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