Field Testing Transgenic Grapevine for Bacterial and Fungal Disease Resistance 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
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
Why Grape Research in the Subtropics? Florida is the US s # 2 consumer of wine & grape products Existing muscadine-based industry satisfies less than 1% of market Therefore a large untapped local market exists 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 Conventional breeding cannot be used to create resistant versions of desirable varieties
Pierce s s Disease Susceptible and Tolerant Vines in Central Florida V. vinifera Chardonnay Vitis rotundifolia Carlos
Genetic Engineering of Grapevine Insertion of genes for disease resistance into otherwise desirable varieties might result in grapevines that can be grown in Florida and address the existing market Agrobacterium-mediated genetic transformation of certain Vitis vinifera varieties and rootstocks is now routine & efficient Li et al. 2006 In Vitro Cell. Dev. Biol. Plant 42:220-227 Dhekney et al. 2007 ACTA Hort 738:749-753 Therefore all needed technology is in place to evaluate use of genetic transformation in grapevine improvement
The Genes Plants have been genetically engineered with either: Proprietary lytic peptide genes for PD resistance or V. vinifera thaumatin-like protein gene (VVTL-1) for fungal disease resistance Plants also contain an nptii/egfp fusion marker gene Simultaneously allows selection with kanamycin and observation of green fluorescent protein expression Genes are expressed via a proprietary Bidirectional Promoter System (US patent 7,129,343, 2006)
Green Fluorescent Protein Expression in Vitis vinifera & Vitis rotundifolia A A. Embryos B. Leaf/stem/tendril C. Flowers E. Anther/stigma B A-C = V. vinifera Thompson Seedless D-E = V. rotundifolia Alachua C D E
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 Transgenic Vines Control Vines w Lytic Peptide Thompson Seedless Resistant Control Tampa
Powdery Mildew Resistant Transgenic Grapevines Selected in Greenhouse at MREC 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 1 is an endogenous gene from grapevine 5 resistant lines have been selected for field trials Susceptible Thompson Seedless Transgenic Thompson Seedless
Selected Transgenic Plants Propagated For Field Trial
The Virgin Islands Field Site 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 Approved through USDA APHIS notification process in October 2006
The Virgin Islands Field Site University of the Virgin Islands, St. Croix Established January 2007 Two Thompson Seedless lines containing VVTL-1 gene (June 2007)
The Florida Field Site 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%) Approved through USDA APHIS notification process in October 2006
The Florida Field Site UF/IFAS Mid-Florida Research & Education Center
The Florida Field Site Trellises constructed in March 2007
The Florida Field Site Planting April 2007
The Florida Field Site Thompson Seedless, June 2007
The Florida Field Site July 6, 2007
Status and Future 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 2008-09
Screening Endogenous Genes for use in Powdery Mildew Resistance Non-transgenic Transgenic Syrah before veraision
Screening Endogenous Genes for use in Powdery Mildew Resistance Non-transgenic Transgenic Syrah ripe
Selected Research Articles Dhekney, S.A., Z.T. Li., M. Van Aman, M. Dutt, J. Tattersall, and D.J. Gray, Genetic transformation of embryogenic cultures and recovery of transgenic plants in Vitis vinifera, Vitis rotundifolia and Vitis hybrids, ACTA Hort. 738, 2007, 743-748. Dutt, M., Z.T. Li, K. Kelley, S.A. Dhekney, M. Van Aman, J. Tattersall, and D.J. Gray, Transgenic rootstock protein transmission in grapevines, ACTA Hort. 738, 2007, 749-753. Li, Z.T., S. Dhekney, M. Dutt, M. Van Aman, J. Tattersall, K.T. Kelley and D.J. Gray, Optimizing Agrobacterium-mediated transformation of grapevine, In Vitro Cell. Dev. Biol. Plant. 42, 2006, 220-227, 2006. Gray, D. J., S. Jayasankar and Z. Li, Vitaceae (Grape Vitis spp.), Chap. 22, In: R. E. Litz (Ed.), Biotechnology of Fruit and Nut Crops, Biotechnology in Agriculture Series, No. 29, CAB International Wallingford, U.K., 2005, pp. 672-706. Gray, D. J., Z. Li and J. Subramanian, Tissue-specific expression of lytic peptides in transgenic grapevines via use of a GFP/NPTII fusion marker, ACTA Hort., 692, 2005, 125-130. Li, Z. and D. J. Gray, Isolation by improved TAIL-PCR and characterization of a seed-specific 2S albumin gene and its promoter from grape (Vitis vinifera L.). Genome 48, 2005, 312-320. Li, Z., S. Jayasankar and D. J. Gray, Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Trans. Res. 13, 2004, 143-154. Li, Z., S. Jayasankar and D. J. Gray, Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera), Plant Sci. 160, 2001, 877-887. Jayasankar, S., Z. Li and D. J. Gray, In vitro selection of Vitis vinifera Chardonnay with Elsinoe ampellina is accompanied by fungal resistance and enhanced secretion of chitinase, Planta 211, 2000, 200-208. Scorza, R., J. M. Cordts, D. J. Gray, D. Gonzalves, R. L. Emershad and D. W. Ramming, Production of transgenic 'Thompson Seedless' grape (Vitis vinifera L.) plants, J. Amer. Soc. Hort. Sci. 121, 1996, 616-619. Printed version on request or contact djg@ufl.edu
Enabling US Patents Regeneration system for grape and uses thereof. US Patent 6,455,312, 2002. D. J. Gray, J. Subramanian, and R. E. Litz Disease resistance in Vitis. US Patent 6,232,528 B1, 2001. R. Scorza and D. J. Gray Disease resistance in Vitis. US Patent 7,151,203 B2, 2006. R. Scorza and D. J. Gray Pathogen resistant grape plants. US Patent 6,995,015, 2006. J. Subramanian and D. J. Gray Bi-directional dual promoter complex with enhanced promoter activity for transgene expression in eukaryotes. US Patent 7,129,343, 2006. Z. T. Li and D. J. Gray Nucleotide sequences of the 2S albumen gene and its promoter and uses thereof. US Patent 7,250,296, 2007. Z. T. Li and D. J. Gray
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