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Am. J. Enol. Vitic. 60:3:285-292 (2009)
Copyright © 2009 by the American Society for Enology and Viticulture.
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Factors Influencing Genetic Transformation and Plant Regeneration of Vitis

Sadanand A. Dhekney1, Zhijian T. Li1, Thomas W. Zimmerman2 and Dennis J. Gray1,*

1 Mid-Florida Research and Education Center, University of Florida/IFAS, 2725 Binion Road, Apopka, FL 32703; and 2 Biotechnology & Agroforestry, University of the Virgin Islands, RR 1, Box 10,000, Kingshill, St. Croix, VI 00850.

Acknowledgments: This research was supported by the USDA Tropical & Subtropical Agriculture Research Program, the Florida Department of Agriculture and Consumer Services Viticulture Trust Fund, and the Florida Agricultural Experiment Station.

The authors thank Seenivasan Natarajan for assistance with statistical analysis.

* Corresponding author (email: djg{at}ufl.edu; tel: 407 884-2034; fax: 407 814-6186)

The effect of callus induction media, culture protocols, embryogenic culture age and cotyledon excision treatment on the production of transgenic embryo and plant lines of Vitis was studied. Embryogenic cultures initiated from leaves or stamens and pistils were transformed with Agrobacterium containing an enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. The production of transgenic embryo lines on different culture media was genotype-dependent. Vitis vinifera produced the most transgenic embryo lines (7.5 to 26%) when cultured on DM and X6 medium, whereas Vitis champinii and certain Vitis hybrids produced the best response on NB and X6 medium (5 to 12.5%). Among Vitis vinifera genotypes, Merlot, Superior Seedless, and Thompson Seedless produced transgenic embryo lines irrespective of 4, 8, or 12 month culture ages (11.6 to 36%), whereas Cabernet franc, Cabernet Sauvignon, and Shiraz produced embryo lines only from 4 month cultures (4.1 to 16%). The effect of cotyledon excision of germinated embryos on recovery of transgenic plants varied with variety. The treatment resulted in better plant recovery in Vitis vinifera and Vitis riparia (26.7 to 73.3%), whereas it resulted in a lower plant recovery in Vitis champinii and Vitis hybrids. Transgenic plants were recovered from 19 Vitis genotypes comprised of the species Vitis champinii, Vitis riparia, Vitis rupestris, Vitis vinifera, and Vitis interspecific hybrids. PCR and quantitative real-time PCR confirmed the presence and copy number of the egfp transgene in selected transgenic plants. Transgenic vines established in the field exhibited normal vegetative and reproductive growth compared to non-transformed vines. Stable GFP (green fluorescence protein) expression patterns in mature plant parts of transgenic plants have been observed for over 4 years indicating no transgene silencing.

Key words: grapevine, Agrobacterium, somatic embryos, transgenic plants, EGFP






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Copyright © 2009 by the American Society for Enology and Viticulture.