OptimizingAgrobacterium-mediated transformation of grapevine

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Abstract

A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl−1 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75 mgl−1 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome: 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.

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