An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.) |
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Authors: | Zhijian T Li S A Dhekney M Dutt D J Gray |
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Institution: | (1) Grape Biotechnology Core Laboratory, Mid-Florida Research and Education Center, University of Florida/IFAS, 2725 Binion Road, Apopka, FL 32703-8504, USA;(2) Present address: Citrus Research and Education Center, University of Florida/IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850, USA |
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Abstract: | An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP
and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing
period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin
added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic
plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on
MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic
plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated
to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’. |
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Keywords: | Agrobacterium tumefaciens Cocultivation EGFP GUS expression Transgenic plants Vitis vinifera |
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