Shoot apical meristem explants of Vitis vinifera "Thompson Seedless" were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L(-1) in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L(-1) kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.
A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l(-1) in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l(-1), which permitted the proliferation of more non-transformed cells. Transgenic plants of "Alachua" and "Carlos" were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.
Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of 'Supreme' and unopened leaves and petioles of 'Delicious'. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of 'Alachua' and 'Carlos' cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in 'Carlos' (379.3) followed by 'Alachua' (350.0) and 'Delicious' (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 lM Benzyladenine (BA). Although high embryo germination rates (80-100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6-47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.
Factors influencing Agrobacterium-mediated transformation of 15 Vitis rotundifolia, Vitis rupestris, Vitis vinifera and Vitis hybrids were explored. A green fluorescent protein/neomycin phosphotransferase II (GFP/NPT II) fusion gene was used to measure transient and stable transgene expression levels. Embryogenic cell cultures at different developmental stages were co-cultivated with Agrobacterium to determine the best stage for transformation. The effect of antioxidant treatment on recovery of transgenic embryo and plant lines was also studied. Transient and stable GFP expression varied widely among the species. For example, 1-81% of V. vinifera somatic embryos exhibited transient GFP expression, depending on the cultivar. Similarly, 0-60% of co-cultivated embryos produced stable transgenic lines. Proembryonal masses and cotyledonary stage somatic embryos were best for transformation of V. rotundifolia, V. rupestris and certain Vitis hybrids, whereas only cotyledonary stage somatic embryos were best for transformation of V. vinifera. Agrobacterium-induced browning/ necrosis was reduced by adding 1 g L-1 dithiothreitol (DTT) antioxidant to the post cocultivation wash medium. Transgenic plants have been recovered from V. rupestris 'St.
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