Articles
GENETIC TRANSFORMATION OF EMBRYOGENIC CULTURES AND RECOVERY OF TRANSGENIC PLANTS IN VITIS VINIFERA, VITIS ROTUNDIFOLIA AND VITIS HYBRIDS
Article number
738_98
Pages
743 – 748
Language
English
Abstract
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 co-cultivation wash medium.
Transgenic plants have been recovered from V. rupestris St.
George, V. rotundifolia Alachua and Carlos, V. vinifera Cabernet Franc, Chardonnay, Merlot, Pinot Noir, Sauvignon Blanc, Shiraz, Superior Seedless, Thompson Seedless and Zinfandel, and Vitis hybrids Conquistador, Seyval Blanc and Freedom. Research to optimize transformation conditions of additional Vitis species and hybrids is ongoing.
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 co-cultivation wash medium.
Transgenic plants have been recovered from V. rupestris St.
George, V. rotundifolia Alachua and Carlos, V. vinifera Cabernet Franc, Chardonnay, Merlot, Pinot Noir, Sauvignon Blanc, Shiraz, Superior Seedless, Thompson Seedless and Zinfandel, and Vitis hybrids Conquistador, Seyval Blanc and Freedom. Research to optimize transformation conditions of additional Vitis species and hybrids is ongoing.
Authors
S.A. Dhekney, Z.T. Li, M. Van Aman, M. Dutt, J. Tattersall, K.T. Kelley, D.J. Gray
Keywords
grape, Agrobacterium, somatic embryo, green fluorescent protein, browning, antioxidant
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