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Improved Agrobacterium Mediated Transformation in Cowpea Vigna unguiculata L. Walp.

Keywords: legume , hpt gene , Transgenic plants , L-cysteine , Acetosyringone

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Abstract:

Agrobacterium tumefaciens strain LBA4404 carrying binary vector pCAMBIA1305.1, with a hygromycin phosphotransferase gene (hpt) and a β-glucuronidase (GUS) gene interrupted with an intron, was used for transformation of Vigna unguiculata cotyledonary node explants. High concentration of acetosyringone (200 μM) in the Agrobacterium culture and co-cultivation medium with 1 g L-1 L-cysteine, 250 mg L-1 Na-thiosulphate and 150 mg L-1 dithiothretiol (DTT) proved to be indispensable for successful transformation. Three days old cotyledonary nodes were used for transformation studies. Binary vector pCAMBIA 1305.1 proved to be very efficient for transformation. Stable transformation with 1.61% efficiency was achieved using optimized conditions. Transformed green shoots were rooted on medium containing hygromycin. Transformed shoots tested positive for hpt gene by polymerase chain reaction. GUS activity was detected in cotyledonary nodes and leaves of the putative transformants. Southern analysis of putative transformants showed the integration of hpt into the plant genome.

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