STUDY ON AGROBACTERIUM-MEDIATED AND PEG- MEDIATED TRANSFORMATION OF HYPOCOTYL PROTOPLASTS OF V1GNA SINENSIS
Li Xue-bao, Bai Yong-yan, Xu Zhi-hong (National Laboratory of Plant Molecular Genetics, Shanghai 200032)
The transformed calli from cowpea (Vigna sinensis) hypocotyl protoplasts were selected on MS medium containing 20 mg/L kanamycin or 50 mg/L chloramphe-nicol after the protoplasts were coeultiva-ted with Agrobacterium tumefaciens (strains PGV 2260(pBI 121) or C 58 C 1 (pBZ 6111)). and confirmed by analysis of neo-mycin phosphotransferase Ⅱ(NPT Ⅱ), glu-curonidase (GUS) and nopaline synthetase (NOS) activity. The experimental result that transient expression of GUS activity in cowpea cells markedly increased indicated that lower pH, phosphate starvation and phenolic compounds could activate vir genes of A. tumefaciens and facilitate T-DNA transfer into cowpea cells in some extent. Higher pH and concentration of Ca2+ promoted transient expression of GUS gene in PEG-mediated DNA direct transfer. The activity of transient expression of GUS gene was positively correlated with the amount of foreign plasmid DNA in the range of 10-100 μg. 48 hours after DNA uptake was the optimal by flurometric assay. In the same experimental condition, expression of GUS gene under the control of MAS-CaMV 35S chimeric promoter was more effective than that under the control of CaMV 35 S promoter.