农杆菌介导佛手遗传转化主要影响因素的研究

Agrobacterium-mediated transformation of Citrus medica L.var.sarcodactlis was performed by leaf disc.The binary vector harbors the gene of interest TPS and NPTII gene driven by CaMV 35S promoter.The presence of TPS gene integration in the genome was detected and confirmed by GUS assay and PCR,respectively. Transformation parameters optimized were bacterial concentration,pre-culture period,immersion time, concentrations of antibiotics(cefotaxime,Cef and carbenicilline,Cb)and reagents(acetosyringone,AS and L-cysteine,L-Cys).Results were obtained based on the percentage of GUS positive transient expression.A. tumefaciens strain LBA4404 at OD600 nm 0.6 showed the highest virulence on the leaf explants of C.medica with 20 min of immersion.Two to three days of pre-culture on MT medium in dark and 3 days of co-cultivation were optimum for C.medica.The mixture containing 250 ml L-1 Cef and 250 ml L-1 Cb gave the highest rate of callus induction for C.medica.Addition of 100μmol/L AS and 400 mol/L L-Cys enhanced the transformation efficiency of C.medica.Under the optimal condition,the percentage of GUStransient expression was 5.9%.