Factors Effecting the Transformation of Citrus medica L.var. sarcodactlis Mediated by Agrobacterium

Abstract:

Agrobacterium-mediated transformation of Citrus medicaL, var. sarcodactlis was performed by leaf disc. The binary vector harbors the gene of interest TPS and NUFII 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. tumefaeiens strain LBA4404 at OD_{600 nm} 0.6 showed the highest virulence on the leaf explants of C. medica with 20 rain of immersion. Two to three days ofpre-culture on MT medium in dark and 3 days of co-cultivation were optimum for C. medica. The mixture containing 250 ml L^-1 Cefand 250 ml L^-1 Cb gave the highest rate of callus induction for C. medica. Addition of 100 μ mol/L AS and 400 mol/L L-Cys enhanced the transformation efficiency of C. medica. Under the optimal condition, the percentage of GUS transient expression was 5.9%.transformation; Trehalose synthase gene.