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

ZHOU Chun-li1,3, GUO Wei-dong2*, LU Mei2, CHEN Jin2, LI Yu-ping1 ( 1. College of Life Sciences, Jiangxi Normal University of Science and Technology, Nanchang 330013, China; 2. Academy of Life and Environment Science, Zhejiang Normal University, Jinhua 321004, China; 3. College of Horticulture, Northwest Agricultural and Foresty University, Yangling 712100, China)  
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 100 μmol/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%.
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