Abstract:

In order to establish transformation system based on embryogenic suspension cultures in rubber tree (Hevea brasiliensis Mu?ll. Arg), cell aggregates from embryogenic suspension cultures were co-cultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hpt II genes. Effect of co-cultivation time and A. tumefaciens concentration on transient expression of gusA was examined by GUS assay. The sensitivity of cell aggregates to hygromycin (Hyg) added to the medium was also tested in order to determine the optimal concentration of this antibiotic in the selective medium. The results suggested that 3 days was an optimal co-cultivation time, a desired OD600 of strain EHA105 was 0.4-0.6, and 15 mg/L concentration of Hyg was found to be enough to inhibit the growth of cell aggregates. A total of 53 Hyg-resistant embryogenic calli were obtained after transformation and co-cultivation. At last, 7 somatic embryos were further formed. GUS assay and PCR analysis indicated that gusA had been integrated into the genome of rubber tree. In all, this study laid the groundwork for efficient Agrobacterium tumefaciens mediated transformation using embryogenic suspension cultures in rubber tree.