An efficient method for sonication assisted Agrobacterium-mediated transformation of coat protein (CP) coding genes...

An efficient method for the production of transgenic papaya was developed via Sonication Assisted Agrobacterium-mediated Transformation (SAAT) of somatic embryos. The plasmid pGA482G was modified to contain gene PTi-Epj-TL-PLDMV with CP coding sequence of PLDMV Japan strain and chimeric gene PTi-NP-YKT with multiple CP coding sequences from PRSV Taiwan strain, PRSV Hawaii strain and PRSV Thailand strain, respectively. Disarmed Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pGA482G with the CP genes and nptII gene was used to transform embryo calli of papaya variety Sunset to produce transgenic papaya plants. The experiment was focused on the screening of effective transformation method. The engineered Agrobacterium grown overnight was diluted with an infection media of high osmotic pressure (1/2 MS medium contain 6% sucrose and 1% glucose, pH 5.7) and adjusted to optical density OD600nm = 0.15-0.20, embryonic calli were immerged in it for 30 min and treated with 5 s, 15 s, and 20 s sonication respectively during the infection. Results indicated that 15 s sonication treatment improved the transformation efficiency dramatically. After 15 s sonication treatment on embryo calli loaded in 15 ml sterile plastic tubes, 21 putative transgenic lines were produced from 80 pieces embryonic calli (26.3%) transformed by Agrobacterium [pGA482G/CPG] and 8 putative transgenic lines was produced from 48 pieces embryonic calli (16.7%) transferred by Agrobacterium [pGA482G/CPB], while only a single line came out of 64 pieces embryonic calli (1.6%) transformed by Agrobacterium [pGA482G/CPG] and none from 25 pieces embryonic calli transformed by Agrobacterium [pGA482G/CPB] in the non-treatment control. Results also showed that the best concentration of selection antibiotic was 120 mg/L kanamycin. A total of 42 resistant shoots were produced from 421 pieces of original embryonic calli in 9 months. The presence of the CP genes in the transgenic plants and their integration into the papaya genome were confirmed by PCR and Southern hybridization respectively.