EFFICIENT TRANSFORMATION OF PAPAYA BY COAT PROTEIN GENE OF PAPAYA RINGSPOT VIRUS-MEDIATED BY AGROBACTERIUM FOLLOWING LIQUID-PHASE WOUNDING OF EMBRYOGENIC TISSUES WITH CABORUNDUM

Generation of transgenic papaya (Carica papaya L.) has been hampered b y the low rates of transformation achieved by conventional Agrobacteri um infection or microprojectile bombardment. We describe an efficient Agrobacterium-mediated transformation method based on wounding of cult ured embryogenic tissues with carborundum in liquid phase. Embryogenic tissues were obtained from cultured immature zygotic embryos collecte d 75-90 days after pollination. The expressible coat protein (CP) gene of a Taiwan strain of papaya ringspot virus (PRSV) was constructed in a Ti binary vector pBGCP, which contained the NPT-II gene as a select ion marker. The embryogenic tissues were vortexed with 600 mesh carbor undum in sterile distilled water for 1 min before treating with the di sarmed A. tumefaciens containing the pBGCP. Transformed cells were cul tured on kanamycin-free medium containing 2,4-D and carbenicillin for 2-3 weeks and then on the kanamycin medium for 3-4 months. The develop ed somatic embryos were transferred to the medium containing NAA, BA a nd kanamycin and subsequently regenerated into normal-appearing plants . Presence of the PRSV CP gene in the putative transgenic lines was de tected by PCR and the expression of the CP was verified by Western blo tting. The transgene was nuclearly inherited as revealed by segregatio n analysis in tile backcrossed R-1 progeny. From five independent expe riments, the average successful rate of transformation was 15.9% of th e zygotic embryos treated (52 transgenic somatic embryo clusters out o f 327 zygotic embryos treated), about 10-100 times higher than the ava ilable methods previously reported. Thus, wounding highly regenerable differentiating tissues by carborundum vortexing provides a simple and efficient way for papaya transformation mediated by Agrobacterium.