New method for obtaining transgenic papaya plants by Agrobacterium-mediated transformation of somatic embryos

摘要

Here we describe a new method for efficient genetic transformation and rapid regeneration of transgenic papaya (Carica papaya L.) plants within six months. Agrobacterium LBA 4404 cells were transformed by electroporation with the Ti binary vector pBI 121 carrying the uidA and nptII genes and activated by acetosyringone. Somatic embryos obtained from liquid-culture were wounded by vortexing with tungsten M-15 in half-strength MS liquid medium and then co-cultivated with the transformed and activated Agrobacterium for three days. The infected somatic embryos were transferred to callus initiation medium containing 600 mg/L of carbenicillin for three weeks and then to selection medium with 300 mg/L of kanamycin for three weeks and another selection medium with 150 mg/L of kanamycin for six weeks. The kanamycin-resistant embryos were selected and cultured on maturation medium for one week and then on regeneration medium for 3-5 weeks. The germinated embryos were transferred to rooting medium for 2-4 weeks and thereafter acclimatized for one week in a growth chamber and established in a greenhouse. Forty-five kanamycin-resistant lines were produced from five initial zygotic embryos. Forty-four (97.8 percent) of them developed roots, 42 (93.3 percent) were X-gluc positive, and 41 (91.1 percent) regenerated normal plants and were established in a greenhouse. The transgenic lines where the nptll and uidA genes were integrated into their genomes were further confirmed by PCR detection and Southern hybridization. The method described here should be very valuable for generation of transgenic papaya plants with different genetic constructs.