Development of transgenic tea plants from leaf explants by the biolistic gun method and their evaluation

The PDS1000-He biolistic gun was used to bombard plasmid DNA harbouring gus and nptII genes into tender young leaves of in vitro grown shoots of Camellia sinensis (tea). Out of a total of 500 bombarded leaves, 217 (43.4 %) showed callusing after 5 weeks on selection medium containing 1.71 A mu M kanamycin. Only 15 of these regenerated into indirect shoot buds. Only 7 out of 15 putative transformants showed the expected 400 bp signal with gus gene specific primers during PCR analysis. On the other hand, all the 15 putative transformants tested positive with nptII gene specific primers. In Southern hybridization with nptII specific gene probe, all the six randomly selected PCR positive plants showed stable integration of nptII gene. Both the transgenic and not-bombarded control plants showed phenotypic similarity under polyhouse conditions. Although their growth parameters were significantly at par, significantly lesser shoot height was recorded in transgenic plants. The reproductive behavior of the transgenics was also depressed. Thus, floral bud and flower abscission, fruit drop as well as empty seed production was higher in the transgenics as compared to control. Viability and germination of transgenic seeds was also significantly lower than control. Survival of the transgenic seedlings was also negligible (about 3 %). Hence, the chances of germ-line transmission of the transgenes were remarkably reduced in case of gus-transgenics. Tea being a vegetatively propagated plant, the method described in the present paper is an important approach for developing transgenics of elite tea plants from leaf explants.