Agrobacterium-mediated Genetic Transformation and Regeneration of Transgenic Plants in Eucalyptus tereticornis

Transgenic plants were regenerated from transformed embryogenesis tissue of Eucalyptus tereticornis Sm. Friable nodular callus initiated from mature zygotic embryos on MS agar medium supplemented with 2mg/l NAA were transferred to MS medium containing 0.5 mg/l BAP and active embryo masses were started to appear after 3-4 weeks. The embryogenic tissues were inoculated by pricking and cells of disarmed Agrobacterium strain carrying the Beta-glucorinidase (uid A) and neomycin phosphotransferase II (nptII) reporter genes on the binary vector pBI121 were directed at the wound sites. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to selection medium containing Cefotaxime and Kannamycin. After one week on selection medium, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Embryogenic callus with putative transgenic embryos were allowed to develop into rooted plants in MS medium containing 50mgl-1 kanamycin and 500mgl-1 cefotaxmine. Further elongation of the regenerated transgenic plants was carried out on MS liquid medium containing 50mgl-1 kanamycin and 500mgl-1 cefotaxmine. The transformation status was analysed by the growth in the presence of Kanamycin, GUS assays, and PCR amplification of the GUS and nptII gene.