AGROBACTERIUM-MEDIATED PRODUCTION OF TRANSGENIC PLANTS IN TRICYRTIS HIRTA(LILIACEAE)

Abstract

A system for producing transgenic plants has been developed for the Liliaceous geophyte Tricyrtis hirta via Agrobacterium-mediated transformation. Tepal-derived embryogenic calluses were co-cultivated with A. tumefaciens EHA101/pIG121Hm, which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing β-glucuronidase (GUS-intron) genes in the T-DNA region. The duration of co-cultivation and acetosyringone (AS) treatment during co-cultivation affected the frequency of transient expression of the GUS gene: the best result was obtained when embryogenic calluses were co-cultivated for 7 days in the presence of 50 mg l -1 AS. Following co-cultivation, the calluses were transferred to a medium lacking plant growth regulators (PGRs) but containing 40 mg l -1 hygromycin and 300 mg l -1 cefotaxime, on which several hygromycin-resistant (Hyg r ) somatic embryos were produced 8 weeks after transfer. These embryos were transferred to the same medium but containing lower concentrations of antibiotics (20 mg l -1 hygromycin and 100 mg l -1 cefotaxime) to promote germination. Finally, Hyg r embryo-derived plantlets were established on a medium without both PGRs and antibiotics. Most of them were verified to be stable transformants by GUS histochemical assay and PCR analysis.