ISHS Acta Horticulturae 673: IX International Symposium on Flower Bulbs
AGROBACTERIUM-MEDIATED PRODUCTION OF TRANSGENIC PLANTS IN TRICYRTIS HIRTA(LILIACEAE) |
| Authors: | Y. Adachi, S. Mori, M. Nakano |
| Keywords: | Agrobacterium tumefaciens, embryogenic calluses, Liliaceous geophyte |
| 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  -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 (Hygr) 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, Hygr 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.
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