Genetic transformation of Curcuma alismatifolia Gagnep. using retarded shoots

抄録

A protocol for regeneration and genetic transformation was established for Curcuma alismatifolia Gagnep. ‘Chiang Mai Pink’ using retarded shoots as explants. In vitro retarded shoots were cut into 0.5×0.5×0.5 cm blocks and co-cultivated with Agrobacterium tumefaciens strain AGLO harboring the binary vector, pBI121 or pBI121-Ca-ACS1. The explants were incubated in the bacteria suspension for 30 min. The explants and bacteria were cultured on MS medium for 2 days in darkness at 25°C for co-cultivation. Then, the explants were transferred onto MS medium containing 0.1 mg l⁻¹ IAA, 4 mg l⁻¹ IMA, 0.5 mg l⁻¹ TDZ, 50 mg l⁻¹ kanamycin and 500 mg l⁻¹ vancomycin. The explants were subcultured every 2 weeks. After 4 weeks in culture, the explants with small shoot buds were transferred onto MS medium containing 50 mg l⁻¹ kanamycin. Within 4 weeks, the shoots were separated and subcultured every 2 weeks on MS medium containing 0.1 mg l⁻¹ IAA and 50 mg l⁻¹ kanamycin. PCR analysis, histochemical GUS assay and Southern blotting of the regenerated plants confirmed transformation events. We obtained transformed plants within 3 months after co-cultivation with the bacteria and the transformation frequency exceeded 14%, which is suitable for practical use.