Genetic transformation of Curcuma alismatifolia Gagnep. using retarded shoots

Abstract

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 0.5x0.5x0.5 cm blocks and cocultivated 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-1IAA, 4mg l-1IMA, 0.5 mg l-1TDZ, 50 mg l-1kanamycin and 500 mg l-1vancomycin. 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-1kanamycin. Within 4 weeks, the shoots were separated and subcultured every 2 weeks on MS medium containing 0.1 mg l-1IAA and 50 mg l-1kanamycin. 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. Copyright © 2006 The Japanese Society for Plant Cell and Molecular Biology.