Genetic transformation of Curcuma alismatifolia Gagnep. using retarded shoots
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A protocol for regeneration and genetic transformation was established for <i>Curcuma alismatifolia</i> Gagnep. 'Chiang Mai Pink' using retarded shoots as explants. <i>In vitro</i> retarded shoots were cut into 0.5×0.5×0.5 cm blocks and co-cultivated with <i>Agrobacterium tumefaciens</i> strain AGLO harboring the binary vector, pBI121 or pBI121-<i>Ca-ACS1</i>. 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<sup>−1</sup> IAA, 4 mg l<sup>−1</sup> IMA, 0.5 mg l<sup>−1</sup> TDZ, 50 mg l<sup>−1</sup> kanamycin and 500 mg l<sup>−1</sup> 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<sup>−1</sup> kanamycin. Within 4 weeks, the shoots were separated and subcultured every 2 weeks on MS medium containing 0.1 mg l<sup>−1</sup> IAA and 50 mg l<sup>−1</sup> 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.
- Plant tissue culture letters
Plant tissue culture letters 23(2), 233-237, 2006-03-01
Japanese Society for Plant Cell and Molecular Biology