Direct somatic embryogenesis and plant regeneration from leaf sheath explants of mango ginger (Curcuma amada Roxb.)

An efficient and simple procedure was systematically developed for inducing direct somatic embryogenesis and plantlet regeneration from leaf sheath explants of Curcuma amada Roxb. A two-step culture system was used to induce somatic embryogenesis. The optimized procedure resulted in direct somatic embryogenesis from 93.3% explants after 3-wk culture. Leaf sheath explants were incubated for 2 wk on medium containing 2.24 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine to initiate direct somatic embryogenesis. Thereafter, these explants were transferred to a medium containing 9.10 μM thidiazuron and 1.33 μM α-naphthaleneacetic acid. Elongated somatic embryos obtained from these cultures germinated readily, and the optimal frequency of plantlet development (86.7%) was achieved when embryos were cultured in darkness on 1/2 strength Murashige and Skoog medium containing 1.44 μM gibberellic acid. Histological and scanning electron microscopic studies showed that the initial cell divisions that led to embryo formation occurred in epidermal and subepidermal cells, followed by the development of globular and elongated structures that appeared to be somatic embryos. The presence of a clear protoderm in the globular structures and procambial strands in the elongated structures confirmed that these structures were true somatic embryos. Plantlets derived from somatic embryos were acclimatized successfully to ex vitro conditions at a survival rate of 87.43% and developed with normal phenotypes.