Abstract
Crinum malabaricum, Lekhak & S.R. Yadav (Amaryllidaceae) is a critically endangered bulbous plant endemic to India. Establishing an efficient plant regeneration protocol is necessary for its conservation and large-scale propagation. In the present study, regeneration was achieved through somatic embryogenesis from callus produced on MS media supplemented with various different concentrations of 2,4-D alone and in combination with N6-benzyl-adenine (BA). Different advanced stages of embryo development (globular, torpedo and cotyledonary) and maturation were obtained on MS medium, with combinations of picloram and thidiazuron (TDZ). A high rate of somatic embryos (55.89 ± 0.60) was obtained after eight weeks. SEM examinations showed the occurrence of cell clusters (embryogenic) which converted to somatic embryos. Well-developed cotyledon embryos were successfully germinated on PGRs free full and half strength MS medium, however, 94.03% of somatic embryos germinated on half-strength MS medium supplemented with 1.0 mg L−1 gibberellic acid. The true-to-type genetic conformity of regenerated plants was examined by SCoT, ISSR and RAPD primers based molecular analysis. The amplification bands produced by SCoT, ISSR and RAPD primers applied were monomorphic across all the regenerated plants. This confirmed their genetic homogeneity compared to the mother plant and also demonstrated the reliability of our somatic embryogenesis system for C. malabaricum. A rapid method for phytochemical analysis based on LC-ESI/MS exhibited better separation and analysis of galanthamine and lycorine from methanolic extracts of in vitro raised plants derived from somatic embryogenesis. The protocol developed should be helpful in reintroduction, restoration and ex situ conservation of this valuable plant in the natural condition as well as in the pharmaceutical sectors.
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