Somatic embryogenesis and genetic homogeneity assessment of regenerated plants of Crinum brachynema (Amaryllidaceae): an endemic critically endangered medicinal plant

Crinum brachynema is a bulbous plant belonging to the family Amaryllidaceae which is restricted to Western India. Due to its high rarity and low distribution range, it has been classified as "critically endangered". Establishing an efficient and unprecedented somatic embryogenesis protocol is necessary for its conservation and large-scale propagation. In this study, regeneration was achieved through somatic embryogenesis using bulb explants on MS media supplemented with various concentrations of 2,4-D alone and in combination with N-6-benzyl-adenine. Different advanced phases with maturation of somatic embryo were obtained on MS medium with different ratios of picloram and thidiazuron (TDZ). The highest number of somatic embryos (50.33 +/- 1.52) was obtained after eight weeks in the medium supplemented picloram (2.0 mg L-1) in combination with TDZ (0.5 mg L-1). MS medium with reduced concentration of salts in combination with GA(3) (1.0 mg L-1) was used for somatic embryo germination. The maximum embryo germination frequency (82.22) was recorded on half-strength MS medium fortified with 1 mg L-1 GA(3). The genetic true-to-typeness of regenerated plants was confirmed by ISSR, SCoT and RAPD primers based molecular analyses. This confirmed their genetic homogeneity compared to the mother plant and it also demonstrated the reliability of our somatic embryogenesis system for C. brachynema. The protocol developed may facilitate efforts in reintroduction, restoration, and ex situ conservation of C. brachynema in its natural habitat and its potential commercial utilization.