Assessment of genetic stability in somatic embryo derived plantlets of Roxb. using inter-simple sequence repeat analysis.

An efficient plantlet regeneration protocol using immature zygotic embryos (IZEs) via somatic embryogenesis has been developed in Roxb. The regenerated plantlets were evaluated for their genetic stability. IZEs were incubated on Murashige and Skoog (MS) media augmented with 1.07-16.11 μM naphthalene acetic acid (NAA) or 0.90-13.97 μM 2,4-dichlorophenoxyacetic acid. The optimum callus induction (96.6%) was observed on MS medium augmented with 5.37 μM NAA. Induction of somatic embryos (SEs) was observed after sub-culture of calli on medium with decreased concentrations of NAA (0.54-5.37 μM), either alone or 2.69 μM NAA in combination with 2.22-8.90 μM benzyladenine (BA) or 2.32-9.30 μM Kinetin. Maximum number (33.4 ± 0.85) of SEs occurred on MS medium augmented with 2.69 μM NAA + 4.40 μM BA + 3% sucrose. Highest percentage (67.3 ± 0.37) of SEs matured and developed into cotyledonary stage by subsequent subculture on the same medium. SE formation and maturation decreased when sucrose concentrations were higher than 3%. Seventy percent of mature somatic embryos developed into plantlets on half strength MS medium augmented with 5.80 µM gibberellic acid. The various stages of development during somatic embryogenesis include  globular, heart, torpedo and mature stages as revealed by the stereomicroscopic and histological studies of explants. Plantlets derived from SEs were successfully acclimatized in the greenhouse with a survival rate of 78%. Among the survived plantlets, 9 plantlets were randomly selected for inter-simple sequence repeat (ISSR) analysis. Of the 13 primers used, 8 produced reproducible and monomorphic bands. ISSR analysis revealed a homogenous amplification profile for all regenerated plantlets analyzed validating the genetic stability of somatic embryo derived plantlets.