Encapsulation of Rauvolfia tetraphylla microshoots as artificial seeds and evaluation of genetic fidelity using RAPD and ISSR markers

Abstract

Synseeds of Rauvolfia tetraphylla were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Woody Plant Medium (WPM) (Lloyd and McCown, 1981) and 100 mM calcium chloride. Effect of different medium on in vitromorphogenic response of synseed was evaluated. The maximum frequency (90.3%) of conversion of encapsulated beads into plantlets was achieved on WPM containing 7.5 μM 6-benzyladenine (BA) and 2.5 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. Encapsulated nodal segments stored at 4°C for 1 to 8 weeks also showed successful conversion, followed by development into complete plantlets when returned to regeneration medium. Healthy root formation was achieved in plantlets following 4 weeks of their transfer to Murashige and Skoog medium containing 0.5 μM indole-3-butyric acid (IBA). Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. Genetic stability of synseed-derived plantlets was assessed and compared with mother plant using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis. No changes in RAPD and ISSR profiles were found among the regenerated plantlets. This synseed protocol could be useful for conservation, short-term storage and exchange of germplasm of this endangered medicinal plant between national as well as international laboratories. Key words: Encapsulation, germplasm storage, regeneration, synthetic seeds, random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR).