In vitro regeneration technique in Rauwolfia serpentina and quantification of reserpine

Abstract

The reliable technique for the indirect regeneration of R. serpentina was standardised in the present investigation. The secondary metabolites extracted from callus using methanol and analysed using Thin Layer Chromatography (TLC) and UV-Vis Spectrophotometer. The in vitro induction of callus influenced by several factors such as media composition and explants quality. From the various media combinations of 2, 4-D and BAP the frequency of highest callus induction (94.67%) was observed on MS + 2.0 mgL-1 2.4-D + 1.0 mgL-1 BAP when leaf disc used as explant. Throughout the organogenic callus induction, different calli were observed having variation in colour and texture. The shoot regeneration frequency was observed highest (98.33%) in 4.5 mgL-1 BAP + 0.5 mgL-1 NAA. Maximum shoots (15.33 per callus) were observed in leaf callus. The rooting was induced (95.33%) in in vitro regenerated shoots in Murashige and Skoog (MS) medium which contain 1.0 mgL-1 NAA + 0.1 mgL-1 BAP + 1 gL-1 activated charcoal (AC). The in vitro regenerated plantlets through callus culture were hardened in the greenhouse with 75 per cent establishment. The quantity and quality of reserpine was measured in callus and root of R. serpentina. The TLC reveals the accumulation of reserpine at callus as well as roots of cultivated plant. Spectrophotometric estimation showed that concentration of reserpine observed in callus extract was higher (6.8 µgml-1) than the root extract (6.4 µgml-1).