Plant regeneration thrugh somatic embryogenesis and RAPD analysis of regenerants in Tylophora indica.

Abstract

A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2.4-D; 0.03–3 mg l−1; 0.0–13.56 μM) and kinetin (Kn; 0.01 mg l−1; 0.05 μM). The best response for callus induction was obtained on MS medium containing 2 mg l−1 (9.04 μM) 2.4-D and 0.01 mg l−1 (0.05 μM) Kn. After two subeultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5–3 mg l−1; 2.22–13.32 μM) and 2-isopentenyladenine (2ip; 0.53 mg l−1; 2.46–14.76 μM) along with 0.01 mg l−1 (0.05 μM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg l−1 (9.84 μM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic, DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.