Plant regeneration via organogenesis and somatic embryogenesis in Tylophora indica- an important medicinal plant

Abstract

The present investigation was carried out on an important endangered medicinal plant Tylophora indica with a view to develop a reproducible protocol for its clonal propagation under in vitro conditions. This plant is commonly known as “Dama-Bel” or ‘Antmool’ and is of great medicinal value. Leaf and stem explants were excised from an elite field grown plant and thereafter planted on variously supplemented Murashige and Skoog’s medium for callus induction, direct and indirect organogenesis and somatic embryogenesis.

Tylophora exhibited good degree of propensity for de novo adventitious shoot formation directly from leaf segments on MS medium supplemented with NAA(29.4µM) and Kn (4.65µM) in about 30% cultures forming 20-25 shoots after 8 weeks of culturing. Direct rooting from the leaf explant was observed on MS medium supplemented with different concentrations of NAA with K or BA.

Induction of callus from leaf and stem explants was observed on both NAA+ Kn and 2,4-D supplemented media but best results were obtained on MS medium with NAA(29.4µM) and Kn (4.65µM), hereby, designated as NK medium. the callus formed was green and was capable of sustained growth on subsequent subcultureing. The callus on 2,4-D (9.74- 19.48µM) supplemented medium took longer time to initiate and was higly frible and pale yellow in color. The spectrum of induced differentiation from calli was wide and included xylogenesis, rhizogenesis, caulogenesis and embryogenesis.

Root differentiation from leaf and stem calli occurred on MS medium supplemented with α-naphthalene acetic acid (29.4 μM) + kinetin (4.65 μM) or IBA supplemented medium where the roots formed were thick, white having dense root hairs. High frequency of shoot differentiation from all the calli was observed on both solid and liquid MS media supplemented with either 6-benzyladenine or kinetin alone. Leaf callus when transferred onto MS containing 8.8 μM benzyladenine resulted in excellent shoot induction in 85% of the cultures.

For the induction of embryogenesis, 2, 4- dichlorophenoxy acetic acid at a concentration of 19.48 μM and 3 % sucrose proved very effective in inducing somatic embryos in 95 % of the cultures. Detailed stereozoom microscopic studies revealed the occurrence of various stages of development of embryos like globular, heart shaped, torpedo and cotyledonary. Among the numerous globular shaped embryos formed, only 60% developed into cotyledonary embryos after 6-7 weeks of culturing which further developed into tiny plantlets with distinct root and shoot axis.

Regenerated shoots thus obtained were rooted on MS medium supplemented with IBA. Rooted plantlets of T. indica were transferred successfully to the field conditions through successive hardening stages showing 90% survival .