Cloning of Tylophora Indica through Forced Axillary Branching and De Novo Adventitious Shoot Formation

Abstract

The present investigation was carried out on an important medicinal plant Tylophora indica belonging to family Asclepiadaceae with an aim to establish an efficient and reproducible micropropagation protocol for its mass production. The different vegetative parts i.e. leaf, nodal segment and shoot apices taken from an elite, field grown, 4 years-old-plant and thereafter planted on variously supplemented Murashige and Skoog’s medium for de novo adventitious shoot induction and multiple shoot proliferation. The plant exhibited high degree of propensity of de novo shoot proliferation directly from leaf segments on MS medium supplemented with BAP (8.8 μM) either alone or in combination with adenine sulphate (1.35μM). Nodular meristemoids differentiated from the cut ends of leaf lamina after 8-10 days of culturing and covered the whole surface within 3-4 weeks. Eventually these meristemoids developed into green leafy shoots in nearly 80% of cultures. Initially, fewer shoots were formed but number increased further to 55-60 shoots per flask on subsequent sub culturing in about 90% of the cultures. Tylophora indica exhibited high degree of multiple shoot proliferation from nodal segments and shoot apices taken from in vivo plants. Bud break and axillary shoot proliferation from nodal segments occurred on MS supplemented with 17.6-μM 6-benzyl adenine forming nearly 20-25 shoots/explant after 6 weeks. However, prolific multiple shoot induction from nodal explants occurred on MS medium supplemented with 22 μM 6-benzyladenine in conjunction with α- naphthalene acetic acid (3.67 μM) and L-ascorbic acid (8.4 μM), where 45-50 shoots regenerated from single axillary bud after 5-6 weeks of culturing. Shoot apices also exhibited multiple shoot proliferation when cultured on MS medium supplemented with BAP (17.6-22.0μM) either alone or in combination with NAA (3.6μM) + L-ascorbic acid (8.4μM), where 20-25 shoots were obtained after 6-7 weeks of culturing. The regenerated shoots were rescued from the culture vessels and transferred onto half strength MS medium, full strength MS medium and MS medium supplemented with different concentrations of IAA, NAA and IBA for root induction. The best rooting response (90%) was observed on auxin free half strength MS medium whereas addition of IBA to full strength MS medium also showed good results. Rooted plantlets were then transferred to moist cotton jars covered with plastic bags for initial acclimatization and were kept in growth room for 12-15 days. They were then transferred to the potting mixture of soil: vermicompost (1:1) and plants with newly formed leaves were shifted to green house bench with 90% survival rate.