Micropropagation of Leptadenia reticulata -: A medicinal plant

Leptadenia reticulata (Retz.) Wight. & Arn., an important herbal medicinal plant, belongs to the family Asclepiadaceae. This plant has been known for its medicinal uses since 4500 bc. Presently this is an endangered species. There is a need for applying non-conventional methods of propagation for conservation and sustainable utilization of biodiversity of Leptadenia reticulata. We developed a micropropagation method for mass multiplication of L. reticulata. Explants harvested from greenhouse-maintained and field-grown plants were used to establish cultures of L. reticulata. The nodal shoot segments were surface-sterilized and cultured on Murashige and Skoog (MS) medium along with additives (25 mg l(-1) each of adenine sulfate, arginine, and citric acid, and 50 mg l(-1) ascorbic acid) containing 0.6 muM indole-3-acetic acid (IAA) and 9 muM N-6-benzyladenine (BA). Three to four shoots differentiated from each node within 25-30 d at 26 +/- 2degreesC and 36 mumol m(-2) s(-1) spectral flux photon (SFP) for 12 h d(-1). Shoots were further multiplied by (1) repeated transfer of mother explant on fresh medium containing 0.6 muM IAA and 2.2 muM BA, and (2) subculture of in vitro-differentiated shoots on MS medium with 6.6 muM BA and 0.6 muM IAA. After three or four subcultures, the basal clump with shoot bases was divided into three or four subclumps and multiplied on the fresh medium. From each clump 15-20 shoots regenerated within 25 d. Ninety percent of the in vitro-produced shoots rooted ex vitro if these were pulse-treated with 123 muM each of indole-3-butyric acid and beta-naphthoxyacetic acid. The plantlets were transferred to bottles containing sterile 'soilrite' (soilless compost and soil conditioner) moistened with half-strength MS macrosalts. Ninety-five percent of the plantlets were hardened in the bottles within 15 d. The hardened plants were then transferred to black polybags in the nursery. Field transferred plants are growing normally and have flowered. The protocol developed is reproducible. From a single nodal segment about 1700 hardened plants could be regenerated within 174 d.