A Micropropagation Protocol for Mass Multiplication and Off-Site Conservation of<i>Celastrus paniculatus-</i>

Abstract

Abstract A protocol was developed using nodal shoot segments as explants for mass/clonal propagation of Celastrus paniculatus-an important threatened medicinal plant. Four to five shoots differentiated from nodal region within 15-20 days on Murashige and Skoog's (MS; 1962) medium containing 1.5 mg 1−1 6-benzylamino purine (BAP) + 0.1 mg 1−1 naphthaleneacetic acid (NAA) + additives (50 mg 1−1 ascorbic acid and 25 mg 1−1 each of adenine sulfate, arginine and citric acid) at 28 ± 2°C temperature and 36 µmol m−28−1 photonflux density, 10 h/day photoperiod. In vitro produced shoots were further multiplied by subculturing on modified Murashige and Skoog's (MMS) medium containing 0.8 mg 1−1 BAP + 0.1 mg 1−1 NAA + additives. Five to seven shoots regenerated from each node of subcultured shoots; on subsequent sub-culturing rates of multiplication were increased to 10-15 folds. The mother explant could also be repeatedly transferred onto fresh medium to yield fresh crops of shoots. After 10-12 culture cycles the cultures exhibited hyperhydration and callusing. These could be checked by lowering the BAP concentrations to 0.25 mg 1−1 and that of NAAto 0.01 mg 1 . The clonally-produced shoots were treated with a solution containing 100 mg 1−1 each of indole-3-butyric acid (IBA) and β-naphthoxy acetic acid (NOA). The auxin-pulsed shoots were subsequently treated with 10.0 mg 1−1 solution of chlorogenic acid for 3 minutes. The pulsed shoots were kept in glass bottles containing soilrite in greenhouse at 28 ± 2°C. Of these 83% rooted ex vitro. The ex vitro rooted plantlets were hardened and transferred to polybags/earthen pots containing black soil, fine sand, farm yard manure and soilrite in the ratio of 4:2:l:l(v/v). To date 7500 shoots have been produced from 10 explants originating from a single plant. Two thousand plantlets have been hardened and acclimatized. It is suggested that within a period of 6 months 3000 plants can be produced from single explant of C. paniculatus by this protocol. The protocol developed can be useful for cloning and conservation of germplasm of C. paniculatus.