Indirect shoot organogenesis from leaf explants of threatened medicinal plant Cleistanthus collinus

Abstract

In this study an effective plant regeneration protocol for multiple shoot bud induction was optimized for the micropropagation of plantlets through indirect organogenesis from leaf explants of C. collinus. The young leaves were cultured on Murashige and Skoog (MS) medium with Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), Kinetin (KIN), 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA) at 0.5-3.0 mg/l for callus induction. The induced calli was excised and subcultured on MS medium supplemented with BAP, KIN and TDZ (0.5-4.0 mg/l) in combination with NAA (0.5 mg/l) to find out the optimum hormone concentration for shoot bud induction. For rooting, half strength MS medium with various concentrations (0.5-3.0 mg/l) of Indole 3-acetic acid (IAA), Indole 3-butyric acid (IBA) and NAA was used. Initially, a creamy white callus was formed from the cut ends of leaf explants and gradually these mass of unorganised cells transformed to light green regenerable callus. The maximum callus induction (100%) was observed in MS medium with TDZ at 1.0 mg/l concentration. The highest frequency of organogenesis was observed in the combination of BAP (1.0 mg/l) and NAA (0.5 mg/l) with a mean number of 12.1±1.1 shoots followed by TDZ (1.0 mg/l) and NAA (0.5 mg/l) with 10.2±1.0 shoots per explants. The healthy shoots were rooted best on half strength MS medium supplemented with 0.5mg/l IAA. The well developed plantlets with healthy roots were successfully acclimatized. The development of thriving protocol for the callus induction and regeneration would make possible for the mass multiplication and in vitro production of plantlets with somaclonal variations.