In vitro regeneration of Piper longum L. and comparative RP-HPLC analysis of piperine production of in vitro and in vivo grown plants

Piper longum L. is a well known spice plant belonging to the family Piperaceae with high pharmacognosy potential, but it is becoming threatened due to overexploitation. Thus, this investigation aims to standardize a cost effective protocol for in vitro propagation of this economically important plant. Internodal segments were used as explant for callogenesis in Murashige and Skoog medium with 3% sucrose and 0.8% agar, with NAA or 2,4 D. Optimum callus induction was observed in MS medium with 5.0 mg/L NAA. Calli were subcultured on shoot regeneration media containing different concentrations of cytokinin (KIN/BAP) along with 0.1mg/L NAA. Best shoot regeneration was obtained on MS media supplemented with 2.0 mg/L KIN and 0.1mg/L NAA. Induced shoots were rooted in either NAA or IBA and highest rooting was induced in MS medium enriched with 0.5 mg/L NAA. Rooted plantlets were acclimatized and 88% of hardened plants survived. Field emission scanning electron microscopic (FE-SEM) showed that regeneration from callus had occurred by somatic embryogenesis. A comparative study on identification and quantification of piperine ( the chief alkaloid of the cultivar) were done from root and fruit of both in vitro and in vivo grown plants through Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method. I n vitro grown fruit was found to have the maximum amount of piperine.