Effect of cytokinin combined elicitors (l-phenylalanine, salicylic acid and chitosan) on in vitro propagation, secondary metabolites and molecular characterization of medicinal herb – Coleus aromaticus Benth (L)

Abstract

This study investigated the effect of cytokinin combined elicitors (l-phenylalanine, Salicylic acid and chitosan) on in vitro propagation of Coleus aromaticus. Furthermore, we determined the elicitor induced changes on genetic stability through Random amplified polymorphic DNA (RAPD), secondary metabolites and Phenylalanine ammonia lyase (PAL) gene expression. In in vitro propagation, the explant source of shoot tips and nodal explants were cultured on Murashige and Skoog (MS) medium with 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l of cytokinins (Benzyl amino purine (BAP), Kinetin (KIN)) for shoot bud induction. Further, nodal explants derived shoots cultured on the combination of 1.0 mg/l of BAP with 0.5, 1.0, 1.5, 2.0, 2.5 mg/l of l-Phe, 0.2, 0.4, 0.6, 0.8, 1.0 mg/l of SA and 20, 40, 60, 80 of mg/l of Ch individually. The 79.81% of shoot bud induction was obtained with 1.0 mg/l of BAP supplemented nodal explants. In multiple shoot development, 87% of multiple shoot development was obtained in the combination of 1.0 mg/l BAP and 40 mg/l chitosan supplemented culture. The developed shoots were rooted with 0.5 mg/l of Naphthalene acetic acid (NAA) and transplanted to greenhouse condition with 75% survival rate. In vitro regenerated plants were subjected to RAPD profiling and it was exposed a similar banding pattern with that the mother plant. In secondary metabolites, compared to in vivo control the higher percentage of alkaloids (16), flavonoids (24), saponins (7.5), terpenoids (6.2), total phenolic content (24.4) and tannins (8) with increased PAL gene expression was analyzed in in vitro regenerated plants. Hence, this study shows that the choice of cytokinin combined elicitors supplementation during tissue culture noticeably influences not only multiple shoot development, but also the production of secondary metabolites without genetic modification. Keywords: Coleus aromaticus, Elicitors, Multiple shoot, 6-Benzylaminopurine, PAL gene expression