Statistical optimization of in vitro callus induction of wild and cultivated varieties of Mucuna pruriens L. (DC.) using response surface methodology and assessment of L-Dopa biosynthesis

Abstract

Mucuna pruriens L. (DC.), is a valuable, economically significant medicinal crop, known worldwide for producing the anti-Parkinson's drug Levodopa (L-Dopa). Itching property of the plant poses certain constrains in its conventional breeding, but, in vitro cell culture systems offer advantage in which controlled conditions can be used for the production of valuable compounds. In the current investigation, a simple and efficient method for callus induction from the wild (var. pruriens) and cultivated (var. utilis) varieties of M. pruriens is described. Juvenile leaves and roots obtained from 30 day old aseptically grown seedlings were used as explants for callus induction. The leaves were found to be the best explants for obtaining maximum callus within a short period of time in 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented Murashige and Skoog (MS) medium. White compact root callus was produced from var. utilis in 2,4-D (2.0 mg/L) and indole 3-acetic acid (0.2 mg/L) + benzyl amino purine (2.0 mg/L) containing media. Whereas, callus formation was not observed from root explants of var. pruriens. Central composite design–response surface methodology (CCD-RSM) experiment was designed to analyze the linear-model and quadratic model (including pair-wise and interactive effects) of each variable to study the maximum biomass production from leaf and root explants. In this study, 1.5 mg/L concentrations of BAP as well as 2,4- D in plant tissue culture medium resulted in maximum callus biomass (2.378) and L-Dopa % yield (0.635 %). L-Dopa content in these varieties was investigated by high performance-thin layer chromatography (HP-TLC) using the methanolic extract prepared from callus cultures of both the varieties and compared with natural plant parts. L-Dopa content was found to be higher in seeds obtained from the naturally grown plants of var. pruriens followed by var. utilis (> 0.55 % and 0.494 % respectively). From the result obtained it can also be ascertained that root callus can serve as a source of L-Dopa extraction commercially.