Establishment of Mucuna pruriens (L.) DC. callus and optimization of cell suspension culture for the production of anti-Parkinson’s drug: L-DOPA

Abstract

It has become a huge challenge to satisfy the emerging demand for levo-3,4-dihydroxyphenylalanine (L-DOPA), an anti-Parkinson’s drug in the international drug market. This is attributed to the conventional methods of extraction from the natural sources of Mucuna spp., which has a low germination rate, less viable seeds, and an irritating, itching trichomes on the pods. The need for an alternative method with continuous supply of L-DOPA without affecting the natural biodiversity has been achieved through in vitro procedures. However, there has not been a systematic approach to optimize the cultural conditions for the maximum productivity. Hence, in this study, we aim at optimizing the cultural conditions for high biomass and L-DOPA production. Various plant growth regulators such as auxins (indole acetic acid, indole butyric acid, picloram [Pic], naphthalene acetic acid, and 2,4-Dichlorophenoxyacetic acid), cytokinins (kinetin, benzylaminopurine, 2-isopentenyl adenine, and thidiazuron), and their combinations have been experimented to figure out the best combination to induce callus. At the same time, various factors such as growth kinetics, different media (MS, Gamborg’s-B5, Chu’s-N6, and Nitsch and Nitsch), media strength (0.5, 1.0, and 2.0X), effect of different macro elements and their strength (0, 0.5,1, 1.5, 2, and 3X), inoculum density, different hydrogen ion concentration (pH), ammonium/nitrate concentration, different sucrose concentrations (0–10%), and other carbon sources have been investigated in detail for optimizing the cell suspension culture. It was found out that 0.5 mg/L Pic gave the best results for callus induction. With respect to biomass, 6-week growth period (135.7 g/L fresh weight [FW]), 1.0X MS media (126.87 g/L FW), 1.5X magnesium sulfate (266.3 g/L FW), ammonium/nitrate ratio of 21.57/18.8 mM (131.4 g/L FW), pH of 6.0 (129.47 g/L FW), 100 g/L of inoculum (222.2 g/L FW), 3% sucrose concentration (125.6 g/L FW), and 3% glucose (183.4 g/L FW) as other carbon sources were found to give the highest biomass. In terms of L-DOPA production, 3-week growth period (5.90 mg/g dry weight [DW]), 0.5X B5 medium (4.27 mg/g DW), 2.0X calcium chloride (5.06 mg/g DW), ammonium/nitrate ratio of 21.57/18.8 mM (3.44 mg/g DW), pH 6.5 (4.02 mg/g DW), inoculum density of 30 g/L (4.79 mg/g DW), and 2% sucrose (5.17 mg/g DW) resulted in a higher L-DOPA yield.