Feasible biosynthesis of biologically active metabolites in in vitro culture of Macrotyloma uniflorum

Macrotyloma uniflorum, commonly called “Horse gram” is an underutilized pulse crop recognized for its great nutritional significance and a broad range of biological properties. There have been no in vitro studies for the biosynthesis of enhanced bioactive metabolites in the callus culture of M. uniflorum. In the study reported here, we have designed a feasible protocol for high-frequency callus induction and maintenance utilizing a leaf as an explant grown on MS media enriched with various concentrations of different plant growth regulators (PGRs) including α-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and thidiazuron (TDZ) either alone or in combination. Among all the tested PGRs, NAA alone resulted in high-frequency callus induction (97%), and maximum biomass accumulation (Fresh weight (FW): 200 g/L: Dry weight (DW): 20.4 g/L). Moreover, hormonally optimized callus cultures exhibit maximum production of phenolic compounds (166.6 mg/L) and flavonoid compounds (351.6 mg/L). The antioxidant potential of calli extracts was also determined by utilizing various antioxidant activities. Maximum antioxidant activities ((2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) ABTS = 497.9; TEAC μM; (Ferric reducing antioxidant power) FRAP = 823 TEAC μM; (Cellular Antioxidant Activity) CAA: 58.2%) were recorded in leaf-derived calli supplemented with different PGRs treatments. High-performance liquid chromatography (HPLC) analysis further revealed maximum biosynthesis of caffeic acid (1.63 mg/g DW), gallic acid (8.92 mg/g DW), kaempferol (0.71 mg/g DW), myricetin (0.39 mg/g DW), apigenin (0.64 mg/g DW) at 10 mg/L BAP and isorhamnetin (0.68 mg/g DW) at 1 mg/L TDZ + 10 mg/L NAA. The objective of this study was to explore in vitro biosynthesis of enhanced bioactive metabolites in the callus culture of M. uniflorum, leveraging a feasible protocol for high-frequency callus induction and maintenance. The results showcase the remarkable efficacy of NAA in callus induction and biomass accumulation, highlighting the hormonally optimized callus cultures as a potent source for enhanced biosynthesis of bioactive metabolites, paving the way for further applications in pharmaceutical and commercial industries.