Bioprocess development for enhancing cell biomass, differential picrosides accumulation, and gene expression profiling at shake flask levels in suspension cultures of Picrorhiza kurroa

Abstract

Plant cell suspension culture is an alternative and perpetual source for large-scale metabolites production in significantly less time. However, this approach has not yet been executed in Picrorhiza kurroa for their specialized metabolites production. Therefore, the present study aimed to develop the suspension culture process from the rhizome and leaf callus of P. kurroa by optimizing the physical cultivation parameters. Furthermore, the established cultures were subjected to sequential scale-up at 250, 500, 1000, 2000, and 5000 mL shake flask levels to evaluate growth, metabolites accumulation, and gene expression profiling of picrosides biosynthesis. Concurrently, the growth, metabolites content, and gene expression patterns were also assessed at the 4th, 8th, and 12th months of the incubation period. The investigation results revealed that the cell biomass in suspension cultures significantly improved solely by varying the culture parameters. The initial inoculum density (3 g/75 mL), subculture by pouring method, shaking speed (80 rpm), and pH (5.8) were suitable for enhanced production of cell biomass in rhizome and leaf suspension cultures in a 250 mL shake flask. During sequential scale-up, the cell biomass was reached in rhizome suspension (335 g FW; 13.80 g DW) and leaf suspension (263.33 g FW; 10.87 g DW) under optimized parameters at 5000 mL shake flask level. However, a maximum increase in the cell growth index and cell biomass (DW) in g/L of the medium was observed in a 250 mL flask compared to other shake flask volumes. In rhizome and leaf suspension, total picrosides (PI, PII, and PIII) (rhizome: 2.36–2.48 mg/g DW; leaf: 2.50–2.67 mg/g DW) and total precursors (vanillic acid, caffeic acid, cinnamic acid, catalpol, and aucubin) (rhizome: 1.35–1.37 mg/g DW; leaf: 1.20–1.45 mg/g DW) content range was observed during sequential scale-up at 250–5000 mL shake flask levels. Hence, no significant level of variation was observed in picrosides content (PI, PII, and PII in mg/g DW) with respect to different shake flask volumes (250–5000 mL) in rhizome and leaf suspension culture, respectively. Nevertheless, the productivity of metabolites content in mg/L of medium with respect to cell growth in g/L of medium showed a significant level of variation. The 250 mL shake flask volume culture of rhizome and leaf cell suspension accumulated the maximum total picrosides (31.07 and 31.20 mg/L of medium) and total precursors (17.04 and 16.94 mg/L of medium) content with cell biomass (12.53 and 11.69 g/L of medium), respectively. The results also revealed that the shikimate/phenylpropanoid pathway (EPSPS), mevalonate pathway (PMK and GDPS), non-mevalonate pathway (ISPD, DXPS, and ISPE), and iridoid pathway (GS, G10H, UGT, F3D, SQM, and ACT) genes showed upregulated expression in the rhizome and leaf suspension cultures. Analyzing the 1st month cultures with 4th, 8th, and 12th month cultures, the study depicted that growth index, cell viability, metabolites content, productivity in mg/L of the medium, and gene expression pattern were decreased with the age of cultures under one year of incubation. The present investigation delivered a simple yet feasible suspension culture process for metabolites enriched cell biomass production in P. kurroa.