Media Composition Effects on Hairy Root Biomass and Tetrandrine Production in Stephania tetrandra

Stephania tetrandra S. MOORE, a medicinal plant, is recognized for tetrandrine production, which is extensively accepted for its therapeutic benefits. However, the slow growth of S. tetrandra limits tetrandrine's availability, which can be addressed by in vitro hairy root cultivation using Rhizobium rhizogenes and optimization of medium components. The present study attempted the three-step optimization of three components of woody plant medium (ammonium nitrate, calcium nitrate, and sucrose), including two-level factorial design, path of steepest ascent (PSA), and central composite design (CCD) to obtain high hairy root biomass and tetrandrine production. The CCD-based optimization for enhanced hairy root biomass resulted in a hairy root biomass of 9.75 g dw/L at optimal concentrations of ammonium nitrate (NH₄NO₃), calcium nitrate (Ca(NO₃)₂), and sucrose of 631.96 mg/L, 651 mg/L, and 41.35 g/L, respectively. The optimal concentration of 550.31 mg/L, 862.88 mg/L, and 25.89 g/L for NH₄NO₃, Ca(NO₃)₂, and sucrose, respectively, obtained after CCD analysis for enhanced tetrandrine production led to the maximum tetrandrine of 70.48 mg/L. Medium optimization resulted in a 1.47-fold increase in hairy root biomass and a 1.37-fold increase in tetrandrine production under individually optimized conditions. The present study findings confirmed the important role of process optimization for enhanced product yield.