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

<i>Stephania tetrandra</i> S. MOORE, a medicinal plant, is recognized for tetrandrine production, which is extensively accepted for its therapeutic benefits. However, the slow growth of <i>S. tetrandra</i> limits tetrandrine's availability, which can be addressed by in vitro hairy root cultivation using <i>Rhizobium rhizogenes</i> 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.