OPTIMIZATION OF HAIRY ROOT PROLIFERATION IN LIQUID MEDIUM USING RESPONSE SURFACE METHODOLOGY

Abstract

Aim: Arbuscular mycorrhizal (AM) fungi are valuable tools for sustainable agriculture, but their large-scale production is limited by the lack of optimized in vitro host root systems; therefore, this study used Response Surface Methodology (RSM) to optimize key physicochemical factors affecting hairy root proliferation in liquid culture for efficient AMF multiplication. Methodology: Rhizobium rhizogenes strain MTCC 2364 was used to generate hairy roots in carrot (Daucus carota) explants. The effects of culture medium, pH, and sucrose content on fresh weight, root length, and number of lateral branches were assessed using a Box-Behnken experimental design. ANOVA and second-order polynomial regression were used to analyze the data. Results: Hairy root induction occurred within 16–17 days after inoculation, achieving a high transformation efficiency of 94%. RSM analysis identified culture medium, pH, and sucrose concentration as key factors influencing root growth, with optimized conditions (Modified M medium, pH 5.5, and 12.5 g L⁻¹ sucrose) predicting 3.30 g fresh weight, 37.05 cm root length, and 47.38 lateral branches (R² > 0.88). Interpretation: The resulting methodology contributes to the optimization of hairy root growth which can be used for a scalable, repeatable method of AMF inoculum production.