Abstract
Withania somnifera (L.) Dunal, an Ayurvedic herb rich in withanosides, has anti-cancer and health-rejuvenating properties. These bioactive chemicals, primarily found in the roots, are of significant interest for production in hairy root cultures. In this study, researchers investigated a hairy root line produced by Rhizobium rhizogenes (ATCC 43057), varying cell density, infection period, and acetosyringone concentrations. The most successful root induction frequency (31.66% ± 2.98%) occurred at a bacterial cell density of OD600 = 0.8 combined with a 20-min infection period and 200 µM acetosyringone. After 25 days, the hairy root culture achieved its highest biomass (5.20 ± 0.15 g/50 mL flask fresh weight (FW)) in MS liquid medium with 3% sucrose. At 30 and 25 days, the greatest amounts of withanolide A (0.824 ± 0.01 mg/g DW) and withaferin A (1.292 ± 0.02 mg/g DW) were observed. Hairy roots were further treated with methyl jasmonate (MJ) and salicylic acid (SA) at various concentrations (25, 50, and 100 µM). Notably, the maximum content of withanolide A (2.90 ± 0.08 mg/g DW, 3.86-fold) and withaferin A (3.40 ± 0.01 mg/g DW, 2.61-fold) was obtained in the hairy roots treated with MJ at 100 µM. After 48 h, the content of withanolide A reached 3.16 ± 0.03 mg/g DW (6.72-fold), and withaferin A peaked at 4.32 ± 0.05 mg/g DW (4.59-fold) when treated with 50 µM SA + 100 µM MJ. This study demonstrated that W. somnifera hairy root culture enhances large-scale production of withanolide A and withaferin A.