Influence of Biotic and Abiotic Elicitors on Rosmarinic Acid Accumulation in Hairy Root Cultures of Dracocephalum kotschyi Boiss.

<i>Dracocephalum kotschyi</i> Boiss., an endangered and endemic medicinal plant of Iran belonging to the Lamiaceae family, is a valuable source of methoxylated flavonoids with proven anticancer activity. In this study, hairy roots were effectively induced from two-week-old hypocotyl explants using <i>Agrobacterium rhizogenes strain</i> ATCC 15834. Biomass optimization showed that maximum seedling growth occurred in ¼-strength MS medium, while the highest hairy root induction (76.55%) was achieved in ½-strength MS medium supplemented with 1 mM L-arginine. Root induction varied significantly with explant type and age, with the lowest response (14.66%) observed in five-week-old leaf explants. To increase rosmarinic acid (RA) accumulation, transgenic hairy roots were treated with yeast extract (200 mg L^-1) as a biotic elicitor and titanium dioxide (TiO₂) nanoparticles (200 and 400 mg L^-1) as abiotic elicitors for 24 and 48 h. HPLC analysis revealed that treatment with yeast extract (200 mg L^-1 for 48 h) resulted in the highest accumulation of rosmarinic acid (5.65 mg g^-1 DW), corresponding to a 26% increase over the control, accompanied by a substantial enhancement of antioxidant activity (63.4%). Yeast extract application also elevated total protein content and glutathione peroxidase (GPX) activity, while markedly suppressing ascorbate peroxidase (APX) and polyphenol oxidase (PPO) activities. In contrast, TiO₂ nanoparticles, particularly at a concentration of 400 mg L^-1 for 48 h, augmented APX and PPO activities, indicating the induction of oxidative stress. These findings demonstrate that yeast extract and low concentrations of TiO₂ nanoparticles can serve as effective elicitors to enhance rosmarinic acid accumulation in <i>D. kotschyi</i> hairy root cultures, providing valuable insights for applications in plant biotechnology.