Abstract
Dracocephalum kotschyi 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 Agrobacterium rhizogenes strain 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 (TiO(2)) 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, TiO(2) 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 TiO(2) nanoparticles can serve as effective elicitors to enhance rosmarinic acid accumulation in D. kotschyi hairy root cultures, providing valuable insights for applications in plant biotechnology.