Effect of elicitation on antioxidant activity and production of tropane alkaloids in Hyoscyamus reticulatus hairy root cultures

Background and objectives: Hyoscyamus reticulatus contains two distinguished tropane alkaloids, hyoscyamineand scopolamine and both of the compounds possesspotential acute or chronic toxicity. In the present study, a simple and efficient transformation system was established for in vitro hairy roots induction in Hyoscyamus reticulatus. Methods: Effect of different factors including Agrobacterium rhizogenes strains (A7, 15834, A13 and D7), various explant types (cotyledon, hypocotyl, two weeks old leaf, four weeks old leaf, two weeks old internode and four weeks old internode), two inoculation methods (immersion and injection) and four types of culture media (MS, ½ MS, ¼ MS and B5) on hairy roots induction efficiency in Hyoscyamus reticulatus were tested. In the second part of the experiments, elicitations with different concentrations of colchicine (0, 0.01, 0.03 and 0.05% w/v) and different UV-B exposure time (0, 3, 6 and 9 min) were used to analyze hyoscyamineand scopolamine production.  Transgenic status of hairy roots was confirmed by PCR using specific primers of the rolB gene. The total antioxidant activity was evaluated by DPPH) method. Results: Induction of hairy roots in H. reticulatus was affected by bacterial strain and explant type. A7 strain and cotyledon explants were detected as the best strain and explants for induction of hairy root in H. reticulatus. Hairy roots growth was significantly affected by medium type. The highest fresh weight was produced in MS and B5 medium.Fresh and dry weight of hairy root reached 1.44 and 0.134 mg at 0.05 percentage of colchicine after 48 h, respectively but in UV-B treatment fresh weight was decreased. In addition, antioxidant activity of hairy root samples treated with colchicine and UV-B increased to 27% (0.05 colchicine) and 26% (UV-B 9 min), respectively compared to the antioxidant activity level in non-transgenic roots (12%) and transgenic roots (18%). The highest amount of hyoscyamine and scopolamine (0.58% and 1.9 %) found in elicited hairy root cultures was 3.2 and 5.1 folds higher than the non-transformed roots (0.18% and 0.37%), respectively. B5 and MS medium were detected as the best appropriate medium for growth of H. reticulatus hairy roots. Antioxidant activity in elicited hairy roots with elicitors increased in comparison to the antioxidant activity level in transgenic and non-transgenic hairy roots. Conclusion: Hairy root lines developed and elicited in this study can be used to investigate the production of pharmaceutically important metabolites of H. reticulatus.