Chitosan nanoparticles as a strategy for enhancing h6h gene expression and tropane alkaloid production in Hyoscyamus reticulatus hairy roots

Hyoscyamus reticulatus L. is a rich source of tropane alkaloids (TA) which is widely used in medicine. In this study, hairy roots were obtained from two-week cotyledon explants of H. reticulatus L. by using the A7 strain of Agrobacterium rhizogenes. The effects of different concentrations of chitosan nanoparticles (CNP) (0, 10, 30, 50 and 100 mg L^{- 1}) as a signaling molecule with two exposure times (24 and 48 h) on the growth rate, antioxidant enzymes activity, phenol content, TA contents and hyoscyamine-6-beta-hydroxylase (h6h) gene expression levels were investigated. After extraction of TA from hairy roots, High-performance liquid chromatography (HPLC) was used to measure the amount of hyoscyamine and scopolamine. The results showed that the highest amount of hyoscyamine (1300.3 µg g^{- 1} FW) and scopolamine (918.06 µg g^{- 1} FW) accumulated in hairy roots treated with 100 mg L^{- 1} CNP at 48 h and 10 mg L^{- 1} CNP at 24 h, respectively. In the present study, the effect of CNP on the expression of the h6h gene was also investigated using semi-quantitative RT-PCR. The highest expression of this gene was observed at a concentration of 10 mg L^{- 1} in 24 h and the lowest was observed in the control sample. Based on these results, it is concluded that CNP can be used as an effective stimulant for the production of plant secondary metabolites, including TA, probably due to the stimulation of gene expression effective in the production of TA.