Genetic engineering of tropane alkaloid biosynthesis of hyoscyamus reticulatus L. hairy roots by pmt gene overexpression and feeding with putrescine

The use of biotechnology strategies to increase the production of natural products in medicinal plants, can be very useful and commercially profitable. In this study, control and transgenic hairy roots were obtained from Agrobacterium rhizogenes strain A4 and Agrobacterium tumefaciens strain C58C1(pRiA4-pBMI) carrying the pmt gene C58C1, respectively. The effect of pmt gene overexpression on growth, biochemical traits, and phyto-chemical ingredients were determined in Hyoscyamus reticulatus hairy roots, which were also fed with different concentrations of the precursor compound putrescine (0, 1, 2, 3, and 4 mM) for two exposure times (24 and 48 h). The growth rate was higher in transgenic hairy roots. The maximum total content of phenol, flavonoid, and alkaloid were obtained in transgenic hairy roots fed with putrescine (4, 3 and 1 mM, respectively) after 48 h. According to the half maximal inhibitory concentration (IC50) values, the antioxidant activity was lower in the control than transgenic hairy roots. High performance liquid chromatography indicated that the hyoscyamine content (125.09 mu g g(-1) DW with 3 mM putrescine after 24 h; 203.83 % compared with the control) was higher in control hairy roots, whereas scopolamine levels (161.88 mu g g(-1) DW with 3 mM putrescine after 48 h; 207.34 %) was higher in transgenic hairy roots. The highest increase in the expression level of the pmt (2.6-fold with 3 mM putrescine) and h6h (2.83-fold with 2 mM putrescine) genes occurred after 48 h in both transgenic and control hairy roots, respectively. The combination of genetic engineering and precursor feeding strategies improved the accumulation of secondary metabolites in vitro.

Automated summary

The study demonstrated that using biotechnology and precursor feeding strategies for transgenic engineering of hairy roots in Hyoscyamus reticulatus resulted in higher growth rate, increased content of phenol, flavonoid and alkaloid, and improved antioxidant activity. High levels of hyoscyamine were found in control hairy roots, while scopolamine levels were higher in transgenic hairy roots. The expression of pmt and h6h genes significantly increased after 48 hours in both transgenic and control hairy roots, leading to enhanced accumulation of secondary metabolites in vitro.