Optimization of induction and hairy root culture establishment in two mullein species, Verbascum erianthum and Verbascum stachydiforme

The genus Verbascum, belonging to the family Scrophulariaceae, has a significant center of diversity in Iran. Two of its species, V. erianthum and V. stachydiforme, originate in the Iranian-Turanian region, but no studies have been conducted on the induction of their hairy roots. This genus is a valuable source of biologically active compounds such as iridoid glycosides and flavonoids. Hairy root culture is a suitable technique for the production and accumulation of secondary metabolites. Three different studies were conducted to optimize the induction and establishment of hairy roots. In the first experiment, the influence of explant type (leaf and hypocotyl), six infection methods, and co-culture time (48 and 72 h) on the efficiency of hairy root induction was investigated. The results showed that the highest hairy root induction (68.18%) was observed in the leaf explants inoculated by direct infection with three wounds within 72 h co-culture time. In the second experiment, the effect of four Agrobacterium rhizogenes strains (ATCC 15834, A4, A7, and A13) and leaf age (14, 21, and 28 days) on transformation efficiency and some morphological traits examined in both species were studied. The high transformation efficiency of hairy root (80.55%) was detected in the 21-day-old leaf explant of V. erianthum species that was inoculated with the A13 strain. The transformed hairy root colons were confirmed by PCR using rolB gene-specific primers. To optimize hairy root growth and avoid tissue browning, hairy roots were cultured in various media containing different antioxidants and improver agents (including ascorbic acid, citric acid, and NAA). The results showed that the highest fresh growth index (20.42) and the lowest tissue browning (9%) as well as total phenol (8.51 mg GA/g DW), and total flavonoid content (4.42 mg QUE/g DW) were obtained in medium B5 with 1.5 mg/l NAA.