Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 mu M). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 mu M AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC-ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g(-1) dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g(-1) dry wt. and 9.97 mg g(-1) dry wt., respectively) comparable to those found in root tubers.