ORIGINAL PAPER Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes: iridoid and phenylethanoid glycoside accumulation in hairy root cultures Renata Gra ˛bkowska • Aleksandra Kro ´licka • Wojciech Mielicki • Marzena Wielanek • Halina Wysokin ´ska Received: 12 May 2009 / Revised: 7 December 2009 / Accepted: 28 December 2009 / Published online: 9 January 2010 Ó Franciszek Go ´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako ´w 2010 Abstract A genetic transformation method using Agro- bacterium 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 lM). 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 lM 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 ana- lyzed for accumulation of iridoid and phenylethanoid gly- cosides. 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 ana- lyzed 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. proc- umbens. 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. Keywords Harpagophytum procumbens Á Hairy roots Á Harpagoside Á Verbascoside Á Isoverbascoside Introduction Harpagophytum procumbens (Burch.) DC. ex Meisn. (family Pedaliaceae), commonly known as Devil’s claw, is a herbaceous plant, growing in Kalahari savannas of South Africa and Namibia (Hachfeld and Schippmann 2000; Wichtl 2004). The secondary roots (tubers) of the species have been widely used in South African folk medicine for treatment of digestive disorders, as a bitter tonic, in blood disorders, as an antipyretic, as an analgesic and for pain relief during pregnancy (Wichtl 2004). The root tubers of the plant (preferably in the form of dry extracts) are also used as official drug (Harpagophyti radix) in European Pharmacopoeia (2007). Clinical studies of the drug avail- able since the 1950s have reported on its analgesic, anti- phlogistic and antiarthretic activities (ESCOP Monographs 2003; Wichtl 2004). The pharmacological actions of Harpagophyti radix are mainly attributed to the presence of Communicated by P. Wojtaszek. R. Gra ˛bkowska (&) Á H. Wysokin ´ska Department of Biology and Pharmaceutical Botany, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland e-mail: renata.grabkowska@umed.lodz.pl A. Kro ´licka Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland W. Mielicki Department of Pharmaceutical Biochemistry, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland M. Wielanek Department of Plant Physiology and Biochemistry, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland 123 Acta Physiol Plant (2010) 32:665–673 DOI 10.1007/s11738-009-0445-6