Industrial Crops and Products 44 (2013) 643–652 Contents lists available at SciVerse ScienceDirect Industrial Crops and Products journa l h o me page: www.elsevier.com/locate/indcrop High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: Hairy root production and essential oil analysis G. Sujatha a,∗ , S. Zdravkovi ´ c-Kora ´ c b , D. ´ Cali ´ c b , G. Flamini c , B.D. Ranjitha Kumari a a Medicinal Plant Biotechnology Division, Department of Plant Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India b University of Belgrade, Institute for Biologic Research, “S. Stankovic”, Despot Stefan Blvr. 142, 11060 Belgrade, Serbia c Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Via Bonanno 33, 56126 Pisa, Italy a r t i c l e i n f o Article history: Received 18 May 2012 Received in revised form 21 August 2012 Accepted 7 September 2012 Keywords: Artemisia vulgaris Essential oils Hairy roots Camphor 1,8-Cineole a b s t r a c t Agrobacterium rhizogenes-mediated genetic transformation of Artemisia vulgaris was investigated using four bacterial strains: A 4 GUS, R1000, R1601and ATCC15834 and three explant types: shoot tip, leaf and node. The A 4 GUS proved to be more competent than other strains and the highest transformation rates were observed in leaf explant (92.6%). Among 92 hairy root clones acquired, clones AV1 and AV2 exhibited the highest growth. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Out of four media composition, the ½ MS medium was found to be most suitable for biomass production. Biomass accumulation was the highest when the medium was supplemented with 40 g L -1 sucrose. PCR and Southern hybridization analysis revealed both the TL- and TR-DNA integration in the root clones AV1 and AV2. Hairy roots showed higher growth rate and accumulated substantial amount of essential oils than non-transformed roots. Eighty-seven compounds were identified from transformed root essential oils, whereas 77 compounds were identified from non- transformed root essential oils. The main compounds identified were camphor, camphene, -thujone, germacrene D, 1,8-cineole and -caryophyllene. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Artemisia vulgaris L. (mugwort) belongs to the family Asteraceae and is a tall aromatic threatened perennial herb, which grows in the hilly districts of India in areas up to 2400 m elevation. The plant has been known not only as an edible plant (mostly as a spice) but also as a folk medicine resource. The investigations on mugwort extracts indicated a hepatoprotective activity and validated the traditional use of this plant for various liver disorders (Gilani et al., 2005). Mugwort is commonly used in traditional European medicine as choleretic and for amenorrhea and dysmen- orrhea (Teixiera da Silva, 2004), for the treatment of diabetes and epilepsy, and in combination for psychoneurosis, depression, irri- tability, insomnia and anxiety stress (Walter et al., 2003). Several Abbreviations: B5 medium, Gamborg medium; DIG, digoxigenin; FW, fresh weight; GC–MS, gas chromatography–mass spectrometry; GUS, -glucuronidase; MS medium, Murashige and Skoog medium; NT, non-transformed; PCR, polymerase chain reaction; PGR, plant growth regulator; SH medium, Schenk and Hilderbrandt medium; TR, transformed roots; YMB, yeast mannitol broth. ∗ Corresponding author. Present address: Department of Botany, Government Arts College for Women, Pudukkottai 622 001, Tamil Nadu, India. Tel.: +91 4322 222202; fax: +91 4322 229931. E-mail address: sujathagovindaraj@gmail.com (G. Sujatha). medicinally active components of A. vulgaris have been identified including coumarins, sesquiterpene lactones, volatile oils and inulin (USDA-ARS-NGRL, 2004). Mugwort essential oil is used for its insecticidal, antimicrobial and antiparasitical properties (Judzentiene and Buzelyte, 2006). The essential oil of A. vulgaris was reported to exhibit 90% repellency against Aedes aegypti, a mosquito that transmits yellow fever (Hwang et al., 1985). Repel- lant and fumigant activity of A. vulgaris against Musca domestica and the stored-product insect pest Tribolium castaneum (Herbst) were also reported (Judzentiene and Buzelyte, 2006; Wang et al., 2006). Various strategies have been employed for the production of useful secondary plant compounds in in vitro systems (Bourgaud et al., 2001). In vitro production of bioactive compounds as an alter- native source has not yet been attempted in this valuable medicinal plant. However, in vitro cell cultures generally tend to be geneti- cally unstable and synthesize very low levels of useful secondary metabolites. On the other hand, the in vitro cultures of conventional roots are slow growing and have short life span (Giri and Narasu, 2000). Agrobacterium rhizogenes-mediated gene transfer is being widely used to obtain hairy root lines with high production of useful metabolites (Canel et al., 1998; Ali et al., 2012). Agrobacterium- mediated hairy roots are fast-growing, genetically stable which can also be successfully cultured in large scale bioreactors and have been intensively utilized to produce a stable and high production 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.09.007