BIOLOGIA PLANTARUM 52 (1): 26-35, 2008 26 Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonoids in hairy roots R.K. TIWARI*, M. TRIVEDI, Z.-C. GUANG, G.-Q. GUO* and G.-C. ZHENG Institute of Cell Biology, School of life Sciences, Lanzhou University, Lanzhou-73000, Gansu, P.R. China Abstract Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A 4 GUS, R1000 LBA 9402 and ATCC11325. The A 4 GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15 - 20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B 5 medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A 4 GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1 - 30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %). Additional key words: baicalin, genetic transformation, medicinal plants, wogonin. Introduction Scutellaria baicalensis Georgi (Lamiaceae) is widely distributed in different regions of Russia, Japan, Korea and Mongolia. High biological activity of huang-qin root extract is determined by the presence of almost 70 flavonoids such as baicalin, baicalein, wogonin, wogonin 7-o-glucuronide, oroxylin A, and oroxylin A 7-o-glucuronide (Wang et al. 1994). Out of these, baicalin and wogonin are the most studied components (Zhou and Gu 1991, Jang et al. 2003). They are antioxidants and free radical scavengers (Shieh et al. 2000) and possess anti-allergic, anti-bacterial, anti-HIV, anti-hepatitis-B and anti-tumor activities. The natural plant population of this extremely important medicinal plant is over exploited for pharmaceutical preparations and therefore in vitro production as an alternative source has been attempted (Yamamoto et al. 1986, Morimoto et al. 1995). However, the cultures tend to be genetically unstable and synthesize very low levels of useful secondary metabolites (Rhodes et al. 1990). In contrast, Agrobacterium rhizogenes mediated hairy root cultures exhibit stable and fast growth rate comparable to cell suspension culture, genetic and biochemical stability and increased production of secondary compounds (Giri and Narasu 2000). Moreover, roots can also be successfully cultured in large-scale bioreactors (Flores et al. 1999, Sévon and Oksman- Caldentey 2002). These transformed hairy root, can be produced by inoculation of plants with agropine strains of A. rhizogenes containing a T-DNA, divided into two regions, T L and T R (Huffman et al. 1984). The T L region of Ri plasmid contains eighteen open reading frames ⎯⎯⎯⎯ Received 2 January 2006, accepted 31 July 2006. Abbreviations: ags - agropine synthase; B5 medium - Gamborg B 5 medium; FM- fresh mass; HPLC - high performance liquid chromatography; MS medium - Murashige and Skoog medium; NT- non-transformed; PCR - polymerase chain reaction; Ri - root-inducing plasmid; T L - left-terminus DNA; T R - right-terminus DNA; YEB - yeast extract broth. Acknowledgments: We thank Prof. D. Tepfer, INRA, France for kindly providing the Agrobacterium rhizogenes A 4 GUS strain. We also thank Dr. Simon Deroles, New Zealand Institute for Crop & Food Research, New Zealand, Dr. Mark Tepfer, International Center for Genetic Engineering and Biotechnology, Italy and Dr. S.Z. Korac, Institute for Biological Research, Belgrade for stimulating discussions. Financial assistance in the form of Post-doc fellowship provided by the China Post-Doctoral Research Foundation (No. 224-415-203) and PDF fund of Lanzhou University (No. 505-415-104), Lanzhou, China is gratefully acknowledged. 1 Corresponding authors; fax: (+86) 931 8912561, e-mail: rajeshktindia@rediffmail.com and gqguo@lzu.edu.en