ORIGINAL PAPER A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum Ali Sharafi • Haleh Hashemi Sohi • Amir Mousavi • Pejman Azadi • Khadijeh Razavi • Valentine Otang Ntui Received: 4 June 2012 / Accepted: 13 October 2012 / Published online: 19 October 2012 Ó Springer Science+Business Media Dordrecht 2012 Abstract An efficient and reliable transformation system for a very important medicinal plant Papaver bracteatum was developed through optimization of several factors that affect the rate of effective A. rhizogenes-mediated trans- formation and growth rate of hairy root. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, and three explants types, hypocotyls, leaves and excised shoots were examined. The highest frequency of transfor- mation was achieved using LBA9402 strain in the excised shoots. Several inoculation and co-cultivation media and different concentration of arginine were evaluated using LBA9402 strain and the excised shoots as explant. Inter- estingly, a drastic increase in the frequency of transfor- mation (47.3 %) was observed when Murashige and Skoog medium containing 1 mM arginine and lacking NH 4 NO 3 KH 2 PO 4 , KNO 3 and CaCl 2 was used. The effect of sucrose concentration and the ratio of NH 4 ? : NO 3 - on hairy root biomass was examined. Maximum biomass was obtained in 30 g/l sucrose and 20:10 mM ratio of NH 4 ? to NO 3 - on MS medium. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and Southern hybridization. Keywords Agrobacterium rhizogenes  Transformation  Co-cultivation  Macro elements  Hairy root  Papaver bracteatum Introduction Papaver bracteatum (Iranian poppy) is a medicinal plant of great interest because of its benzylisoquinoline alkaloids (Le Flem-Bonhomme et al. 2004). This plant is naturally distributed in the Alborze mountains north of Tehran and in the Iranian Kurdistan. Iranian poppy CVs. produce mor- phinan alkaloids including thebaine, codeine and mor- phine. However, thebaine is the main alkaloid in this plant (Tisserat and Berhow 2009). A similar pathway for the- baine biosynthesis was shown in P. somniferum and P. bracteatum (Milo et al. 1990). Thebaine can be converted to codeine, oxymorphone and oxycodone (Carolan et al. 2002). In the 70s, the United Nations requested for studies on the use of P. bracteatum as a source of thebaine for possible replacement for opium poppy (P. somniferum), a major source of morphinan alkaloids. However, the perennial nature of P. bracteatum makes it uneconomical for this replacement. Therefore, research is focused on the potential of using in vitro cell culture technology for morphinan alkaloid production. Also, metabolic engineer- ing has facilitated the development of plant cell and tissue system as alternative production platforms that can be scaled up in a controlled condition (Leonard et al. 2009). In plants, benzylisoquinoline alkaloids are synthesized in the roots and accumulate in specialized internal secretary systems (Kutchan 1998). Numerous studies have been done A. Sharafi  H. H. Sohi (&)  A. Mousavi  K. Razavi Plant Biotechnology Group, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran e-mail: halehsohi@gmail.com P. Azadi Tissue Culture and Gene Transformation Department, Agricultural Biotechnology Research Institute of Iran (ABRII), P. O. Box 31535-1897, Karaj, Iran e-mail: azadip22@gmail.com V. O. Ntui Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo City 271-8510, Chiba, Japan 123 Plant Cell Tiss Organ Cult (2013) 113:1–9 DOI 10.1007/s11240-012-0246-2