ORIGINAL PAPER An efficient transformation method for estrogen-inducible transgene expression in Catharanthus roseus hairy roots Noreen F. Rizvi • Miglia Cornejo • Kassi Stein • Jessica Weaver • Erin J. Cram • Carolyn W. T. Lee-Parsons Received: 14 July 2014 / Accepted: 28 August 2014 / Published online: 6 September 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Efficient genetic engineering of the medicinal plant, Catharanthus roseus, is essential in improving the production of the pharmaceutically important anticancer compounds, vinblastine and vincristine. Here we optimize several steps in the Agrobacterium-mediated transforma- tion of C. roseus, including antibiotic selection and Agro- bacterium elimination parameters. With the optimized protocol, stable transgenic hairy root cultures expressing green fluorescent protein (GFP) under control of the estrogen-inducible XVE system were established with a transformation efficiency of 33 %. The estrogen-inducible system has not been previously tested in C. roseus hairy roots but offers several advantages as an inducible system. Prior to induction, low basal levels of Gfp are observed. Upon induction with 5 mM 17b-estradiol, Gfp transgene expression is highly and rapidly induced within 1 h, increasing after 24 h. Strongest GFP expression is observed in the meristematic root tips and is sustained over 5 days in the presence of estradiol. Neither the 17b-estradiol induc- tion nor the estrogen-inducible system elicits a defense response in C. roseus hairy roots. Keywords Catharanthus roseus Á Agrobacterium- mediated transformation Á Genetic engineering Á Estrogen- inducible Á Hairy roots Introduction The Catharanthus roseus plant is the source of several pharmaceutically valuable terpenoid indole alkaloids (TIAs), including the two anticancer drugs, vincristine and vinblastine. These secondary metabolites are produced at extremely low levels (0.0002 wt%), making commercial production expensive (Noble 1990). It is not currently possible to use other host systems for production since the TIA pathway is complex and not fully characterized (van der Heijden et al. 2004). Therefore, whole plants or plant tissue cultures are currently the only feasible routes for producing these valuable TIAs. Methods such as media optimization and elicitation have increased TIA produc- tion, but genetic engineering of C. roseus is a promising strategy to further improve metabolic flux and TIA concentrations. Several enzymes and transcription factors involved in alkaloid biosynthesis in C. roseus have already been over- expressed through Agrobacterium-mediated genetic trans- formation (Zarate and Verpoorte 2007; Zhao et al. 2013). Initial transgene expression strategies used the constitutive cauliflower mosaic virus 35S promoter to drive transgene expression. An inducible system, however, is beneficial for several reasons. The timing of transgene expression can be controlled and expressed at the desired developmental stage, the uninduced condition serves as the negative control, and deleterious effects associated with constitutive expression such as growth retardation can be minimized. N. F. Rizvi Á M. Cornejo Á K. Stein Á C. W. T. Lee-Parsons (&) Department of Chemical Engineering, Northeastern University, Boston, MA 02115, USA e-mail: ca.lee@neu.edu J. Weaver Á E. J. Cram Department of Biology, Northeastern University, Boston, MA 02115, USA C. W. T. Lee-Parsons Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA 123 Plant Cell Tiss Organ Cult (2015) 120:475–487 DOI 10.1007/s11240-014-0614-1