An efficient transformation method for estrogen-inducible transgene expression in Catharanthus roseus hairy roots

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 transformation of C. roseus, including antibiotic selection and Agrobacterium 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 17 beta-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 17 beta-estradiol induction nor the estrogen-inducible system elicits a defense response in C. roseus hairy roots.