Agrobacterium rhizogenes-mediated transformation of Rubia peregrina L.: in vitro accumulation of anthraquinones

An Agrobacterium rhizogenes-mediated transformation system for Rubia peregrina L. has been established by co-cultivation of callus cultures or by direct infection of explants with A. rhizogenes LBA 9402 harbouring the binary vector pMON 9703 containing gus and npt-II genes as markers. The putative transformed roots were selected on medium containing kanamycin (25 mg l^-1). Antibiotic resistant root clones were subjected to histochemical analysis for the localisation of -glucuronidase activity. Polymerase chain reaction was used to confirm the presence of gus, npt-II and T _L border sequences in the transformed root clones. Spontaneous regeneration of shoots was observed from 30 day-old transgenic roots. Total anthraquinone and alizarin contents of transgenic root cultures were measured by spectrophotometry and by high performance liquid chromatography. The accumulation of total anthraquinones in transformed roots was found to be approximately 2-fold higher than that found in one year-old field grown roots (2.12±0.12 and 1.23±0.12 mg g^-1 dry weight, respectively). Alizarin was found to be the major anthraquinone in transformed root cultures and was found to be approximately 3-fold higher than in field grown roots.Abbreviations BA 6-benzyladenine - B5 Gamborg B5 medium - gus -glucuronidase gene - GUS -glucuronidase - HPLC high performance liquid chromatography - MS Murashige and Skoog medium - NAA -naphthalene acetic acid - npt-II neomycin phosphotransferase II gene - OD₆₀₀ optical density at 600 nm - PCR polymerase chain reaction - T _L left border sequence of T-DNA - vir D1 virulence D1 gene - YMB yeast mannitol broth