A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum

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 transformation 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 transformation 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. Interestingly, a drastic increase in the frequency of transformation (47.3 %) was observed when Murashige and Skoog medium containing 1 mM arginine and lacking NH4NO3 KH2PO4, KNO3 and CaCl2 was used. The effect of sucrose concentration and the ratio of NH4 +: NO3 − on hairy root biomass was examined. Maximum biomass was obtained in 30 g/l sucrose and 20:10 mM ratio of NH4 + to NO3 − on MS medium. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and Southern hybridization.