Investigation of factors in improving Agrobacterium-mediated gene transfer in Ruellia tuberosa L. and evaluation of α-glucosidase inhibitory activity in established hairy roots

Abstract

Ruellia tuberosa (family Acanthaceae) is widely known in traditional medicine in Asian countries for the treatment of diabetes and other diseases. Its roots were demonstrated to possess a hypoglycemic ability in diabetic animal models. In this study, an original induced procedure was investigated to establish hairy root (HR) from R. tuberosa. With the aim of increasing the transformation rate, some induced factors (acetosyringone (AS) dosage, type of explant, age, infection time, bacterial density, co-cultivation duration) were individually examined. As a result, an improved procedure was implemented: ten-day-old in vitro cotyledon explants were injured and then immersed in the bacterial suspension (OD600 nm = 0.4) added 200 µM AS during 10 min. The infected explants were co-cultivated for 4 days in the Murashige & Skoog (MS) medium before transferring to the medium containing cefotaxime for bacterial elimination. After thirty days of culture, the improved procedure revealed a synergistic effect by enhancing the rooting rate and number of secondary roots per explant up to 4.4- and 8.0-fold, respectively, in comparison with the original procedure. The R. tuberosa HR was then cultured in liquid MS medium and achieved the highest biomass production at the late exponential growth phase (3rd week). Its ethanol extract was also higher 2.0-fold in α-glucosidase inhibitory activity than that of the natural root. In conclusion, the α-glucosidase inhibitory activity of HR inducing by the improved procedure may offer an effective and reliable substitute for the utilization of this herbal plant.