Induction Of Hairy Roots And Preliminary Establishment Of Cell Suspension Culture Of Rehmannia Glutinosa Libosch

Objective: Radix Rehmanniae deriving from the fresh or dry root of Rehmannia glutinosa Libosch, belong to the big Chinese medicinal materials in common us. Catalpol, one of the main active components of Radix Rehmanniae, has the pharmacological activities of hypoglycemic, diuresis and diarrhoealysis, et al. Recently, with the increasing of the market demand of Radix Rehmanniae, the area of artificial cultivation is growing,it is imperative to seek a sustainable development way to solve the supply problem of Radix Rehmanniae. In this study, the induction of hairy root and the establish of suspension cell cultures system of Radix Rehmanniae was studied in order to provide the references for the large-scale culture of hairy root and suspension cell and the industrial production of active substances of Radix Rehmanniae.Methods: Agrobacterium Accc10060 used to induce leaf of Radix Rehmanniae to produce hairy root, and PCR was used to identify hairy root. To discuss the effects of light, bacteria liquid concentration, incubation time, infection time and AS on the inductivity of hairy root. The effects of plant hormone, carbon, pH, inoculum size and culture time on suspension cell culture system were studied, and catalpol content of suspen sion culture cells was measured by HPLC.In addition, the influence of exogenous elicitor on the biomass and catalpol content were also discussed.Results: The hairy root of Radix Rehmanniae leaf could be induced by agrobacterium Accc10060, and PCR result showed masccline. The optimal induction condition of hairy root was the OD600 of bacterial concentration as 0.6, infecting for 10 min, co-culturing for 4 d and acetosyringone concentration as 200 μmol·L-1. Cell suspension culture of Radix Rehmanniae was established, and the optimal induction conditions was sucrose(45 g·L-1) used as carbon source, pH at 6.0, inoculation size for 0.6 g, liquid volume for 40 mL, with 2.0 mg·L-12,4-D and 0.5 mg·L-16-BA. The growth curve of suspension culture cell showed “S”. Biomass of suspension culture cell reached maximum in the 15 th day, while catalpol content reached maximum in 20 th day. In addition, catalpol content were increased under the treatment of SA at 1.0 mg·L-1, YE at 10 mg·L-1 and CH at 400 mg·L-1. Conclusion: Hairy root of Radix Rehmanniae could be induced by Accc10060, cell suspension culture of Radix Rehmanniae was established, adding elicitor can increase the content of catalpol in suspension culture of Radix Rehmanniae. This study established the basis for the large-scale culture of hairy root and suspension cell and the industrial production of catalpol and other active substances of Radix Rehmanniae.