Research On Secondary Metabolist Accumulation Of Psammosilene Tunicoides Hairy Root Liquid Culture

Psammosilene tunicoides is an endemic medicinal plant distributed in the southwesternregions of China. Because of long-term excessive collection, together with the destruction ofits habitats, P. tunicoides has become a rare and endangered species, consequently wasenlisted on the secondary-class conservation plant species in China. So, it’s important todevelop specific biotech for the production of artificial cultures in place of wild P.tuniocoides resources.In this study, agrobacterium rhizogenes (ACCC10060) was employed to induce hairyroot from the young leaves of P. tunicoides. A kind of white-color and fast growing hairy rootwas obtained on the solid medium with500mg·L-1Cefotaxime. Then a liquid culture systemfor hairy root was established through primary screening of several factors including liquidmedium, temperature, shaking speed, pH, carbon sources. On this basis, to study differentculture medium composition, concentration of phytohormone and the elicitors on the growthand accumulation of total saponins of P. tunicoides hairy root. In addition, the relationshipbetween hairy root biomass and the changes in culture conductivity, reducing sugars wereexamined in the process of liquid culture. The main results are as follows:1The suitable formula of basic liquid conditions for hairy root was represented as:1/2MS,25±1℃,pH5.8,100rpm. The hairy root period was about28d, and showed an “S”type.2Through addition of different carbon sources and modulations of the concentration ofcarbon sources, nitrogen sources, phosphate sources, the growth and accumulation of totalsaponins of P. tunicoides hairy roots were studied. The results showed that30g/L sucrosewere favorable for the biomass and saponins accumulation. A ratio of1∶2of NH4+to NO3-was found to be suitable for the growth and saponin accumulation. The phosphateconcentration equal to1/2MS proved to facilitate the growth and saponins accumulation.3The effects of various growth regulators on the growth and accumulation of saponin ofhairy roots were investigated. Additions of0.5mg·L-1NAA, IAA, IBA separately to the media,all exhibits active effects on the biomass and saponin accumulation, and the yield of saponin was increased by53.42％,25.55％,79.60％respectively. Although additions of6-BA and2,4-D could improve the accumulation of saponin, both slow down the growth of hairy root.4The effects of elicitors on the P. tunicoides hairy roots growth and saponinaccumulation were studied. It showed that addition of100ug·mL-1salicylic acid, an inorganicelicitor, was proven to be beneficial to the growth and saponin accumulation, contrarily,extract of yeast restrain the growth and saponin accumulation of hairy roots. The biomass andsaponins accumulation both were better in the presence of2.0mmol·L-1Ca2+.5The total reducing sugars in the medium increases and then decline during culture ofhairy root. During logarithm growth period, the conductivity decreased linearly relative to thebiomass accumulation. So the conductivity and reducing sugars could be used as indicators ofhairy root biomass during the large scale culture.