Studies On Scaling Up Culture Of Psammosilene Tunicoides Hairy Roots And Plantleting Regeneration

Psammosilene tunicoides is an endangered species of China and has long been used as a precious medicine in treating pain-relieving, anti-inflammatory and blood-stopping and so on. In recent years, natural resources of P. tunicoides are in short supply. Using the method of liquid culture of hairy root from the Agrobacterium rhizogenes-infected explants is of great significance for alleviating crisis of medicinal plants resources drying up.In this study, Agrobacterium rhizogenes-transformed hairy roots from the aseptic seedlings of Psammosilene tunicoides were sampled for studying the kinetics of biomass, saponin content and pH value, as well as the the consumption of carbon, nitrogen and phosphor resources in media, The hairy roots suspension culture period was about 32 days by which the maximum biomass (DW) and the saponin content reached to 12.33g/L and 0.97%, respectively. The accumulation of saponines was in a semi-coupling mode with hairy roots growth, and they reached their peak of 1.00 mg/(L-d) and 0.36 g/(L-d) on the 30th and 22th day of culture, respectively. By the 22th d of culture, the contents of both carbon and phosphorus in the media declined to the lowest; however, a maximum biomass of hairy roots was harvested at the same time. Moreover, a rapid consumption of nitrogen source was found at the static stage of hairy roots growth (0-4 d), and its content declined to blank by the 22th d of culture (period of exponential growth). The above results suggest that the extending of P. tunicoides hairy roots growth period would be realized by feeding of necessary nutrients during culture.To study the different ratios of nitrate nitrogen and ammonium nitrogen influence of growth and secondary metabolites accumulation of hairy root. The result showed that NO3- is essential for hairy root growth, and it can promote the hairy root growth and branch elongation within a certain range. Also, the secondary metabolites accumulation were promoted by increase the proportion of ammonium nitrogen.Investigate the efforts of different inoculations and culture medium volume on hairy root growth and secondary metabolites. The result showed that inoculation amount is larger, the faster growth rate, the amplification of biomass and secondary metabolites accumulation of hairy root were smaller. The biomass of hairy root which cultured in 2 L simple bioreactor is 11.33 g/L, and reached 5 times higher than inoculation, and increased 40.40% and 27.59% than the biomass and saponins of hairy root cultured in 500 mL flasks, respectively.Investigate the plantlets regeneration of hairy root. The result showed that the effective induction scheme is 1/2 MS medium with the dosage ratio of 6-BA:IBA:NAA is 5:6:10.