The Key Technique Of The Expanded Culture Of Psammosilene Tunicoides Hairy Roots

Psammosilene tunicoides(P.tunicoides)is a unique medicinal plant in the south of China,and it is a national second-level protected plant.It is mostly used for clinical treatment of rheumatic pain,bruises,trauma bleeding,stomachache and other diseases for its analgesic,anti-rheumatic,and anti-stasis effects.P.tunicoide are one of the main components of "Yunnan Baiyao".Due to the limited natural growth area of P.tunicoide and the serious phenomenon of chaotic harvesting,the natural survival conditions of P.tunicoide are particularly harsh.Therefore,it is of great significance to optimize the cultivation conditions of P.tunicoides and realize the industrial production of it.The root of the P.tunicoide are the main part of the active ingredient.Different nutrients and growth conditions have an important influence on the growth rate and final biomass of hairy roots induced for wild P.tunicoide.Therefore,this experiment explores different cultivation methods,optimizes cultivation conditions,simulates industrial production,and finally measures its effective components to explore the best seed conservation and production conditions,which will provides a powerful theoretical fundation for large-scale industrial production.In this study,the hairy roots of wild P.tunicoide is excised.We infect the roots with Agrobacterium rhizogenes to get the P.tunicoides hairy roots.The main research contents are as follows:(1)To study the effect of different basic culture media on solid activation of P.tunicoides hairy roots;(2)Using response surface analysis optimize liquid culture of P.tunicoides hairy roots;(3)Determine the relationship between the growth of P.tunicoides hairy roots and the accumulation of effective components;(4)Optimizing the environmental conditions of liquid culture P.tunicoides hairy roots in bioreactor;(5)Bioreactor liquid culture P.tunicoides hairy roots product,sugar consumption and final ash mass fraction analysis;(6)Bioreactor liquid culture P.tunicoides hairy roots detection of the composition of saponins by HPLC.(7)Research on the anti-oxidation of flavonoids in the liquid culture of P.tunicoides hairy roots in the bioreactor.The main conclusions are as follows:1.P.Tutnicoides hairy roots can reach the maximum biomass of 12.79 g / L(DW),the growth rate is 0.39 g / d,the total saponin content is 96.30 mg / L,when the culture condition is B5 medium,the sucrose concentration is 30 g / L,the initial When the inoculation amount is60 g / L and the growth cycle is 32 d.The number of root growth points per unit length in the morphology of P.Tutnicoides hairy roots is 2.73 ± 0.59,the number of side root growths per unit length is 2.33 ± 0.52,and the maximum number of growth stages can be 3.2.The optimal conditions for optimizing liquid culture by response surface method are that initial p H is 5.86,sucrose concentration is 32.06 g / L,and IBA concentration is 1.25 mg /L.Under the simulated conditions,the liquid-cultured gold iron lock hairy roots can obtain a final biomass of 11.89 g / L(DW)with a growth rate of 9.72% and a total saponin content of96.37 mg / L.3.The characteristics of the growth phase of the hairy root of the golden iron lock after liquid expansion culture optimization: 0-20 d is the lag phase,20-24 d is the logarithmic growth phase,the maximum growth rate reaches 0.18g(L / d),24-30 d is the stable phase,After 30 days,it entered a period of decay.When the golden iron lock hairy root liquid was cultured to 30 days,the final biomass was 12.03 g / L(DW),the growth rate was 9.66%,and the total saponin content reached 96.19 mg / L.4.The liquid culture conditions of the P.tunicoides hairy roots bioreactor after optimization: the initial p H 5.86,the initial enhancement is 2.20 k Pa,the ventilation rate is100 m L / min,the medium is based on B5,and 32.06 g / L is added Sucrose and 1.25 mg / L IBA.5.After optimizing the liquid culture of the P.tunicoides hairy root in the bioreactor,the total saponin content was 98.36 mg / L,the biomass was 14.18 / L(DW),and the final sugar consumption rate was 94.73%.The ash mass of the hairy root of the golden iron lock is 0.17± 0.04 g / g.The ash content of wild gold iron roots,stems and leaves were 0.19 ± 0.06 g /g,1.52 ± 0.02 g / g and 1.11 ± 0.03 g / g,respectively.6.The total concentration of saponins of wild P.tunicoides and P.tunicoides hairy roots cultured in bioreactor are 0.076 mg / m L and 0.098 mg / m L,respectively.The hairy roots of wild golden iron locks are 22.44% higher.7.The total flavonoids of P.tunicoides hairy roots have a certain reducing ability,and the reducing ability shows a positive correlation with the total flavonoid concentration of P.tunicoides hairy roots.Total flavonoids scavenging DPPH free radical scavenging rate is positively correlated with the concentration of total flavonoids in P.tunicoides hairy roots,of which the IC50 of total flavonoids in P.tunicoides hairy roots are 0.040 mg / m L.The total flavonoid scavenging rate of-OH free radicals is positively correlated with the total flavonoid concentration of P.tunicoides hairy roots,and the IC50 of the total flavonoids of P.tunicoides hairy roots are 0.042 mg / m L.