The Establishment Of Cell Suspension Culture System And Optimization Culture Conditions Of Taxus Chinensis Var.mairer

In this study,cell suspension culture system that induced from the one-year-old branches of Taxus chinensis var.mairer was established.The dynamics of suspension cell growth,paclitaxel accumulation and changes of major nutrient components were analyzed.At the same time,the effects of physicochemical factors on cell growth and paclitaxel content in suspension cell was investigated.The results showed as follows:1.The methods of explant disinfection and callus induction was designed by disinfectant combinations,concentration and ratio of phytohormone.Through single-factor experiments and orthogonal experiments,medium type,callus inoculum size,initial pH,phytohormones types and concentration of suspension culture of Taxus chinensis var.mairer were selected and optimized.The results showed that the most suitable disinfection method was 75%alcohol soaked 0.5 min,and then soaked with 0.1%mercuric chloride 8 min.MS+1.3mg/L 2,4-D+1.0mg/L NAA+1.3mg/L6-BA+sucrose 30 g/L was the best medium of callus inducement.The optimal media for Taxus chinensis var.mairer cell suspension culture was B5 medium supplemented with 0.4 mg/L 2,4-D,0.3 mg/L NAA,1.2 mg/L 6-KT and sucrose 30 g/L.The optimum callus inoculum size was 0.09g/L and the optimum initial pH of medium was 5.8.During the culture cycle of 27 days,the biomass accumulation exhibited"S"curve,and the highest biomass reached 7.66 g/（30 mL）yielded at 15th day after inoculation.2.Paclitaxel was extracted from the callus of Taxus chinensis var.mairer with ultrasonic.The influence of extraction solvent,times,temperature,time and ratio of solvent to material on the content of paclitaxel was evaluated by single factor test and orthogonal experiment.The results showed that the best solvent was methanol,and extraction times were 3 times.The optimum extraction conditions of paclitaxel from callus was as follows:extraction temperature 55℃,extraction time 30 min,ratio of material to liquid 1:40.3.The kinetic relationships of Taxus chinensis var.mairer cell growth,paclitaxel synthesis and nutrient consumption were also studied,and kinetics model was built.During the culture cycle of27 days,the results showed that sugar,phosphor,ammonia-N and nitrate-N in the culture medium were nearly used up on the 18th day.The growth law of Taxus chinensis var.mairer cell was consistent with Logistic growth model and the maximum specific growth rate was 0.14365 d^-1.The substrates of consumption and paclitaxel synthesis was described by Luedeking-Piret model.The growth of suspension cell and the accumulation of paclitaxel belonged to partial growth coupling type.4.The effects of carbon,nitrogen,phosphorus,Ca²⁺and Mg²⁺concentration on the growth and paclitaxel synthesis of Taxus chinensis var.mairer cell was studied.The results showed what was suitable for cell growth when sucrose concentration was 30 g/L and it was favorable to the synthesis of paclitaxel,when sucrose concentration was 40 g/L.The achieved biomass of cell and synthesis of paclitaxel was highest when ammonia-N/nitrate-N was 3:24.It was advantageous to cell growth when the concentration of total nitrogen was high,and low concentration nitrogen was favorable to paclitaxel synthesis.It was suitable for cell growth when phosphorus concentration was 1 mmol/L and it was favorable to the synthesis of paclitaxel,when phosphorus concentration was 0.25mmol/L.The achieved biomass of cell and synthesis of paclitaxel were highest when CaCl₂ was 2mmol/L.It was advantageous to cell growth when MgSO₄ concentration was 3 mmol/L,and MgSO₄ concentration that was kept at 2 mmol/L was favorable to paclitaxel synthesis.