| Taxus cuspidata cells contain a kind of terpenoid called taxol which has significant curative effect on various cancer diseases.Taxol is a kind of natural anticancer drug that is expensive and the most widely used on the market today.However,it is difficult to extract taxol efficiently,and Wild Chinese yew is the national resource focus of the conservation.Therefore,it is necessary for us to use biotechnology-method of cell efficient induction to extract taxol,so that we can solve the problem of insufficient raw materials fundamentally,to meet the growing needs of mankind.Therefore,in this paper young stems of Taxus cuspidata are selected as materials mainly,we get an optimal process of cell suspension culture to extract taxol efficiently from Taxus cuspidata,through the induction of the callus of Taxus cuspidata under suitable(fill light)environmental conditions,subculture constantly for the callus of Taxus cuspidata and selection stable callus of the high content of taxol,screening of high yield cell lines and optimization on cell suspension culture conditions(including the elicitor).At the same time,a method of cryopreservation of the callus of Taxus cuspidata is proposed,which has a significant effect on the long-term preservation of the callus.The optimization process has an important guiding significance for cell efficient induction to extract taxol from Taxus cuspidata,and lay a solid basis for the preliminary work for industrialization of cell culture to produce taxol of Chinese yew.In addition,the optimization process is also conducive to the efficient development of medicinal plant resources utilization,has the important practical significance and far-reaching social influence.The innovative conclusions obtained from the experimental study are as follows:(1)In the experiment,young stems of Taxus cuspidata are selected as explants,the modified B5 medium is chosen,under certain environmental conditions induced,when healing time is 10-11 days,we can obtained callus in good condition of growth.Through the single factor experiment of light environment,optimal light environment is obtained:(with LED as light source)light quality is the red light(λ =660nm),photoperiod is 12h/d,and light intensity is 10μmol·m·S-1.The photoperiod,light intensity,temperature and humidity are regarded as independent variables,Box Behnken compound factors test is conduct,the results show that the optimal optical environment conditions for the induction of callus of Taxus cuspidata were photoperiod of 11.82h/d,light intensity of 9.77μmol·m-2·s-1,the temperature of 24.83 ℃and humidity of 85.00%.At this time,induction rate of the callus was 95.61%.(2)The single factor experiment of the pre culture conditions of the callus of the super low temperature preservation was conduct.The optimal pre culture conditions were as follows:the temperature was 5℃,the sucrose concentration was 6%,and the time was 10d.Time(X1),sucrose concentration(X2),temperature(X3)were regarded as independent variables,the test results of Box Behnken complex factors showed that the best pre culture conditions were the time of 10.47d,concentration of sucrose of 5.50%,temperature of 3.37℃.At this time,relative survival rate of the callus cells of Taxus cuspidata reached maximum,was 62.30%.For cryoprotectant test of super low temperature preservation in callus of Taxus cuspidata,10%sorbitol and 10%DMSO were chosen as cryoprotectants,relative survival rate of the cells reached the highest,was 65.73%;Freezing method experiment on the callus in super low temperature preservation showed that callus were treated with the method of slow freezing and method of step-by-step freezing,both of which had little effect on callus cells after thawing,and at the time,the relative survival rates of the cells were high,67.23%and 65.12%respectively.(3)Subculture test of callus of Taxus cuspidata through 11 times showed that fresh weight and taxol content of callus tended to be stable for subculture after nine generations.Taxol contents in the ninth generation callus were 259.14 μg/g,was increased 2.1 times compared with that of the direct extraction method.Therefore,callus of the ninth generation can be used for cell suspension culture to obtain high-yield cell lines of the test.10 strains of high production cell lines were screened from the multi clonal cell strains,and pass through the stability screening.It was found that the fresh weight growth rates of cell clone in the liquid culture medium were higher than those of the solid culture medium,but taxol contents of cells for No.5,No.12 and No.21 remained relatively stable in the two kinds of culture medium.(4)It was found through the experiment and the analysis that increasing sucrose concentration in the cell suspension culture medium of Taxus cuspidata,is conducive to the synthesis and accumulation of taxol content,however,the growth rate of cells would be inhibited.Therefore,sucrose supplemented with concentration of 30g/L was more appropriate in the early suspension culture medium.Compared with not adding sugar,the cell fresh weights were significantly improved when adding sugar in the middle suspension,and sugar of the best effect to improve cell fresh weight and taxol content was sucrose.The single factor test showed that taxol content were higher with the condition of culture temperature of 24 ℃ and shaking speed for 120r/min in the process of suspension culture.The suspension culture conditions were optimized by orthogonal test,and considering not only cell growth,but also the accumulation of taxol content,the final choices of suspension culture were that sucrose was added with the concentration of 30g/L in the early culture time,and then in the middle culture time sucrose with the concentration of lOg/L was supplemented,temperature was for 24 ℃ and speed was for 120r/min.When adding inducing substances and sucrose with a certain amount to suspension culture medium,fresh weight and taxol content of cells were the highest,reaching up to 72.025g/L and 5.652mg/L respectively,increased by 8.23%and 25.96%,compared with the induced group. |
PDF Full Text Request