Studies On Optimizatio Of Conditions Of Taxal Production In Taxus Madia Supension Culture

In this experiment, take the stable Taxus media callus after subculture more than fivetimes the in laboratory as test material to study the conditions of Taxus cell suspensioncultures by the single factor experiment and orthogonal method, to explore the sucroseconcentration, the initial concentration, the effect of plant growth regulators andanti-browning substances on cell growth; the effect of natural additives in Taxus cell growthand intracellular taxol content; the effect of carbon source in Taxus media cell growth andintracellular paclitaxel content; the effect of inducing substance in Taxus media cell growthand intracellular paclitaxel content. In hope to get the best culture medium, screening out theoptimal ratio of anti-browning agents through the establishment of culture conditions and toestablish a stable Taxus cell suspension culture system through the above test. Get the besttypes of natural additives by the study of natural additives in Taxus cell growth andintracellular paclitaxel content; determine the best type of carbon source and the optimumconcentration of carbon source, and its add time by the study of carbon source in Taxus cellgrowth and cell paclitaxel content; determine the the best type of inducing substance and itsbest add time by the study of inducing substance in Taxus cells growth and intracellular taxolcontent. Through these experiments, to create a stable suspension culture system and inducethe production of paclitaxel, to provide a basis for the subsequent in a large-scale cultivation.Experimental study achieved the following results:1.Taxus cell suspension cultures:The effect of different sucrose of concentration, inoculum,the combination of plant growth hormone and anti-browning ratio of reagents of Taxus cellgrowth, Determine the conditions of Taxus cell suspension cultures by the single factorexperiment and orthogonal experiment screening. Results are: B5medium+2.4-D0.6mg/L+NAA0.8mg/L+KT1.0mg/L+citric acid0.22g/L+PVP0.35g/L+LH0.45g/L+sucrose30g/L, pH=5.8the inoculum size0.1g/mL, the incubator speed110r/min,25°C,dark culture. Subculture time is15d.2. Natural additives on Taxus cells: banana juice, potato juice, apple juice are choosen asnatural additives materials. We found that three kinds of natural additives have effects on Taxus cell growth and intracellular taxol, which potato juice on Taxus cell proliferation has asignificant role in promoting and intracellular paclitaxel content has a significant role, itsoptimal dosage of75g/L. Three kinds of natural substances on callus proliferation indescending order: potato juice, banana juice, apple juice. The three kinds of naturalsubstances taxol Taxus cells played an important role in its Taxus cells Taxol content indescending order: apple juice, potato juice, banana juice.3. Carbon source on Taxus cells are: sucrose, maltose, lactose, fructose, glucose, sorbitol,mannitol,75％sucrose+25％fructose,75％fructose+25％sorbitol and75％+25％sucrose,sorbitol10carbon source for the materials are tested by single factor test to determine the besttype of carbon source and dosage. Taxus cell growth and taxol content of intracellular greatestimpact on carbon sources sucrose, The optimum concentration of40g/L, and adding whenstarting the culture at a concentration of sucrose30g/L, when the culture to15d and add10g/L sucrose..In the carbon source experiments, the polysaccharide substances on cell growthpromoting effect than the single sugars and alcohols to be large, the content of the impact ofTaxol, alcohol influence bigger, but alcohols can not support cell growth and proliferation.4. Inducing substance on Taxus cells: AgNO3, sodium acetate, salicylic acid, phenylalanineand glycine as inducing substance, determined by single experiments and orthogonalexperiments, Taxus cells to produce optimal induction ratio of paclitaxel: AgNO33mg/L+sodium acetate4mg/L+salicylic acid15mg/L+phenylalanine10mg/L+glycine10mg/L. Add inducing substance in time of the18d of culture. Five kinds of inducing substanceson Taxus cell production of taxol in a decreasing order: Phenylalanine、glycine、silver nitrate、acetate、salicylic acid.5. The results of verification tests: The ratios of proliferation was3.21; the taxol content was93.57μg/mL.