Studies On Regulation Optimization Of Callus Growth And Taxol Production In Taxus Chinensis Var. Mairei Callus Culture

Taxus chinensis var. mairei is a variation of Taxaceae Taxus in china, and it containsPaclitaxel (Taxol), which is a kind of anticarcinogen, in its branch, bark, needle, root andpulp. In order to production oftaxol in callus cultures of Taxus chinensis var. mairei,the inducement culture of callus from the young stem, bark of maturer young stems,cambium tissue, immature needle, and apical meristem explant of Taxus chinensis var.mairei, callus culture optimization, regulation optimization of callus growth and taxolbiosynthesis quantity were systematically studied in this dissertation with the methodof HPLC, weighing callus, measuring callus diameter and orthogonal experiment.Callus inducement, MS+1.2mg/L2,4-D+4mg/LNAA+2.8mg/L6-BA is the bestinducing medium, and young stem and apical meristem are the best explant, the next isimmature needle. B₅+0.4 mg/L 2,4-D+3.2 mg/L NAA+0.1 mg/L KT and B₅+0.4 mg/L2,4-D+3.2 mg/L NAA+0.8 mg/L KT is the optimal culture medium of benefit to growthmultiplication of T. chinensis var. mairei callus, and 0.8 mg/L2,4-D+2.0 mg/L NAA+0.2mg/L KT+0.1 mg/L CH is of benefit to biosynthesis taxol in the callus, and B₅+1.2 mg/L2,4-D+3.2 mg/L NAA+0.1 mg/L KT+0.1 mg/L CH is of benefit to either the growth of thecullus or increase of the taxol content in the callus(the growth rate of the callus is 3.24times/period, a period is about 45 d～50 d, and the content of taxol in the callus is0.0187%). More taxol is synthesized in the callus of young stem and leaf than inthe callus of others.When use chlorogenic acid in Eucommia ulmoides Oliv to regulate, if in order togrowth of callus, need to choose the calluse that is initially growing stages, and shouldselect the callus that is Logarithmic phase or Stationary phase to increase the taxol contentin the callus. When Logarithmic phase callus was cultured on the subculture mediumsupplemented with 1 mg/L 95% chlorogenic acid, the taxol content in the callus reached0.065%, was 100 times of the lowest content in the experiment team sample. There is atrend that taxol content in Stationary phase callus is increase following increase of the concentration of chlorogenic acid in the subculture medium, when the concentration wasincreased to 10 mg/L, taxol content in the callus went up to 0.025%, was 4.3 times of thecontent in antitheses sample. T. chmensis var. mairei callus was cutltured in the mediumsupplemented with 0.1 mg/L chlorogenic acid, 0.1 mg/L saliglic acid, 1.7 mg/L silvernitrate, the contend of taxol in the callus reached 0.0735%, was about 100 times ofantitheses sample.Taxol content in the callus that was harvested heightened 15 times more thanantitheses sample after the callus was fermented and transformed, the process was sensitiveto the capacity of fungi fluid inoculated and the fermented and transformed time, thenimiety of the capacity or the time is too long all resulted in the taxol content to reduce. Itis better treating to inoculate 10 mL fungi liquid in 20 g callus sample, to ferment and totransform in 0.5 d.The differences of callus growth amount that was leaded by differences of explantoriginals and the difference of plant growth regulator in culture medium would beeliminated by continuing subculture again and again, there are many combination ofplant growth regulators benefit to callus growth of Taxus chinensis var. mairei. Butthere is the most markedness difference between the isogeny callus with differentcontent and its combination of plant growth regulators and nonisogeny callus withthe same plant growth regulator. It is helpful for the synthesizing of taxol to useexclusively certain amount(2 mg/L) of 2,4-D, or to use it along with certainamount of KT,6-BA,KT+GA, whereas unhelpful by NAA.The growth periods of T. chinensis var. mairei callus is about 45 days, thereinto, thearrearage phase is about 5 days, the logarithm phase is the sixth day to the thirty-fifth, thenthe callus starts to enter stationary phase and to change caducity and dark at about fiftiethday.there is higher content of related taxanes compound in the callus. There is crossregulating action for 2,4-D and NAA to either the growth of the callus or the thebiosynthesis of taxol in that, 0.4 mg/L 2,4-D+3.2 mg/L NAA is the best beneficial to thegrowth of the callus and 0.8 mg/L 2,4-D+2.0 mg/L NAA is of benefit to biosynthesis oftaxol in the callus. CH is of disbenefit to the growth of T. chinensis var. mairei callus.The results that were sdutied and obtained with weighing-method of callus and measuring-method of callus diameter were relatively identical, it showed that both ofmethods could be applicable, but measuring-method of callus diameter is more simpler andeasier than orther method because the method can avoid a series of asepsis process.When inoculated with the callus of Lag phase, there was a current that the growth rateof the callus was going up as the concentration of chlorogenic acid increased, but thecontent of taxol was in reverse, and the growth rate of the callus wasn't go up again as itincreased when it exceeded 1.0 mg/L.Chlorogenic acid could also thin the colour of T. mairei callus compared to antitheses,the result suggested that a certain concentration of chlorogenic acid can reduce darking ofT. mairei callus.The kinds of the related taxanes that were higher in T. mairei callus was reduced as thecallus was fermented and transformed by T. mairei mycorrhiza fungi and those taxaneswere changed 5-6 kinds into 2-3 kinds, that effect was sensitive to fermenting-time, theprocess would be reversible if the fermenting-time was too long.