Effects Of Light Culture On Efficient Taxol Induction By Taxus Cuspidata

Taxus is a rare natural anti-cancer plant that is recognized to be endangered in the world,and it is the first-class protected plant of the country.Taxol,a terpenoid compound,is contained in its bark,which has a unique anti-tumor mechanism and a significant anti-tumor effect,and has an excellent effect on the treatment of ovarian cancer and breast cancer.However,due to the slow growth rate of Taxus and the low content of Taxol,relying solely the method of extracting Taxol from the skin of natural Taxus can not only not meet the growing market demand,but also cause great damage to the resources.Therefore,this paper mainly uses young stems of buds of semi-lignized explants of Taxus cuspidata as the test material.In order to obtain callus with good growth status,the explants are immersed in growth hormone,and the light culture conditions?CO₂ release concentration,light intensity and light cycle?on the tissue culture effect of Taxus cuspidata explants,and finally obtain a set of optimal schemes for light culture of Taxus cuspidata explants.At the same time,the callus after successive transfer for the high-yield cell callus suspension culture,explore natural substances on suspension cell proliferation and the Taxol production.Optimal experiments were performed on the addition of inducers and precursors to select suitable natural additives materials and inducers,optimized suspension cell culture to produce Taxol,and laid the foundation for large-scale production of Taxol by Taxus cuspidata suspension cells.The research conclusions are as follows:1.The growth hormone immersion treatment was performed on Taxus cuspidata explants,and the effects of growth hormones NAA,IBA,6-BA,and immersion time on the effects of the initial culture of the explants were investigated,and to compare and screen the effects of explant tissue culture using orthogonal methods.Finally,the optimal growth hormone immersion scheme was determined as follows:NAA 20mg/L+IBA 15mg/L+6-BA 10mg/L+immersion time 20min,at this time,the explant callus induction rate reached 96.67%,the initial healing time was shortened by 5 days,the bud germination rate was 93.75%,and callus fresh weight increment ratio of 1.78 times.2.Tissue culture studies of Taxus cuspidata explants under light culture conditions showed that when the sucrose concentration in B5 solid medium was reduced to 10g/L,the explant induction rate and growth status were the best.At this time,the callus induction rate reached 83.50%and the browning rate decreased to8.33%.Using orthogonal test method and taking CO₂ release concentration,light intensity and light cycle as test factors,the optimal combination of light culture conditions suitable for tissue culture of Taxus cuspidata explants was obtained as follows:CO₂ release concentration 4500 PPM+light intensity 50?mol·m^-2·s^-1+light cycle 14h/d.At this time,the explant callus induction rate reached 95.83%,the initial healing time was shortened by 6 days,the bud germination rate was 91.67%and the bud germination time shortened 4 days,the bacterial infection rate was reduced to6.67%,callus fresh weight increment ratio of 2.68 times,and the Taxol yield was1.06mg/L,which was 2.83 times that of the control group.3.Using high-yielding cell callus suspension culture study found that the addition of coconut juice,potato juice,and apple juice have different degrees of promotion function of the fresh weight increment and Taxol yield of suspension cells of Taxus cuspidata.However,the effect of coconut juice on the suspension cells of Taxus cuspidata was the largest.When the concentration of coconut juice was 10mL/L,the Taxol yield reached the maximum of 1.534mg/L,which was 1.23 times higher than the blank control group.The suspension cell fresh weight increment ratio can reach 1.64 times,and can make cells rapidly into the logarithmic growth cycle.4.During the suspension culture of Taxus cuspidata,the concentration combination of the inducer and precursor was optimized.The results showed that when methyl jasmonate of 105.98?mol/L,salicylic acid of 21.01mg/L,phenylalanine of 392.16mg/L and glycine of 8.93mg/L,the fresh weight increment ratio reached a maximum of 2.74 times;when methyl jasmonate of 108.61?mol/L,salicylic acid of16.47mg/L,phenylalanine of 381.12mg/L and glycine of 10.67mg/L,the maximum Taxol yield reached 1.959mg/L,which was 1.58 times higher than the blank control group.