Efficient Induction Of Taxol And Metabolic Regulation In Suspension Cells Of Taxus Cuspidata

Taxus Cuspidata is a precious tree species that is on the verge of extinction in the world,and is listed as a first-class protected plant in my country.Its branch bark contains a terpenoid compound-taxol,which has obvious anti-cancer effect.Therefore,the rational use of Taxus cuspidata to produce taxol is a hot research topic in my country in recent years.Due to the very low content of taxol in its bark,it is of great practical significance for us to use the bioengineering technology of cell suspension culture to efficiently produce taxol.This paper mainly uses young stems of buds of semi-lignified explants of Taxus cuspidate as the test material.In order to obtain callus with good growth status,a certain amount of 5-ALA was added to the medium and treated with different light,and the optimum concentration of 5-ALA was obtained under the best light environment.At the same time,the well-grown callus was subjected to suspension culture after multiple subcultures.To investigate the effects of inorganic salts,accelerators and organic solvents assisted ultrasound on the fresh weight proliferation of suspended cells and the yield of taxol,and to carry out reasonable optimization experiments.The optimal concentration and time of adding substances were selected,and the suspension cell culture production scheme was optimized to provide a certain theoretical basis for the industrialization of Taxus cuspidata suspension cell production.The research conclusions are as follows:1.The callus of Taxus cuspidata was used as the experimental materials,and different concentrations of 5-ALA were added to the improved B₅ medium for callus culture.The results showed that 25 d was the best time for the callus subculture of Taxus cuspidata and the determination of taxol.The light intensity of 1000 Lux,the light quality of red light,and the photoperiod of 12 h/d were the best light conditions for the growth of Taxus cuspidata callus and the production of taxol;At this time,the addition of 5-ALA at a concentration of 1μM significantly promoted the growth of callus and the production of taxol.The fresh weight of callus increased by 2.335 times,and the content of taxol was 0.0353%.2.By adjusting the inorganic salt ion concentration in the B₅ medium,the optimal combination of inorganic salt concentrations suitable for the suspension culture of Taxus cuspidata cells to produce taxol is as follows:under the condition of ammonia nitrogen:nitrate nitrogen=3:24（total nitrogen concentration is 27 mmol/L）,when KNO₃ of 1.5 g/L,NH₄SO₄ of 0.268 g/L,Ca Cl₂ of 0.3 g/L,Mg SO₄ of 0.35 g/L,and Na H₂PO₄ of 0.05 g/L,the maximum taxol yield reached 1.468 mg/L,which was 1.3times higher than the CK.3.The fresh weight of the suspension cells of Taxus cuspidata in one cycle was measured,and it was concluded that the growth of suspension cells was roughly"S"-shaped.During the suspension culture of Taxus cuspidata cells,the addition time combination of promoters was rationally optimized,and the results showed that:add 2mg/L gibberellin on day 9,add 20 mg/L salicylic acid on day 12-13,add 400 mg/L phenylalanine on day 18,add 100μM/L methyl jasmonate on day 18-19.This was the optimal condition.At this time,the taxol content was 4.149 mg/L,which was about2.68 times higher than the CK.4.Using oleic acid as the organic solvent for the two-phase culture,adding a certain concentration of sucrose and assisting with ultrasound,the conditions suitable for the growth of Taxus cuspidata suspension cells were obtained as follows:in the early stage of culture,the concentration of oleic acid was 10%and the concentration of sucrose was 30 g/L;After one week of suspension culture,sonication was performed for 2 min,and 10 g/L sucrose was added at the later stage of culture.This was the optimal condition.At this time,the taxol content was 7.549 mg/L,which was about 5 times higher than the CK.