罗布麻悬浮细胞生长特性及产绿原酸的初探

目的]探索罗布麻(Apocynum ventetum Linn.)悬浮细胞生长特性和产绿原酸量的最佳激素浓度,为进一步以罗布麻悬浮培养的细胞生产绿原酸奠定前期基础.方法]以罗布麻悬浮细胞系为材,确定最佳接种量和细胞生长曲线,培养获得的材料经干燥、粉碎后经超声波法提取,以高效液相色谱法(HPIC)测定其中的绿原酸(CGA)量.以绿原酸量为指标,进行方差分析,确定最佳的植物生长调节剂的浓度.结果]将罗布麻细胞悬液抽滤,以1.0 g(FW)/10 mL的接种量接种在MB(硝酸钾加倍,硝酸铵减半)+2.0 mg/L 2,4-D+0.05mg/L 6-BA+50g/L蔗糖(pH 5.9)培养基上,对数生长期为4～8 d.以上述接种量和5 d培养周期为条件,将获得的罗布麻细胞接种在不同NAA和KT浓度的培养基上培养,产绿原酸量最大的培养基为MB+0.3 mg/LNAA+0.3 mg/L KT+50g/L蔗糖(pH5.9),产量达0.183 mg/g(DW),与其它培养基相比在P=0.05水平上差异显著.结论]通过罗布麻悬浮培养的细胞产绿原酸最佳培养基为MB+0.3 mg/L NAA+0.3 mg/L KT+50g/L蔗糖(pH5.9).

Primary Investigation on the Growth Characteristic and the Yield of Chlorogenic Acid from Suspension Cells of Apocynum venetum

【Objective】The cell growth characteristic of Apocynum venetum and the optimum concentration of plant growth regulators(PGR) for the yield of chlorogenic acid(CGA) were explored,and this will prompt the further study and production of CGA from the suspension cells.【Method】 The optimal inoculative quantity and growth curve were determined with suspension cells as material.The culture cells were dried and smashed,and then ultra-sonicated for the extraction of chlorogenic acid.The amount of CGA was determined with high-performance liquid chromatography(HPLC).As the index of CGA,the fine concentration of PGR in media was determined by analysis of variance.【Result】 The filtered cells were inoculated in medium: MB(double of KNO3 and half of NH4NO3) + 2.0 mg/L 2,4-D + 0.05 mg/L 6-BA + 50 g/L sucrose(pH 5.9) with 1.0 g(FW) /10 mL inoculative quantity,the logarithmic phase of the cell was 4-8 days.The obtained cells were inoculated in media supplemented different concentration of NAA and KT and cultured,the optimum medium for CGA production from the cells was MB + 0.3 mg/L NAA+ 0.3 mg/L KT + 50 g/L sucrose(pH 5.9) as condition of above inoculation quantity and 5 d culture-period,the yield reached 0.183 mg/g(DW),the difference was significant compared with others media at P=0.05 level.【Conclusion】 The optimum medium for CGA production derived from the cells was MB + 0.3 mg/L NAA+ 0.3 mg/L KT + 50 g/L sucrose(pH 5.9).