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[摘要]
目的 通过愈伤组织细胞生长及其紫杉醇代谢动力学的研究,筛选确定南方红豆杉愈伤组织培养生产紫杉醇的最佳培养基配方与最佳收获期。方法 以改良MS为基本培养基添加不同质量浓度的IBA、6-BA、GA3组合诱导培养南方红豆杉愈伤组织,通过测定愈伤组织鲜质量进行生长动力学研究,采用HPLC法检测不同生长阶段愈伤组织中紫杉醇的积累量,进行紫杉醇代谢动力学研究。结果 改良MS+0.2 mg/L IBA+0.05 mg/L 6-BA+0.3 mg/L GA3、MS+0.2 mg/L IBA+0.01 mg/L 6-BA+0.7 mg/L GA3及MS+0.2 mg/L IBA+0.1 mg/L 6-BA+0.5 mg/L GA3培养条件均较有利于南方红豆杉愈伤组织的诱导培养,且紫杉醇的积累量也较高,最高可达0.037 76%。结论 在添加IBA及6-BA的基础上适当增补GA3可使南方红豆杉愈伤组织诱导提前启动,缩短出愈时间,促进生长,降低褐变程度,增加愈伤组织中紫杉醇的积累量。愈伤组织的生长曲线呈S型,紫杉醇的积累呈线型增加,是一个逐渐积累的过程,但积累到一定程度就不再增加,且伴随着愈伤组织不断褐变。
[Key word]
[Abstract]
Objective Through study on the growth of Taxus chinensis var. mairei callus culture and its metabolic dynamic research on the paclitaxel, the optimum media and the best harvest time for the callus of T. chinensis var. mairei to produce paclitaxel were screened and determined. Methods T. chinensis var. mairei callus were induced and cultured by taking the improved MS as basic medium supplemented with IBA, 6-BA, and GA3, the growth kinetic research of the callus was carried out by measuring fresh weight of the callus, and the paclitaxel accumulation in the callus at different growth stages was detected by HPLC analysis to study the paclitaxel metabolic dynamics. Results The three kinds of media, the improved MS + 0.2 mg/L IBA + 0.05 mg/L 6-BA + 0.3 mg/L GA3, the modified MS + 0.2 mg/L IBA + 0.01 mg/L 6-BA + 0.7 mg/L GA3, and the improved MS + 0.2 mg/L IBA + 0.1 mg/L 6-BA + 0.5 mg/L of GA3 were more favorable to T. chinensis var. mairei callus culture and made the paclitaxel accumulation up to 0.037 76%. Conclusion On the basis of IBA and 6-BA appropriately added to the GA3 in the improved MS medium, the bourgeon of T. chinensis var. mairei explant callus is enabled early to start, the time of culturing callus shorten, the growth and the browning degree promoted, and the amount of the accumulation of paclitaxel in the callus increased. Callus growth curve is S-shaped and the accumulation of paclitaxel showed a linear increase which is a gradual process of accumulation, but no further increase when accumulated to a certain extent, while along with constantly browning of callus.
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[基金项目]
湖南省科学技术厅科技计划项目(2010TP4007-2);湖南省科学技术厅科研条件创新专项(2010TC2002);湖南省高校“林产资源化学与林化产品开发”科技创新团队支持计划资助(湘教通(2010)212号)