红豆杉高产细胞系快速建立和更新的新方法

目的：通过优化培养基和培养条件,建立太行红豆杉速生细胞系,为红豆杉细胞工厂化生产提供原种细胞系和繁殖技术。方法：以太行红豆杉为材料,筛选B5、WPS、MS等3种培养基和6-卞基嘌呤（6-BA）、萘乙酸（NAA）、2,4-二氯苯氧乙酸（2,4-D）等3种激素组合,采用液体、半固体、纸桥共3种培养方法继代培养并筛选高产细胞系。结果：B5＋NAA（1.0mg/L）＋6-BA（0.5mg/L）＋2,4-D（1.0mg/L）为最适愈伤组织诱导培养基,诱导率达100%;B5＋NAA（1.0～1.5mg/L）＋2,4-D（1.5～2.0mg/L）＋6-BA（0.5mg/L）为最佳继代培养基,生长量最高可达0.30g/（g·d）（鲜重）。结论：初步建立了红豆杉细胞系快速建立和繁殖新方法,用半固体培养产生初代细胞系、液体培养将细胞系同步化、纸桥培养快速繁殖细胞系,并通过检测紫杉醇含量不断更新细胞系。该细胞系体系建立方法简单,纯化速度快,易更新,产物细胞系可作为工厂化生产的原种细胞系。

New Method to Rapid Establish and Renovate High Yield Cell Lines of Taxus L.

Objective： To establish rapid growth cell strains of Taihang taxus（belongs to Taxus chinensis） by optimizing medium and culture conditions that serve as production cell line for cell production industrially that is the raw material of taxol. Methods： Taihang taxus was as explant and cultured in three kinds of basic media, hormones and culture modes so as to filtrate the best one of them. Results： The best medium for inducing callus in the experiment was B5＋NAA（1.0 mg/ L）＋6-BA（0.5 mg/L）＋2,4-D（1.0 mg/L）, whose inducing rate reach to 100%; and for subculturing the best of medium was B5＋NAA（1.0-1.5 mg/L）＋2,4-D（1.5-2.0 mg/L）＋6-BA（0.5 mg/L）, whose growth rate reach to 0.30 g/（g.d）. Conclusion： A new culture system for rapid establishment cell line of taxus was as following： firstly, the cell strains formation in solid medium; secondly, by liquid cultrue cell growth cycle could be in-phese; and thirdly, on paper bridge the cell line could be reproduced rapidly which could be called as first generation cell line for in large scale of production taxus. This culture system was simply in establishment, easy purification, and facility to renovate under the examination taxol.