匍枝筋骨草悬浮培养生产蜕皮甾酮的研究

为得到生长迅速、生物量大、蜕皮甾酮含量高的匍枝筋骨草(Ajuga lobata D.Don)细胞系，从固体培养基选择、激素种类筛选出筋骨草颗粒状的胚性愈伤组织，再从培养基类型、激素配比和接种量3个方面优化悬浮培养体系。试验结果表明：添加0.4 mg/L 2,4-D的MS固体培养基可以诱导匍枝筋骨草组培苗的根段产生松散的颗粒状胚性愈伤。比较细胞系在WPM、MS、1/2MS 3种液体培养基中的生长状况，确定MS为最佳的悬浮培养基。2,4-D对细胞生长有显著影响，细胞生物量可达到添加NAA生物量的1.3倍；其与6-BA配比对筋骨草细胞的影响研究结果表明，随着6-BA的浓度增加，蜕皮甾酮的含量随之增加，其中，添加0.5 mg/L 6-BA的筋骨草细胞中蜕皮甾酮含量与不添加6-BA的对照相比，差异显著(P<0.05)；添加1 mg/L 6-BA积累的蜕皮甾酮与对照组差异极显著(P<0.01)。因此，0.4 mg/L 2,4-D+0.5 mg/L 6-BA为悬浮培养的最优激素配比，培养7天后细胞生物量净增长生物量可达到(4.3652±1.0739) g，蜕皮甾酮含量可达到(4.5692±0.2044) mg/g，所形成的细胞系更均一且繁殖速率最快。接种量为15%时，细胞长势好，鲜重增殖倍数达3.45，第7天细胞生长速率达1.109 g/d，为最适的接种量。试验建立了生长迅速、悬浮液均一的匍枝筋骨草细胞悬浮培养体系，适于蜕皮甾酮的大量生产。

A Study on Edysterone Production of Ajuga lobata D. Don Cell Lines in Suspension Culture

In order to acquire the suspension cultivation production that cells growth quickly, biomass liveweight larger and edysterone content higher, the inducing condition for obtaining the granular embryonic callus was studied by chosen culture medium and screened the phytohormone, and the Ajuga lobata D. Don cell suspension culture system was established by optimizing from three aspects, which were the culture medium type, plant hormone species and their proportion, amount of inoculation in this paper. The results shown that： MS solid medium added 0.4 mg/L 2,4-D could induce the Ajuga lobata D.Don＇s root segments to form unconsolidated granular embryogenic callus. Comparing the growth status of cell lines in 3 kinds of liquid culture medium such as WPM, MS, 1/2 MS found that cells were grew best in MS. 2,4-D affected cell growth obviously. The cell biomass was 1.3 times higher than which cultured in culture medium added NAA.Experiments on the effect of phytohormone ratio on the Ajuga lobata D. Don cell suspension culture shown that,with 6-BA concentration increasing, ecdysterone content increased. Compared with that in control group,edysterone content in the cells cultured in medium added 0.5 mg/L 6-BA was significantly higher （P0.05）.The cells cultured in liquid medium added 1 mg/L 6-BA accumulated very significantly higher edysterone than that accumulated in the cells in control group （P0.01）. Though, the most prosper phytohormone proportion was 0.4 mg/L 2,4-D ＋ 0.5 mg/L 6-BA, under which the net cell biomass could reach （4.3652 ± 1.0739） g after cultured 7 days, the ecdysterone content could reach （4.5692±0.2044） mg/g of cell dry weight, the cell lines was more uniform and grew faster. When the inoculation amount was 15%, cell lines grew best, at the 7th day after inoculation, cell growth rate was 1.109 g/d, cell regeneration index could reach 3.45. It was the most proper inoculation quantity. The optimal cell suspension culture system was established, which would benefit the ecdysterone production.