目的建立和优化获得量大而高紫杉醇愈伤组织的培养技术和方法。方法以山西陵川县野生和在西安栽培的南方红豆杉为材料,就最佳母株、外植体种类、基本培养基、植物激素种类及浓度对愈伤组织诱导、生长和紫杉醇的影响等因素进行了系统研究。结果幼茎外植体因诱导的愈伤组织时间早、频率高而最佳;培养基Y 5:M S+2,4-D 4.0 m g/L+K IN 1.0 m g/L或B 5Ⅲ:B 5+2,4-D 3.0 m g/L+K IN 0.1 m g/L+Phe 0.1 m o l/L为最佳诱导愈伤培养基,其诱导频率高达100%。继代培养基中质量浓度2,4-D(0.5~3 m g/L)促进愈伤组织增殖;高质量浓度(8 m g/L)则有抑制作用。当2,4-D质量浓度适宜时,愈伤组织在B 5培养基上较在M S培养基上褐化轻、生长快。HPLC分析表明紫杉醇最高的继代培养基为M SⅢ,其紫杉醇质量分数高达干重的0.004%;野生母株幼芽来源的愈伤组织,其紫杉醇普遍高于栽培母株幼茎来源的愈伤组织。结论以5月份野生红豆杉幼茎为外植体,以Y 5或B 5Ⅲ为诱导培养基和继代培养基,继代8~10代,每代培养30~35 d;用M SⅢ培养基再培养1~2代,收获愈伤组织或细胞培养物,并从中提取紫杉醇是获得大量高紫杉醇愈伤组织的较佳培养程序。

Objective To establish and optimize the technology and method of producing large quantity and high-paclitaxe yield callus of 〖WTBX〗Taxus chinensis var. mairei. Methods Wild maternal tree grown in Lingchuan County of Shanxi Province and cultivated tree grown in Xi′an were used as explant source. And the optimum maternal tree for explant cutting, optimum explant type, basic medium, composition and concentration of growth regulators in medium and so on, which were factors of affecting on callus induction, growth and paclitaxe yield, were examined in a series order. Results The juvenile stem segments were the optimum explants because of their earlier and higher rate callus induction than that of other explants. Medium Y5: MS+2,4-D 4.0 mg/L+KIN 1.0 mg/L or medium B5 Ⅲ: B5+2,4-D 3.0 mg/L+KIN 0.1 mg/L+Phe 0.1 mol/L was confirmed optimum callus induction medium in which callus induction rate had reached to 100%. In callus subculture medium, lower concentration of 2,4-D (0.5—3 mg/L) always increased callus growth, but higher concentration of 2,4-D (8 mg/L) reduced callus growth. When 2,4-D concentration was suitable, callus grown on B5 medium displayed lighter browning and faster tissue growth than that on MS medium. Further more, HPLC analysis confirmed that the paclitaxel yield in callus grown on medium MSⅢ was highest and had reached 0.004% of callus dry weight. In a general condition, the level of paclitaxel in calli derived from juvenile stems of wild maternal tree was higher than that in calli initiated from cultivated maternal tree's juvenile stems. Conclusion The optimization sequence of obtaining a large quantity and high-paclitaxe yield callus of T. chinensis var. mairei are dividing juvenile stem segments from wild maternal tree in May and culturing on medium Y5 or B5 Ⅲ for callus induction. After the calli having been subcultured on the same medium for 8—10 generations, one or two generations are recultured on medium MSⅢ. Finally, the calli with more paclitaxel are obtained by extracting paclitaxel out of it.

山西省自然科学基金资助项目(951030)