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目的研究南方红豆杉愈伤组织的出愈时间、诱导率、生长率、生长状态、褐变程度,比较愈伤组织中紫杉醇等5种紫杉烷二萜类成分的量。方法采用单因素比较、正交试验设计,研究影响南方红豆杉愈伤组织诱导和生长的各种因素;采用HPLC法测定不同来源愈伤组织中10-脱乙酰基巴卡丁Ⅲ(10-DAB)、巴卡亭Ⅲ、三尖杉宁碱、紫杉醇和东北红豆杉素5种紫杉烷二萜类成分的量;采用加权评分综合考虑以上各因素优选组织培养条件。结果外植体脱分化形成愈伤组织的能力,以完整叶片及茎段最好;暗培养有利于紫杉醇等5种萜类成分的富集;愈伤组织诱导的最适培养基为B5+2,4-D 3.0mg/L+6-BA 1.0 mg/L+NAA 1.0 mg/L+KT 0.5 mg/L。结论南方红豆杉愈伤组织诱导、培养与外植体种类、培养基种类、光照有较大关联,紫杉醇等5种紫杉烷二萜类成分的富集受植物生长调节剂种类及浓度影响显著。
OBJECTIVE To study the time of emergence, induction, growth rate, growth status and degree of browning of callus from Taxus chinensis var. Mairei and compare the amount of taxane diterpenoid constituents such as paclitaxel in callus. Methods Various factors influencing callus induction and growth of Taxus chinensis var. Mairei were studied by single factor comparison and orthogonal experimental design. The effects of 10-deacetylbaccatin Ⅲ (10-DAB ), Baccatin Ⅲ, cephalomannine, paclitaxel and taxol, respectively. The weight of the five taxane diterpenoids was taken into consideration. The above factors were used to optimize the tissue culture conditions. Results The ability of explants to dedifferentiate to form callus was the best in the whole leaves and stem segments. Dark culture was beneficial to the enrichment of five terpenoids such as paclitaxel. The optimal medium for callus induction was B5 + 2 , 4-D 3.0 mg / L + 6-BA 1.0 mg / L + NAA 1.0 mg / L + KT 0.5 mg / L. Conclusion The induction and culture of callus from Taxus chinensis var. Mairei is closely related to the types of explants, the types of culture medium and the light. The enrichment of five taxane diterpenoids such as paclitaxel is significantly affected by the species and concentration of plant growth regulators .