向日葵Ti T—DNA转化系的原生质体培养和细胞培养

PROTOPLAST CULTURE AND CELL CULTURE FROM Ti T-DNA TRANSFORMED LINES OF HELIANTHUS ANNUUS L.

根癌农杆菌离体感染向日葵子叶、下胚轴外植体形成的Ti T-DNA转化组织在激素条件下长期继代培养后,用来进行原生质体培养和细胞培养。适于B6S3转化系和T37转化系原生质体培养的培养基分别为附加不同激素和糖类的C81V和DPD培养基。用液体浅层法培养3～5天时,原生质体开始分裂。10天后形成细胞团。B6S3转化系还可直接从原生质体产生原胚状结构。转化系的细胞克隆均保持着激素自主型生长特性和冠瘿碱合成酶合成特性。

The Ti T-DNA (Transferred DNA from tumor-inducing plasmid) transformed tissues of sunflower have been obtained by invitro infecting cotyledons and hypocotyls with strains B6S3 or T37 of Agrobacterium tumefaciens. Following subculture on hormone-free medium for nearly one year, the Ti T-DNA transformed tissue lines were utilized to carry out protoplast culture and cell culture. The culture medium suitable for protoplast culture of B6S3-and T37-transformed lines were respectively C81V (Dutitz et al., 1977) and DPD (Durand et al., 1973) medium, in which diffenent hormones and carbohydrates were supplemented. After culture in thin-liquid layer of culture medium for 3-5 days, the cultured protoplastsunderwent sustained divisions, and for 10 days, the protoplast-derived colonies were formed. Interestingly, the B6S3-transformed lines could also give rise to proembryo-similar structure directly from protoplast-regenerated cells. The cell colonies from cell culture of transfomed lines were still retained the traits of both phytohormone autotrophy and opine synthetase activity, which are encoded for by Ti T-DNA.