摘要
参考 Horsch 等(1985)建立的叶圆片转化法,建立了一个适合于杨树的遗传转化系统。本文所用的根癌农杆菌菌株含有一个经过改建的 Ti 质粒,这个质粒载有 T-DNA 的4号基因和 CAT 基因。利用毛白杨(Popular tomentosa)的叶外植体经该菌株感染后,培养3-4个星期后,在没有外源激素的 MS_u 培养基上,从叶外植体的边缘处再生出一些不正常的苗。这种苗呈典型的畸胎芽,把这种苗从外植体上切下并转到生根培养基上,在苗的基部生成大量的愈伤组织,但并不形成根,同时在愈伤组织上产生大量的芽。气相色谱法测定内源细胞分裂素的结果表明,转化苗的异戊烯基腺苷的含量是正常苗的14倍。纸电泳结果表明,转化苗均含有特异的胭脂碱。在加有60-100μg/ml 氯霉素的培养基上,转化苗可以正常生长,而未转化的苗在培养约40天后变白死去。用酚类物质水杨酸预处理农杆菌有助于提高转化频率。
The leaf disc method developed by Horsch et al.(1985) has been used for transformation of Populus tomentosa.The strain of Agrobecterium tumefaciens used harbored a reconstructed Ti plasmid which contained gene 4 of T-DNA and the chimeric CAT(chloramphenicol acety- ltransferase) gene.Leaf explants from shoot cultures of Populus tomentosa were co-cultivat- ed with the bacterium.On the hormone free medium,teratoma-like shoots developed from the edge of the leaf explants.When the abnormal shoots were excised from the explants and trans- ferred onto rooting medium,a mass of callus formed at the base of shoots,with new shoots developing,but without root formation.The measurement of endogenous cytokinin showed that the transformed shoots produced 14 times as much iso-pentenyl adenosine as untransformed shoots did.All teratoma-like shoots tested showed the presence of nopaline,and were able to grow well on the medium containing 60—100μg/ml chloromycetin,while normal shoots turn- ed white after 40 days.Pretreatment of A.tumefaciens with phenolic compound,salicylic acid,would increase the frequency of transformation significantly.
基金
国家自然科学基金资助课题
关键词
毛白杨
叶外植体
遗传转化
Populus tomentosa
Agrohacterium tumefaciens
Transformation
