Introduction of Cloning Plasmids into Agrobacterium tumefaciens

Abstract

Most experiments utilizing Agrobacterium tumefaciens as a vector for the introduction of genes into plant cells commence in Escherichia coli. The sheer size and complexity of the Ti-plasmids precludes their direct manipulation. Thus, insertion is usually into a comparatively small binary vector, which is then propagated in E. coli, before being introduced into A. tumefaciens, where the larger, complementing vir plasmid mediates gene transfer to plants. Typically, the binary plasmid will be based on a broad host range replicon, of IncP, IncQ, or IncW derivation, capable of replication in both bacterial hosts. Its construction will have resulted in the binary plasmid possessing the following features:

• 1. A selectable marker for bacterial cells;

• 2. A selectable marker for plant cells;

• 3. A multiple cloning site (MCS) and/or expression site; and

• 4. The Ibft (LB) and right border (RB) from the Ti-plasmid T-DNA, positioned to define a pseudo T-DNA containing the plant selectable marker and the MCS.