Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agro- Warren et al. (1978) showed that the CoEl-encoded mob gene products are involved and that one or more of these mob gene products recognize a special ColE 1 sequence called the bom site. Transfer is then initiated from the origin of transfer at or near the bom site. This system of plasmid mobilization has evolved in several colicin-producing plasmids. Mob and bom are specific within plasmid families, so that, for example, mob and bom of ColE1 and its natural analogue, pMB1, are interchangeable. pBR322 has the pMB1 bom site and can, therefore, be transmitted when the ColE1 mob functions are provided in trans (Finnegan and Sherratt, 1982). Here we report that the ColE1 -type mobilization is extendable to other unrelated Gram-negative bacteria, such as A. tumefaciens.A method for site-specific mutagenesis of Ti plasmids was developed, taking advantage of the efficient transmission from E. coli to A. tumefaciens of pBR322 clones containing mutant pTiC58 fragments, and of the fact that these pBR322 clones cannot replicate in A. tumefaciens. Stable exconjugants to Agrobacterium are therefore only obtained if transfer is followed by co-integration of the mutant clone and acceptor Ti plasmid. Full exchange of mutant and target Ti fragment, resulting from a second recombination, is obtained by screening for the appropriate combination of selectable markers.

The tumor-inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens is the agent of crown gall disease in dicotyledonous plants. The Ti plasmid contains two regions that are essential for the production of transformed cells. One of these regions, termed transfer DNA, induces tumor formation and is found in all established plant tumor lines; the other, termed the virulence region, is essential for the formation but not the maintenance of tumors. Transfer DNA, which transfers to the plant genomes in a somewhat predictable manner, can be increased in size by the insertion of foreign DNA without its transferring ability being affected. The tumor-causing genes can be removed so that they no longer interfere with normal plant growth and differentiation. This modified Ti plasmid can thus be used as a vector for the transfer of foreign genes into plants.

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