Site-specific Insertion of Genes into T-DNA of the Agrobacterium Tumor-inducing Plasmid: an Approach to Genetic Engineering of Higher Plant Cells

This paper presents a method for insertion of genetic material into a specific site in T-DNA, the portion of Agrobacterium tumor-inducing (Ti) plasmid that becomes incorporated into the nuclear DNA of transformed plant cells when crown gall tumors are incited by this plant pathogen. The three stages of our procedure are as follows: 1. A T-DNA subfragment cloned in pBR322 is cleaved by a restriction endonuclease at a unique central site and target DNA (a kanamycin resistance marker) is ligated into this site. 2. The resulting recombinant plasmid is purified and cleaved with EcoRI; the resulting fragment bearing the kanamycin resistance marker is ligated into the unique EcoRI site of pRK290, a wide-host-range plasmid. 3. The pRK290 recombinant plasmid is transformed into an agrobacterium tumefaciens strain containing a wild type Ti plasmid. Double recombination between the altered T-DNA fragment of the clone and its wild-type counterpart in the Ti plasmid is selected for by introduction of R751-pMG2, a plasmid incompatible with pRK290. The approach described here can be adapted for introducing genes into higher plant cells with the Ti plasmid as vector. It can likewise be used for site- or fragment-specific mutagenesis of the Ti plasmid as a means of detailed functional analysis.