Modification of T-DNA of nopaline Ti plasmid by intermediary vector and utilization of agrocin 84 sensitivity as simple criterion of conjugation transfer of modified Ti plasmid.

The chloramphenicol resistance gene from pSa was introduced into T-DNA of pTi T37 of Agrobacterium tumefaciens by cointegration with intermediary plasmid based on pBR322. The resulting intermediary vector was mobilized to A. tumefaciens T37 by conjugative plasmid pRK2. The RK2 plasmid also forms contegrates with pTi due to the Tn3 transposon which was used for the mobilization of modified pTi into plasmid-less A. tumefaciens strain. Transconjugants were selected on the basis of their antibiotic resistance markers and tested for agrocin sensitivity as proof of Ti plasmid transfer. Agrocin sensitivity of tranconjugants together with chloramphenicol resistance was shown to be a sufficient and simple criterion of transfer of modified Ti plasmids. Agrobacterium strains with modified Ti plasmids showed decreased virulence in consequence of the presence of additional borderline sequence inside their T-DNA.