Characterization and host range of five tumorigenic Agrobacterium tumefaciens strains and possible application in plant transient transformation assays

Agrobacterium tumefaciens is a Gram‐negative bacterium. It causes plants to produce crown gall disease because of the transfer, integration and expression of oncogenes encoded by the T‐DNA (transferred DNA) region of the tumour‐inducing (Ti) plasmid. A set of transferred genes directs the production of bacterial nutrients, called opines, formed by condensation of an amino acid and a keto acid or a sugar. Transformed cells synthesize and secrete substantial quantities of particular opines, which A. tumefaciens then uses as a carbon and sometimes nitrogen source. Agrobacterium tumefaciens strains are usually classified on the basis of the opines they can catabolize. Because of the ability to transfer DNA between different kingdoms, A. tumefaciens is also frequently used to generate transgenic plants. This study analyses five poorly characterized wildtype Agrobacterium strains, 1D1108, 1D1460, 1D132, 1D1478 and 1D1487, isolated from Euonymus, cane, cherry, Salix and apple, respectively. Partial Ti‐plasmid sequence analysis demonstrated that the five strains harbour the nopaline‐type Ti plasmid. Tumorigenesis and transient transformation assays of the five analysed and six wildtype A. tumefaciens strains were performed with selected plant species, including two or three species of Brassicaceae, Asteraceae, Solanaceae, Apiaceae and Leguminosae. The A. tumefaciens strains 1D1108, 1D1460 and 1D1478 showed higher transformation efficiencies than the previously characterized A. tumefaciens strains with several economically important crops. These data suggest the potential use of these newly characterized wildtype A. tumefaciens strains in transient transformation assays with certain plant species.     

PCR primers for identification of opine types of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in Japan

The polymerase chain reaction (PCR) is a rapid, precise method for detecting and identifying pathogenic bacteria. In addition to the published primers for identification of Agrobacterium tumefaciens up to species level, two sets of primers were designed to identify the nopaline and octopine types of Agrobacterium tumefaciens. The RBF-RBR primer set designed based on the nopaline type T-DNA right border detected the nopaline type A208 and R225f strains, and the ocsF-ocsR primer set derived from the ocs gene of the octopine type A. tumefaciens detected the octopine type A348 strain. After polymerase Chain reaction (PCR) amplification by the RBF-RBR primers, the A208 and R225f strains could be differentiated from each other by restriction fragment length polymorphism digestion using the restriction enzymes DraI and XbaI. Multiple colonies can be screened at one time in a single PCR tube with satisfactory efficiency, thereby allowing rapid detection of pathogenic A. tumefaciens. Following a rough screening by classical biovar medium and -methyl-d-glucoside medium, the developed PCR system was introduced to identify isolates collected from soil and crown gall samples. Of 42 isolates determined to be A. tumefaciens, 7 were found to be octopine type; all the rest were R225f type.