On the isolation of TI-plasmid from Agrobacterium tumefaciens

An efficient lysis method for Agrobacterium cells was developed, which allows a reproducible isolation of the tumor inducing (TI)-plasmid. The lysis method is based on the sensitivity of this bacterium to incubation with lysozyme, n-dodecylamine,EDTA, followed by Sarkosyl, after growth in the presence of carbenicillin. We also present a procedure for the isolation of the TI-plasmid on a large scale, that might be used for the mass isolation of other large plasmids which like the TI-plasmid, can not be cleared with earlier described procedures. The purity of the plasmid preparations was determined with DNA renaturation kinetics, which method has the advantage that the plasmid need not to be in the supercoiled or open circular form

Supercoiled circular DNA in crown-gall inducing Agrobacterium strains

By alkaline sucrose gradient, neutral sucrose gradient and dye-buoyant density centrifugation a large plasmid was shown to be present in the crown-gall inducing Agrobacterium tumefaciens strain B6-83. Measurements of contour lengths carried out on electron micrographs resulted in a mean length of 54.1 µm, corresponding to a molecular weight of 112 × 10 6. Only one or a few copies of this plasmid are present per bacterial chromosome. Mitomycin C induction has no influence on the amount of plasmid DNA in the cell. At present this plasmid must be considered as cryptic, for no genetic markers on it are known. Furthermore, large plasmids were isolated from crown-gall inducing strains belonging to seven Agrobaeterium groups described by Kersters et al. (1973). Contour length measurements carried out on the plasmids isolated from the various crown-gall inducing strains fell in the range from 54.1 µm to 75.4 µm, depending on the strain examined. We were not able to find such plasmids in eight non-pathogenic strains belonging to four of the same groups. The hypothesis is formulated that the large plasmid present in crown-gall inducing bacteria could be the "tumor-inducing principle"

Transfer of Ti plasmids between Agrobacterium strains by mobilisation with the conjugative plasmid RP4

The P type conjugative plasmid RP4 has been shown to be able to promote the transfer of the Agrobacterium Ti-plasmid. The results provide additional evidence that agrocin 84 sensitivity, exclusion of phage AP1, ability to catabolize the guanidine derivatives octopine and nopaline and tumor inducing ability, are Ti-plasmid determined properties. Furthermore, the results strongly support the notion that at least part of the Ti-plasmid is transferred from the bacterium to the target plant cells, since it was demonstrated that Ti-plasmid linked genes specify the synthesis of octopine or nopaline by crown-gall tumor cells

The use of the Ti plasmid as a vector for the introduction of foreign DNA into plants

Agrobacterium tumefaciens is a Gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called "opines", e.g. "octopine" (N²-(D-1-carboxyethyl)-L-arginine) and "nopaline" (N²-(1,3-dicarboxypropyl)-L-arginine), is transferred and stably maintained and expressed in the transformed plant cells by bacterial plasmid DNA so that the transformed plant cell will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources. By in vivo genetic manipulations, we have recently been able to introduce a bacterial antibiotic resistance transposon, Tn7, in the Ti-DNA segment that is transferable to plant cells. In this way we hope to demonstrate that the Ti-plasmid can be used as a general vector for introducing "foreign" DNA into plants