Figure 6.

Systems for introducing T-DNA harboring transgene expression cassettes into the pgl/picA region of the A. tumefaciens chromosome. A, T-DNA containing a CaMV 35S promoter-bar gene plant selection marker and a fragment of pBluescript, and an aadA gene to the left of the T-DNA left border, has been inserted into the A. tumefaciens C58 chromosome. When a pUC-derived plasmid containing an expression cassette is introduced into this strain, carbenicillin-resistant colonies can only be obtained if homologous recombination occurs, resulting in a cointegrate between this plasmid and the pBluescript region of the T-DNA. B, In this system, a transgene expression cassette is first cloned into a T-DNA region located on a plasmid harboring a ColE1 ori. The plasmid also contains the pgl/picA region of the A. tumefaciens C58 chromosome. When this plasmid is introduced into A. tumefaciens C58-derived strains, carbenicillin-resistant colonies can only be obtained if homologous recombination occurs between the pgl/picA regions of the plasmid and the chromosome. LB, T-DNA left border; RB, T-DNA right border; ori, origin of replication; amp^r, β -lactamase gene conferring ampicillin/carbenicillin resistance upon the bacterium; aadA, spectinomycin resistance gene; 2X35S, double CaMV 35S promoter; bar, gene conferring resistance to Basta/Bialophos/phosphinothricin; P_ocs, octopine synthase promoter; T_ocs, octopine synthase terminator; RCS, multiple rare cutting sites flanking various pSAT vectors (AscI, I-PpoI, I-SceI, I-CeuI, PI-PspI, and PI-TilI). Broken crossed lines indicate homologous recombination events.