pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities

A new plasmid series has been created for Agrobacterium-mediated plant tran sformation; The pBECKS2000 series of binary vectors exploits the Cre/loxP s ite-specific recombinase system to facilitate the construction of complex T -DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore ineffic ient, ligation reactions. The vectors incorporate facilities for the remova l of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A "shuttle" or intermed iate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration rea ction which is mediated by the Cre/loxP site-specific recombinase system. T his reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly usi ng the antibiotic resistance marker carried on the intermediate plasmid. Th is strategy facilitates production of co-integrative T-DNA binary vector fo rms which are appropriate for either(1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) tra nsfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from t he plant genome; or (3) co-transfer of target and marker genes as two indep endent T-DNAs within a single-strain Agrobacterium system, providing the po tential for segregational loss of marker genes.