Selection-expression plasmid vectors for use in genetic transformation of higher plants

Abstract

Plasmid vectors containing both a selectable marker for plant transformation (kanamycin resistance) and a second, directly adjacent, divergent promoter for the transcription of inserted DNA fragments have been constructed. These vectors make use of a small (479 bp) dual-promoter DNA fragment, originally isolated from the T-DNA of Agrobacterium tumefaciens, fused to the neomycin phosphotransferase gene of Tn5. Several unique restriction enzyme cleavage sites, as well as a polyadenylation signal sequence, have been introduced downstream of the open promoter, allowing simple insertional cloning of DNA fragments to be expressed in plants. To test the vectors, the coding region for the chloramphenicol acetyltransferase gene (CAT) from Tn9 was inserted, and the resulting plasmids introduced into tobacco cells. Transformed calli, selected only for Km resistance, contained, in every case tested, both NPTII and CAT activities.