A binary vector plasmid for gene expression in plant cells that is stably maintained in Agrobacterium cells.

Abstract

We constructed a new binary plasmid vector designated pSKl which consists of the replication origin of Ri plasmid from Agrobacterium rhizogenes and the leftand right-border repeats of nopaline type Ti plasmid pTiT37. This plasmid was 23-times stabler than a pBI-derivative plasmid pIG121-Hm which contains the RK2 replication origin in agrobacterium cells. The pSKl carries Xba I and Not I recognition sites between the 35S RNA promoter of cauliflower mosaic virus (P35S) and the poly-adenylation signal of the nopaline synthase gene (Tnos) so that genes of interest can be introduced in the correct orientation and expressed in plant cells. The Agrobacterium harboring a Ti plasmid is a natural machinery for gene transfer from the bacteria to plant cells (for reviews, see ref. 1-3). Using this bacteria, a system with an agrobacterium binary vector has been developed for transformation of both dicotyledonous and monocotyledonous plants. Such a system in general consists of two types of plasmids which must be compatible with each other in agrobacterium cells: one is a binary vector plasmid that can be maintained in both Agrobacterium and Escherichia coli cells and carries a border repeat-flanked T-DNA segment that contains cloning sites for a gene to be transferred, and the other is a virulence plasmid that carries the virulence (vir) region of the Ti plasmid but no T-DNA. As for binary vector plasmids, BIN19, a series of pBI plasmids, and pGA492 (482) were originally made from plasmid RK2 and vectors based on other plasmids such as pSa have also been reported.