Abstract
This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).
Highlights
Agrobacterium tumefaciens is a Gram-negative soil bacterium and plant pathogen causing crown gall disease in angiosperms and gymnosperms [1]
A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery
Developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp)
Summary
Agrobacterium tumefaciens is a Gram-negative soil bacterium and plant pathogen causing crown gall disease in angiosperms and gymnosperms [1]. A smaller pRK2 derivative 10.3 kbp pRK252 was the backbone of a first binary vectors pBin (11.8 kbp) with addition of Streptoccocus faecalis NPTIII gene for bacterial kanamycin resistance, nopaline pTiT37 T-DNA borders, a plant selection marker nos:NPTII:nos, the αcomplementary region of β-galactosidase (lacZ’ locus) for selection, and polylinker site from M13mp19 [16]. PAGS127 (15.0 kbp) has a pRK252 backbone with the T-DNA region consisting of octopine pTiA6 right border and octopine pTiAch left border, a plant selection marker nos:NPTII:ocs, the lac Z’ locus and polylinker, and cos site for a large DNA fragment insertion [19]. A binary vector pOCA18 (24.3 kbp) has the pRK290 backbone and the T-DNA region of octopine pTi right and left borders, a plant selection marker nos:NPTII:ocs, and λcos site for insertion of DNA library of Arabidopsis thaliana [21]. The T-DNA region consists of nopaline pTiT37 right and left borders, nos:BAR:nos plant selection marker gene (Bar, herbicide glufosinate) and pBlueScriptII polylinker for cloning
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