The discovery that the vir genes do not need to be in the same plasmid with a T-DNA region to lead its transfer and insertion into the plant genome led to the construction of a system for plant transformation where the T-DNA region and the vir region are on separate plasmids.
In the binary vector system, the two different plasmids employed are:
- a wide-host-range small replicon, which has an origin of replication (ori) that permits the maintenance of the plasmid in a wide range of bacteria including E. coli and Agrobacterium. This plasmid typically contains:
- foreign DNA in place of T-DNA,
- the left and right T-DNA borders (or at least the right T-border),
- markers for selection and maintenance in both E. coli and A. tumefaciens,
- a selectable marker for plants.
- The plasmid is said to be “disarmed”, since its tumor-inducing genes located in the T-DNA have been removed.
- a helper Ti plasmid, harbored in A. tumefaciens, which lacks the entire T-DNA region but contains an intact vir region.
In general, the transformation procedure is as follows:
- the recombinant small replicon is transferred via bacterial conjugation or direct transfer to
A. tumefaciens harboring a helper Ti plasmid,
- the plant cells are co-cultivated with the Agrobacterium, to allow transfer of recombinant T-DNA into the plant genome, and
- transformed plant cells are selected under appropriate conditions.
A possible disadvantage may ensue from the fact that the stability of wide host range replicons in E. coli and Agrobacterium varies considerably. Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli where both origins are active.
Compared with co-integrated vectors, binary vectors present some advantages:
- No recombination process takes place between the molecules involved.
- Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium.
This vector system is most widely used nowadays. Different types of binary vectors have been devised to suit different needs in a plant transformation process.
Binary vector types
- pGA series vectors, which contain:
- an ori derived from RK2 for replication in E. coli and Agrobacterium,
- a tetracycline resistance gene,
- the cis-acting factor required for conjugal transfer,
- the right (RB) and left (LB) T-DNA borders,
- a neomycin phosphotransferase (nptII) gene, which confers resistance to kanamycin and G418 in transformed plants, and
- a polylinker site (multicloning site).
Specific vectors in this series are designed for cloning large fragments (colE1 origin of replication and phage l cos), analyzing promoters (multiple cloning site immediately upstream of a promoterless cat gene), and expressing a gene of interest (polylinker site between a plant promoter and a terminator).
- pCG series vectors, which contain:
- the origin of replication of the Agrobacterium rhizogenes root -inducing plasmid pRiHRI, which confers more stability in Agrobacterium than the ori derived from RK2, and a ColE1 origin of replication from the vector pBR322 for maintenance in E. coli.
- pCIT series which contain:
- the hygromycin (hph) resistance gene for plants,
- the lambda cos site for cloning long fragments.
- pGPTV (glucuronidase plant transformation vector) series , which have:
- different plant selectable marker genes close to the left T-DNA border. This design overcomes problems inherent with the preferential right to left border transfer of T-DNA and improves the chances of having the gene of interest transferred to the plant cell in cells expressing the selectable marker gene.
- pBECK2000 series, which contain:
- synthetic T-DNA borders and a bar gene, which confers the plants resistance to the herbicide phosphinothricin. Also, the vectors use the phage P1 Cre/loxP site-specific recombinase system, which permits the transfer and integration of a target and marker genes as a single T-DNA unit into the plant genome or as two independent T-DNAs within a single Agrobacterium. It also allows site-specific excision of marker genes from the plant genome after transformation.
- Binary-BAC (BiBAC) vector
- based on a bacterial artificial chromosome (BAC) vector and is suitable for Agrobacterium-mediated transformation of high-molecular-weight DNA
- comprises low-copy number origins of replication for both E. coli and Agrobacterium to ensure replication of the plasmid as a single-copy in both bacteria; and
- a helper plasmid carrying additional copies of vir-genes in order to clone very large T-DNAs (up to 150 kb) into the plant genome.
- pGreen series, small plasmids of around 3.2 Kb containing:
- a broad host range replication origin (ori pSa) and a ColE1 origin derived from pUC,
- a pSa replicase gene (rep A) that provides replication functions in trans and is located in a compatible plasmid (pSoup) in Agrobacterium, and
- multiple cloning sites based on the pBlueScript vector, which allow any arrangement of selectable marker and reporter genes.