Agrobacterium Binary Vectors: Recent Refinements

Two major components, namely, the T-DNA and the vir region are involved for successful Agrobacterium-mediated gene transfer:  The Ti plasmid vectors are called binary because the regions involved in transformation are  resident on two separate plasmids. It is the T-DNA region which carries the alien gene(s) of interest; and the vir region helps the transfer of the foreign gene(s).
In an article entitled, “A guide to Agrobacterium binary Ti Vectors”, published in the October issue of Trends in Plant Science 2000 Oct;5(10):446-5., Roger Hellens roger.hellens@bbsrc.ac.uk , and Philip Mullineaux at John Innes Centre, UK,  and Harry Klee at the University of Florida review the recent refinements in the field of binary Ti vectors. They provide a detailed account how the newly constructed vectors are more user- friendly and suitable for transformation work covering a wide range of crops including cereals.
One popular Ti-vectors is pGREEN which can be used in any Agrobacterium strain such as C58 with a chromosomal background* favoring transformation.
In addition, there are hypervirulent Agrobacterium strains which are capable of mediating the transformation of cereals. These hypervirulent strains contain a number of wild-type and disarmed Ti plasmids** capable of transforming cereals. Thus improvement in the frequency of T-DNA transfer, particularly to the cereals, has been achieved. Another improvement is the development of a method in which  segments of DNA larger than >50kb can be transferred. In this method, an additional plasmid compatible with the binary Ti vectors is used which supplies additional vir functions.
In order not to compromise the efficiency of transformation protocols tailored to suit particular species or genotypes, the authors suggest that new plasmids specific to the requirement of the experiment can be now readilty constructed because of the increased flexibility of modern binary vectors.
The authors point out that the low copy number of broad host range plasmids in E. coli may be a limiting factor for efficient manipulation of DNA. This problem has been solved recently by enhancing  plasmid yields in E. coli by incorporating the colE1 (colicin E1) ori  from pBR322., a plasmid of E. coli origin.

Another recent improvement in the Ti vector has been its reduced sizes. This has been brought about because certain vectors (pPCV001, pMON10098, pGREEN), in which all loci except that representing  the replication origin (OriV), have been transferred either to a separate plasmid or to the Agrobacterium chromosome (such as RK2 replicase and trf genes). Furthermore, direct transfer of  the binary Ti vector from E. coli to Agrobacterium is possible and therefore conjugation functions can be dispensed with.

There has also been an improvement in methods for incorporating new or novel selectable marker genes and when they are available.

Improvement has also been effected in another direction, namely, designing synthetic T-DNA borders. The RB (right border) and the LB (left border) flanking the T-DNA varies little between Ti plasmids.  Placing of  selectable marker genes at the RB,  may sometimes create problems, if T-DNA transfer from the bacterium to the plant is interrupted. In this situation, no genes other than selectable markers are transferred, leaving behind the genes of interest in the bacterium itself. To obviate this difficulty, special Ti vectors with the selectable marker gene nearest to the LB have been constructed so that the passage of the two genes, marker and genes of interest, occur simultaneously.
The authors conclude, that in view of the current situation in which varieties of Ti vectors are available, it will benefit the researchers to shop around and determine which vectors would be most suitable.
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*Besides plasmids, in the cells of Agrobacterium tumefaciens, there are two chromosomes, a 3.0 Mbp circle and a 2.1 Mbp linear. The chromosome in C58 is referred to in this article.
**Disarmed Ti plamids – Agrobacterium strains which no longer contain tumor producing genes are called, ‘disarmed’.
+ori – Regions of DNA that are necessary for its replication to begin, such as pBR322 ori, required for plasmid replication.

 

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