Transfer DNA binary system

A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a binary plasmid and a helper plasmid. The two plasmids are used together (thus binary[1][2]) to produce genetically modified plants. They are artificial vectors that have both been created from the naturally occurring Ti plasmid found in Agrobacterium tumefaciens. The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts (E. coli and Agrobacterium tumefaciens).

Systems in which T-DNA and vir genes are located on separate replicons are called T-DNA binary systems. T-DNA is located on the binary vector (the non-T-DNA region of this vector containing origin(s) of replication that could function both in E. coli and in Agrobacterium tumefaciens, and antibiotic-resistance genes used to select for the presence of the binary vector in bacteria, became known as vector backbone sequences). The replicon containing the vir genes became known as the vir helper. Strains harboring this replicon and a T-DNA are considered disarmed if they do not contain oncogenes that could be transferred to a plant.

There are several binary vector systems that differ mainly in the plasmid region that facilitates replication in Agrobacterium. Commonly used binary vectors include:

Common Helper plasmids include

Plasmid structure

Binary plasmid

The T-DNA portion of the binary plasmid is flanked by left and right border sequences and consists of a transgene as well as a plant selectable marker (PSM). Outside of the T-DNA, the binary plasmid also contains a bacterial selectable marker (BSM) and an origin of replication (ori) for bacteria.[3]

Helper plasmid

The helper plasmid contains the vir genes that originated from the Ti plasmid of Agrobacterium. These genes code for a series of proteins that cut the binary plasmid at the left and right border sequences, and facilitate transduction of the T-DNA to the host plant's cells. The helper plasmid also contains a BSM and an ori for bacteria.[4]

Development of binary vector

The pBIN19 plasmid was developed in the 1980s and is one of the first and most widely used binary vector plasmids. The pGREEN plasmid, which was developed in 2000, is a newer version of the binary vector that allows for a choice of promoters, selectable markers and reporter genes. Another distinguishing feature of the pGREEN plasmid is its large reduction in size (from about 11,7kbp to 4,6kbp) from pBIN19, therefore increasing its transformation efficiency [5]

Along with higher transformation efficiency, pGREEN has been engineered to ensure transformation integrity. Both pBIN19 and pGREEN usually use the same selectable marker nptII, but pBIN19 has the selectable marker next to the right border, while pGREEN has it close to the left border. Due to a polarity difference in the left and right borders, the right border of the T-DNA enters the host plant first. If the selectable maker is near the right border (as is the case with pBIN19) and the transformation process is interrupted, the resulting plant may have expression of a selectable marker but contain no T-DNA giving a false positive. The pGREEN plasmid has the selecable marker entering the host last (due to its location next to the left border) so any expression of the marker will result in full transgene integration.[6]

Notes

  1. ↑ Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J., & Schilperoort, R. A. (1983). A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid.
  2. ↑ "As I remember, the “binary” refers to the function of interest being divided into two parts encoded by two separate plasmids rather than two bacterial hosts: we used the term “shuttle vectors” to refer to the multiple host property." (P. R. Hirsch, personal communication to T. Toal, Feb 27, 2013)
  3. ↑ Slater, Adrian; Scott, Nigel; Fowler, Mark (2008). Plant Biotechnology the genetic manipulation of plants. Oxford University Press Inc., New York.
  4. ↑ Slater, Adrian; Scott, Nigel; Fowler, Mark (2008). Plant Biotechnology the genetic manipulation of plants. Oxford University Press Inc., New York.
  5. ↑ http://www.pgreen.ac.uk/
  6. ↑ Slater, Adrian; Scott, Nigel; Fowler, Mark (2008). Plant Biotechnology the genetic manipulation of plants. Oxford University Press Inc., New York.

References

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