A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a T-DNA binary vector and a vir helper plasmid.[1][2] The two plasmids are used together (thus binary[2][3]) to produce genetically modified plants. They are artificial vectors that have been derived from the naturally occurring Ti plasmid found in bacterial species of the genus Agrobacterium, such as A. tumefaciens. The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts (e.g. Escherichia coli and Agrobacterium).

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 Agrobacterium, 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 plasmid. The vir helper plasmid is considered disarmed if it does not contain oncogenes that could be transferred to a plant.

Binary system components

T-DNA binary vector

There are several binary vectors that replicate in Agrobacterium and can be used for delivery of T-DNA from Agrobacterium into plant cells. The T-DNA portion of the binary vector is flanked by left and right border sequences and may include a transgene as well as a plant selectable marker. Outside of the T-DNA, the binary vector also contains a bacterial selectable marker and an origin of replication (ori) for bacteria.[4]

Representative series of binary vectors are listed below.

Main series of T-DNA binary vectors
Series Vector Year GenBank accession Size (bp) Autonomous replication in Agrobacterium Reference
pBIN pBIN19 1984 U09365 11777 Yes [5]
pPVP pPZP200 1994 U10460 6741 Yes [6]
pCB pCB301 1999 AF139061 3574 Yes [7]
pCAMBIA pCAMBIA-1300 2000 AF234296 8958 Yes [8]
pGreen pGreen0000 2000 AJ007829 3228 No [9]
pLSU pLSU-1 2012 HQ608521 4566 Yes [10]
pLX pLX-B2 2017 KY825137 3287 Yes [11]

Vir helper plasmid

The vir 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 vector at the left and right border sequences, and facilitate transfer and integration of T-DNA to the plant's cells and genomes, respectively.[4]

Several vir helper plasmids have been reported,[12] and common Agrobacterium strains that include vir helper plasmids are:

  • EHA101
  • EHA105
  • AGL-1
  • LBA4404
  • GV2260

Development of T-DNA binary vectors

The pBIN19 vector was developed in the 1980s and is one of the first and most widely used binary vectors. The pGreen vector, 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 pGreen is its large reduction in size (from about 11,7kbp to 4,6kbp) from pBIN19, therefore increasing its transformation efficiency.[13]

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 vector has the selectable 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.[4]

The pGreen-based vectors are not autonomous and they will not replicate in Agrobacterium if pSoup is not present. Series of small binary vectors that autonomously replicate in E. coli and Agrobacterium include:

References

  1. ↑ Lee LY, Gelvin SB (February 2008). "T-DNA binary vectors and systems". Plant Physiology. 146 (2): 325–32. doi:10.1104/pp.107.113001. PMC 2245830. PMID 18250230.
  2. 1 2 Hoekema A, Hirsch PR, Hooykaas PJ, Schilperoort RA (May 1983). "A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid". Nature. 303 (5913): 179–180. Bibcode:1983Natur.303..179H. doi:10.1038/303179a0. S2CID 4343344.
  3. ↑ "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)
  4. 1 2 3 Slater A, Scott N, Fowler M (2008). Plant Biotechnology the genetic manipulation of plants. New York: Oxford University Press Inc.
  5. ↑ Bevan M (November 1984). "Binary Agrobacterium vectors for plant transformation". Nucleic Acids Research. 12 (22): 8711–21. doi:10.1093/nar/12.22.8711. PMC 320409. PMID 6095209.
  6. ↑ Hajdukiewicz P, Svab Z, Maliga P (September 1994). "The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation". Plant Molecular Biology. 25 (6): 989–94. doi:10.1007/BF00014672. PMID 7919218. S2CID 9877624.
  7. 1 2 Xiang C, Han P, Lutziger I, Wang K, Oliver DJ (July 1999). "A mini binary vector series for plant transformation". Plant Molecular Biology. 40 (4): 711–7. doi:10.1023/a:1006201910593. PMID 10480394.
  8. ↑ "List of legacy pCAMBIA vectors – Cambia". Retrieved 2020-08-10.
  9. ↑ Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (April 2000). "pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation". Plant Molecular Biology. 42 (6): 819–32. doi:10.1023/a:1006496308160. PMID 10890530.
  10. 1 2 Lee S, Su G, Lasserre E, Aghazadeh MA, Murai N (May 2012). "Small high-yielding binary Ti vectors pLSU with co-directional replicons for Agrobacterium tumefaciens-mediated transformation of higher plants". Plant Science. 187: 49–58. doi:10.1016/j.plantsci.2012.01.012. PMID 22404832.
  11. 1 2 Pasin F, Bedoya LC, Bernabé-Orts JM, Gallo A, Simón-Mateo C, Orzaez D, García JA (October 2017). "Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins". ACS Synthetic Biology. 6 (10): 1962–1968. doi:10.1021/acssynbio.6b00354. PMID 28657330.
  12. ↑ Hellens R, Mullineaux P, Klee H (October 2000). "Technical Focus:a guide to Agrobacterium binary Ti vectors". Trends in Plant Science. 5 (10): 446–51. doi:10.1016/s1360-1385(00)01740-4. PMID 11044722.
  13. ↑ "pGreen on the Web". www.pgreen.ac.uk.
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