Site-specific insertion of genes into T-DNA of the Agrobacterium tumor-inducing plasmid: an approach to genetic engineering of higher plant cells.
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This paper presents a method for insertion of genetic material into a specific site in T-DNA, the portion of Agrobacterium tumor-inducing (Ti) plasmid that becomes incorporated into the nuclear DNA of transformed plant cells when crown gall tumors are incited by this plant pathogen. The three stages of our procedure are as follows: 1. A T-DNA subfragment cloned in pBR322 is cleaved by a restriction endonuclease at a unique central site and target DNA (a kanamycin resistance marker) is ligated into this site. 2. The resulting recombinant plasmid is purified and cleaved with EcoRI; the resulting fragment bearing the kanamycin resistance marker is ligated into the unique EcoRI site of pRK290, a wide-host-range plasmid. 3. The pRK290 recombinant plasmid is transformed into an agrobacterium tumefaciens strain containing a wild type Ti plasmid. Double recombination between the altered T-DNA fragment of the clone and its wild-type counterpart in the Ti plasmid is selected for by introduction of R751-pMG2, a plasmid incompatible with pRK290. The approach described here can be adapted for introducing genes into higher plant cells with the Ti plasmid as vector. It can likewise be used for site- or fragment-specific mutagenesis of the Ti plasmid as a means of detailed functional analysis.Keywords:
T-DNA Binary system
Ti plasmid
Transfer DNA
In vitro recombination
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INTRODUCTIONArabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. A. tumefaciens is a soil-dwelling bacterium that transforms normal plant cells into tumor-forming cells by inserting a piece of bacterial DNA (the transfer, or "T," DNA) into the plant cell genome. The T-DNA, which is flanked by left- and right-border (LB and RB) sequences, resides on a tumor-inducing (Ti) plasmid. The Ti plasmid also carries many of the transfer functions for mobilizing the T-DNA. This article provides a brief discussion of the principles of T-DNA transformation, including consideration of T-DNA vectors and their hosts.
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Journal Article Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants Get access R. Deblaere, R. Deblaere Laboratorium voor Genetische Virologie, Vrije Universiteit BrusselB-1640 St.-Genesius-Rode, Belgium Search for other works by this author on: Oxford Academic PubMed Google Scholar B. Bytebier, B. Bytebier Laboratorium voor Genetische Virologie, Vrije Universiteit BrusselB-1640 St.-Genesius-Rode, Belgium Search for other works by this author on: Oxford Academic PubMed Google Scholar H. De Greve, H. De Greve Laboratorium voor Genetische Virologie, Vrije Universiteit BrusselB-1640 St.-Genesius-Rode, Belgium Search for other works by this author on: Oxford Academic PubMed Google Scholar F. Deboeck, F. Deboeck Laboratorium voor Genetische Virologie, Vrije Universiteit BrusselB-1640 St.-Genesius-Rode, Belgium Search for other works by this author on: Oxford Academic PubMed Google Scholar J. Schell, J. Schell Laboratorium voor Genetische Virologie, Vrije Universiteit BrusselB-1640 St.-Genesius-Rode, Belgium Search for other works by this author on: Oxford Academic PubMed Google Scholar M. Van Montagu, M. Van Montagu Laboratorium voor Genetische Virologie, Vrije Universiteit BrusselB-1640 St.-Genesius-Rode, Belgium Search for other works by this author on: Oxford Academic PubMed Google Scholar J. Leemans J. Leemans Laboratorium voor Genetische Virologie, Vrije Universiteit BrusselB-1640 St.-Genesius-Rode, Belgium Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 13, Issue 13, 11 July 1985, Pages 4777–4788, https://doi.org/10.1093/nar/13.13.4777 Published: 11 July 1985 Article history Received: 28 March 1985 Revision received: 14 June 1985 Accepted: 14 June 1985 Published: 11 July 1985
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A tumor-inducing (Ti) plasmid from a strain of Agrobacterium tumefaciens that induces tumors on only a limited range of plants was characterized and compared with the Ti plasmids from strains that induce tumors on a wide range of plants. Whereas all wide-host-range Ti plasmids characterized to date contain closely linked oncogenic loci within a single transferred DNA (T-DNA) region, homology to these loci is divided into two widely separated T-DNA regions on the limited-host-range plasmid. These two plasmid regions, TA-DNA and TB-DNA, are separated by approximately 25 kilobases of DNA which is not maintained in the tumor. The TA-DNA region resembles a deleted form of the wide-host-range TL-DNA and contains a region homologous to the cytokinin biosynthetic gene. However, a region homologous to the two auxin biosynthetic loci of the wide-host-range plasmid mapped within the TB-DNA region. These latter genes play an important role in tumor formation because mutations in these loci result in a loss of virulence on Nicotiana plants. Furthermore, the TB-DNA region alone conferred tumorigenicity onto strains with an intact set of vir genes. Our results suggest that factors within both the T-DNA and the vir regions contribute to the expression of host range in Agrobacterium species. There was a tremendous variation among plants in susceptibility to tumor formation by various A. tumefaciens strains. This variation occurred not only among different plant species, but also among different varieties of plants within the same genus.
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This paper presents a method for insertion of genetic material into a specific site in T-DNA, the portion of Agrobacterium tumor-inducing (Ti) plasmid that becomes incorporated into the nuclear DNA of transformed plant cells when crown gall tumors are incited by this plant pathogen. The three stages of our procedure are as follows: 1. A T-DNA subfragment cloned in pBR322 is cleaved by a restriction endonuclease at a unique central site and target DNA (a kanamycin resistance marker) is ligated into this site. 2. The resulting recombinant plasmid is purified and cleaved with EcoRI; the resulting fragment bearing the kanamycin resistance marker is ligated into the unique EcoRI site of pRK290, a wide-host-range plasmid. 3. The pRK290 recombinant plasmid is transformed into an agrobacterium tumefaciens strain containing a wild type Ti plasmid. Double recombination between the altered T-DNA fragment of the clone and its wild-type counterpart in the Ti plasmid is selected for by introduction of R751-pMG2, a plasmid incompatible with pRK290. The approach described here can be adapted for introducing genes into higher plant cells with the Ti plasmid as vector. It can likewise be used for site- or fragment-specific mutagenesis of the Ti plasmid as a means of detailed functional analysis.
T-DNA Binary system
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Transfer DNA
In vitro recombination
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Transformation of Agrobacterium tumefaciens by the freezethaw method is modified. The frequency of transformation is improved, and a modified protocol of transformaing Agrobacterium tumefaciens with plasmid DNA is introduced. Plasmid vectors pBISP1, pBIP1, pBIAP1 and pBISgp120 are transformed into Agrobacterium tumefaciens by this modified protocol.
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Contents: Vectors for the transformation of plant cells using Agrobacterium Transformation of dicotyledenous plant cells using the Ti plasmid of Agrobacterium tumefaciens and the Ri plasmid of A. rhizogenes Transformation of plant cells by DNA-mediated gene transfer The isolation of plant nucleic acids DNA restriction and analysis by Southern hybridization Analysis of gene organization and expression in plants.
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