Patent application filed by Japan Tobacco Inc.

The European application filed by Japan Tobacco discloses methods for co-transforming plants with two T-DNAs. One of the T-DNAs has a gene conferring drug-resistance and the other has a gene of interest and is part of a hybrid plasmid. The hybrid plasmid is constructed by homologous recombination of an acceptor and an intermediate plasmid.

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
EP 687730 A1

  • Earliest priority – 8 December 1993
  • Filed – 6 December 1994
  • Application pending
Title – Method of transforming plant and vector therefor

Claim 1
A method for transforming a plant through a bacterium belonging to the genus Agrobacterium, comprising:A) co-transforming plant cells with a first T-DNA (1) and a second T-DNA (2); and
B) selecting the cells which acquired drug resistance; said first T-DNA (1) containing a gene giving said drug resistance, which function in said plant; said second T-DNA (2) containing a desire DNA fragment to be introduced into said plant, the second T-DNA (2) being contained in a hybrid vector;
said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in said bacterium belonging to genus Agrobacterium;
said acceptor vector containing at least:
(i) a DNA region having a function to replicate a plasmid in said bacterium belonging to the genus Agrobacterium and Escherichia coli,
(ii) a DNA region containing vir B gene and vir G gene in virulence region of Ti plasmid pTiBo542 of Agrobacterium tumefaciens, and
(iii) a DNA region which is homologous with a part of said intermediate vector, which is subjected to homologous recombination in said bacterium belonging to genus Agrobacterium;
said intermediate vector containing at least:
(i) a DNA region having a function to replicate a plasmid in Escherichia coli, which does not function in said bacterium belonging to genus Agrobacterium,
(ii) a DNA region which is homologous with a part of said acceptor vector, which is subjected to homologous recombination in said bacterium belonging to genus Agrobacterium, and
(iii) a DNA region which constitutes at least a part of said second T-DNA.
Claim 16
A hybrid vector comprising a first T-DNA containing: 1) a gene giving a drug resistance, which functions in plant, and2) a second T-DNA giving a restriction site; said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in a bacterium belonging to genus Agrobacterium;
said acceptor vector containing at least:
(i) a DNA region having a function to replicate a plasmid in said bacterium belonging to the genus Agrobacterium and Escherichia coli,
(ii) a DNA region containing vir B gene and vir G gene in virulence region of Ti plasmid pTiBo542 of Agrobacterium tumefaciens, and
(iii) a DNA region which is homologous with a part of said intermediate vector, which is subjected to homologous recombination in said bacterium belonging to genus Agrobacterium;
said intermediate vector containing at least:
(i) a DNA region having a function to replicate a plasmid in Escherichia coli, which does not function in said bacterium belonging to genus Agrobacterium,
(ii) a DNA region which is homologous with a part of said acceptor vector, which is subjected to homologous recombination in said bacterium belonging to genus Agrobacterium, and
(iii) a DNA region which constitutes at least a part of said second T-DNA.

The claims of the application EP 687 730 A1 recite the transformation of a plant with two different T-DNAs via Agrobacterium. The first T-DNA contains a gene conferring drug resistance to the plant and the second one contains a DNA fragment of interest. In the method claim (claim 1), the second T-DNA is part of a hybrid vector formed by homologous recombination between an acceptor vector and an intermediate vector.

A hybrid vector (claim 16) harboring the first T-DNA as described and a second T-DNA with a cloning site is also formed by homologous recombination of the vectors mentioned above.

Elements of the acceptor vector and the intermediate vector are also recited in the claims.

Japan Tobacco Inc.

Note: Patent information on this page was last updated on 2 March 2006.