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Patent granted to Japan Tobacco Inc.

The United States patent granted to Japan Tobacco discloses a method commonly called co-transformation. In this method, two T-DNAs containing genes that encode different products (e.g. gene of interest and selectable marker) are inserted into the plant genome via Agrobacterium. The method results in transformed plants having the gene of interest and lacking the selectable marker gene.

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5731179

  • Earliest priority – 8 December 1993
  • Filed – 8 August 1995
  • Granted -24 March 1998
  • Expected expiry – 7 August 2015
 Title – Method for introducing two T-DNAs into plants and vectors therefor

Claim 1
A method for transforming and cultivating a plant using a bacterium belonging to the genus Agrobacterium, comprising:co-transforming plant cells with a first T-DNA (1) and a second T-DNA (2); and

selecting cells based on a selection marker gene;

said first T-DNA (1) containing a selection marker gene which functions in said plant;

said second T-DNA (2) containing a desired 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 the genus Agrobacterium; said acceptor vector containing at least

(a) a DNA region having a replication origin allowing replication of a plasmid in both a bacterium belonging to the genus Agrobacterium and in Escherichia coli,

(b) a DNA region containing virB gene and virG gene in virulence region of Ti plasmid pTiBo542 of Agrobacterium tumefaciens, and

(c) a DNA region which is homologous with a part of said intermediate vector, which is subjected to homologous recombination in said bacterium belonging to the genus Agrobacterium; said intermediate vector containing at least

(i) a DNA region having a replication origin allowing replication of a plasmid in Escherichia coli, which does not function in said bacterium belonging to the 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 the genus Agrobacterium, and

(iii) a DNA region which constitutes at least a part of said second T-DNA;

obtaining a plant transformed with said selection marker gene and said desired DNA fragment; and

cultivating said plant and selecting a plant in the next generation, which contains said desired DNA fragment but does not contain said selection marker gene.
Claim 16
A hybrid vector comprising:a first T-DNA containing

(1) a selection marker gene which functions in a plant, and

(2) a second T-DNA having a restriction site; wherein there is sufficient distance on said hybrid vector between said first T-DNA and said second T-DNA to allow said first T-DNA and said second T-DNA to be independently inherited, said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in a bacterium belonging to the genus Agrobacterium; said acceptor vector containing at least

(a) a DNA region having a replication origin allowing replication of a plasmid in both a bacterium belonging to the genus Agrobacterium and in Escherichia coli,

(b) a DNA region containing virB gene and virG gene in virulence region of Ti plasmid pTiBo542 of Agrobacterium tumefaciens, and

(c) a DNA region which is homologous with a part of said intermediate vector, which is subjected to homologous recombination in said bacterium belonging to the genus Agrobacterium; said intermediate vector containing at least

(i) a DNA region having a replication origin allowing replication of a plasmid in Escherichia coli, which does not function in said bacterium belonging to the 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 the genus Agrobacterium, and

(iii) a DNA region which constitutes at least a part of said second T-DNA.
Claim 24
A method for transforming and cultivating a plant using a bacterium belonging to the genus Agrobacterium, comprising

  • co-transforming plant cells with a first T-DNA (1) and a second T-DNA (2); and
  • selecting the cells which acquired drug resistance; said first T-DNA (1) containing a gene giving said drug resistance, which functions in said plant; said second T-DNA (2) containing a desired 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 the genus Agrobacterium; said acceptor vector being pSB3 or pSB4; and said intermediate vector being pSB21, pSB22, pSB24, pTOK170, pYS151, pTOK235, pTOK245 or pTOK246;
  • obtaining a plant transformed with said drug resistance gene and said desired DNA fragment; and
  • cultivating said plant and selecting a plant in the next generation, which contains said desired DNA fragment but does not contain said drug resistance gene.
Claim 25
A hybrid vector comprising:a first T-DNA containing
(1) a gene giving a drug resistance, which functions in a plant; and
(2) a second T-DNA having a restriction site; wherein there is sufficient distance on said hybrid vector between said first T-DNA and said second T-DNA to allow said first T-DNA and said second T-DNA to be independently inherited, said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in a bacterium belonging to the genus Agrobacterium; said acceptor vector being pSB3 or pSB4; and said intermediate vector being pSB21, pSB22, pSB24, pTOK170, pYS151, pTOK235, pTOK245 or pTOK246.

The patent US 5731179 claims

  • a method for transforming plants via Agrobacterium by inserting two different T-DNAs. The first T-DNA contains a plant selectable marker gene and the second T-DNA contains a “desired DNA fragment” (claim 1) and may comprise a restriction site. The second T-DNA is contained in a hybrid vector formed by homologous recombination between an acceptor vector and an intermediate vector;
  • a transformed plant with both of the T-DNAs and the subsequent selection of transformed plants in the next generation that contain the desired gene but not contain the selectable marker gene;
  • the elements of the hybrid vector, the acceptor vector, and the intermediate vector.

The independent claims don’t limit the size of the DNA fragment contained by the second T-DNA.

The claims to the hybrid vector state that the T-DNAs are sufficiently far apart to allow them to be independently inherited. The patent disclosure doesn’t describe what minimum distance would allow this but does provide the example, where the borders are separated by an origin of replication and sequences that contain the virB and virG gene (i.e., about 15 kb) and the T-DNAs are independently inherited.  The claims are a bit ambiguous because they don’t require independent segregation to occur at any particular frequency (e.g., 100% of the time? 50%? rarely?).

Some of the hybrid vector claims require specifically named plasmids disclosed in the specification.  Deposit information is not provided in the disclosure of the US patent.

Japan Tobacco Inc.
AU 733623 B2

  • Earliest priority – 8 December 1993
  • Filed – 14 October 1998
  • Granted – 17 May 2001
  • Expected expiry – 5 December 2014
 Title – Method for transforming plants and vector therefor

Claim 1

A method for transforming a plant through a bacterium  belong to genus Agrobacterium, comprising co-transfonming plant cells with a single hybrid vector comprising a first T-DNA (1) and second T-DNA (2); and selecting the cells which acquired drug resistance;

said first T-DNA (1) containing a gene giving said drug resistance, which functions in said plant;

said second T-DNA  (2) containing a desired 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 belong to genus Agrobacterium;

said acceptor vector containing at least

(a) a DNA region having a function to replicate a plasmid in said bacterium belonging to genus Agrobacterium and Escherichia coli,

(b) a DNA region containing virB gene and virG gene in virulence region of Ti plasmid pTiBo542 of Agrobacterium tumefaciens, and

(c) 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 genusAgrobacterium,

(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 13

A hybrid vector comprising a first T-DNA containing
(1) a gene giving a drug resistance, which functions in a plant, and
(2) a second T-DNA having a restriction site;
said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in a bacteria belonging to the genus Agrobacterium;

This granted patent is a divisional of now abandoned AU 11213/95 A1 (OPI of the national phase entry of WO 1995/16031).
AU 771116 B2

  • Earliest priority – 8 December 1993
  • Filed – 22 May 2001
  • Granted – 11 March 2004
  • Expected expiry – 5 December 2014
 Title – Agrobacterium mediated method of plant transformation

Claim 1
A method of Agrobacterium mediated transformation of a plant with a desired DNA fragment comprising:(i) co-transforming plant cells with a vector comprising a first T-DNA (1) that contains a gene that encodes drug resistance in a plant and a hybrid vector comprising a second T-DNA (2) containing a desired DNA fragment that is to be introduced into said plant, wherein said first T-DNA and said second T-DNA are not contained in the same vector; and
(ii) selecting those cells which acquire drug resistance;
and wherein said hybrid vector is prepared by homologous recombination between an acceptor vector and an intermediate vector in Agrobacterium, said acceptor vector containing at least the three DNA regions:
(a) a DNA region that is capable of conferring replication on a plasmid in Agrobacterium and Escherichia coli;
(b) a DNA region containing the virB gene and virG gene in virulence region of Ti plasmid pTiBo542 of Agrobacterium tumefaciens; and
(c) a DNA region that having homology to at least a part of said intermediate vector sufficient for homologous recombination to occur between said intermediated vector and said acceptor vector in Agrobacterium;
and said intermediate vector containing at least the three DNA regions:
(d) a DNA region that is capable of conferring replication on a plasmid in Escherichia coli but not in Agrobacterium;
(e) a DNA region having homology to at least a part of said acceptor vector sufficient for homologous recombination to occur between said intermediate vector and said acceptor vector in Agrobacterium; and
(f) a DNA region that constitutes at least a part of said second T-DNA.

This granted patent is a divisional of now granted AU 733623 B2.
Remarks
  1. National phase entry of WO 1995/16031 in Canada (CA 2155570) is pending.
  2. National phase entry of WO 1995/16031 in Europe (EP 687730) is pending (see next page).
  3. National phase entry of WO 1995/16031 in Japan (JP 3102888) has been granted.

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