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Brassica

Summary

The genus Brassica encompasses crops such as broccoli, brussel sprouts, cabbage and cauliflower. In addition turnips and choy sum, an Asian vegetable, belong to the Brassica genus. Finally mustards, canola or rapeseed and rutabaga are species of Brassica as well.

The patents granted to Calgene in the United States and in Europe are directed to transgenic Brassica cells containing expression cassettes inserted in their genome through co-cultivation with a disarmed Agrobacterium tumefaciens. Brassica explants, such as leaf and hypocotyl tissue, and protocols to achieve transformation are claimed as well as the component elements of the expression cassettes.

One the most limiting aspects of the claimed inventions is the sequences of the expression cassette or construct inserted into the genome of Brassica plants. In this regard the expression cassette must contain the following elements in 5′ to 3′ order:

  • a transcription initiation region;
  • an open reading frame (ORF) or a sequence complementary to an endogenous sequence of a plant;
  • a right T-DNA border; and
  • a structural gene.

AgrEvo (now Bayer Crop Science) has a granted United States patent that is directed to a method to produce a transgenic Brassica microspore using Agrobacterium . The microspore is treated first with a mucolytic enzyme to kill the bacteria and then develops into a haploid or doubled haploid embryo and ultimately a homozygous transgenic plant (Update July 2003).

The University of Helsinki has granted Australian and United States patents and a European application directed to the transformation of turnip rape (Brassica rapa ) with A. tumefaciens. The claims as granted in Australia and in the US and claims as filed in Europe are fairly narrow in scope; the methods comprise very detailed steps, e.g. type of tissue to be transformed and specific, pre-cultivation and co-cultivation conditions.

Brassica – Patents granted to Calgene

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5188958

  • Earliest priority – 29 May 1986
  • Filed – 14 August 1989
  • Granted – 23 February 1993
  • Expected expiry – 22 February 2010
 Title – Transformation and foreign gene expression in Brassica species

Claim 1
Transgenic Brassica species cells and progeny thereof comprising an expression cassette, wherein said cells are characterized as oncogene-free and capable of regeneration to morphologically normal whole plants, and wherein said expression cassette comprises, in the 5′ to 3′ direction of transcription:(i) a transcription initiation region functional in Brassica species cells;
(ii) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product which when expressed will alter the phenotype of said transgenic cells;
(iii) a transcription termination region functional in Brassica species cells;
(iv) a right border of T-DNA; and
(v) a structural gene capable of expression in said Brassica providing for selection of transgenic Brassica species cells;
wherein said expression cassette is capable of altering the phenotype of said Brassica species cells when said cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 8A transformed Brassica plant produced according to the method comprising:

A) co-cultivating Brassica cells with disarmed A. tumefaciens comprising a plasmid containing an insertion sequence resulting from joining in vitro of a transcription cassette to at least the right T-DNA border of a Ti or Ri plasmid whereby said Brassica cells are transformed with said insertion sequence which becomes integrated into the plant cell genome to provide transformed oncogene-free cells;
B) transferring said transformed oncogene-free cells to callus inducing medium, wherein said callus inducing medium contains at least one auxin and a means for selecting for transformed cells as a result of a marker carried on said plasmid whereby callus comprising transformed cells is produced;
C) transferring said callus to regeneration medium containing less than about 2% sucrose or an organic caloric equivalent thereto to produce shoots; and
D) transferring said shoots to a growing medium to produce plants capable of having an altered phenotype when grown under condition whereby a DNA sequence in said insertion sequence is expressed.

Transformation of Brassica with a disarmed A. tumefaciens having a cassette with a right T-DNA border from a Ti or a Ri-plasmid that is free of oncogenic genes. Induction of callus and shoot formation are key steps in the method to regenerate plants having an altered phenotype. The elements of the expression cassette are also recited in the claims.

 Calgene
US 5463174

  • Earliest priority – 29 May 1986
  • Filed – 11 September 1992
  • Granted – 31 October 1995
  • Expected expiry – 22 February 2010
 Title – Transformation and foreign gene expression in Brassica species

Claim 1Transgenic Brassica species cells and progeny thereof comprising an expression cassette, wherein said cells are characterized as oncogene-free and capable of regeneration to morphologically normal whole plants, and wherein said expression cassette comprises, in the 5′ to 3′ direction of transcription:
(i) a transcription initiation region functional in Brassica species cells;
(ii) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product; and
(iii) a transcription termination region functional in Brassica species cells;
wherein at least one of said transcription initiation region and transcription termination region is not naturally associated with said DNA sequence or said nucleic acid sequence; and
wherein said expression cassette imparts a detectable trait when said Brassica species cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 8A method for transforming Brassica species cells to produce morphologically normal whole Brassica plants capable of having an altered phenotype as a result of said transformation, said method comprising:

A) co-cultivating a Brassica leaf explant with disarmed A. tumefaciens comprising a plasmid containing an expression cassette joined to at least a right T-DNA border, whereby said expression cassette becomes integrated into the genome of cells in said Brassica explant to provide transformed oncogene-free cells;
B) transferring said explant to callus inducing medium comprising approximately 1 mg/l of one or more growth regulators selected from the group consisting of 2,4-D, kinetin and zeatin to allow callus to form on said explant;
C) transferring said callus to regeneration medium containing less than about 2% sucrose, or an organic caloric equivalent thereto, and comprising at least one milligram per liter of a cytokinin, and a means for selecting for transformed cells as a result of a marker carried on said plasmid, whereby shoots comprising transformed cells are produced from said callus; and
D) transferring said shoots to a growing medium to produce plants capable of having an altered phenotype when grown under conditions whereby a DNA sequence in said expression cassette is expressed.
Claim 11A transformed Brassica plant produced according to the method comprising:

A) co-cultivating a Brassica leaf or hypocotyl explant with disarmed A. tumefaciens comprising a plasmid containing an expression cassette joined to at least a right T-DNA border, whereby said expression cassette becomes integrated into the genome of cells in said Brassica explant to provide transformed oncogene-free cells, and wherein said expression cassette comprises a neomycin phosphotransferase II gene conferring kanamycin resistance;
B) transferring said explant to callus inducing medium comprising at least one auxin to allow callus to form on said explant;
C) transferring said callus to regeneration medium containing less than about 2% sucrose, or an organic caloric equivalent thereto, and comprising at least one milligram per liter of a cytokinin, and a means for selecting for transformed cells as a result of expression of said neomycin phosphotransferase II gene, whereby shoots comprising transformed cells are produced from said callus; and
D) transferring said shoots to a growing medium to produce plants.
Claim 19A cell culture of stably transformed Brassica species cells, wherein said cells are capable of regeneration into morphologically normal wholeBrassica plants capable of having an altered phenotype as a result of said transformation and regeneration, and wherein said cells are produced according to a method comprising:

A) co-cultivating a Brassica leaf or hypocotyl explant with disarmed A. tumefaciens comprising a plasmid containing an expression cassette joined to at least a right T-DNA border, whereby said expression cassette becomes integrated into the genome of cells in said Brassica tissue explant to provide transformed oncogene-free cells;
B) transferring said tissue explant to callus inducing medium to produce callus comprising stably transformed cells, wherein said callus inducing medium contains at least one auxin and a means for selecting for transformed cells as a result of a marker carried on said plasmid.
Claim 20Transgenic Brassica species cells and progeny thereof comprising an expression cassette, wherein said cells are characterized as oncogene-free and capable of regeneration to morphologically normal whole plants, and wherein said expression cassette comprises, in the 5′ to 3′ direction of transcription:
(i) a transcription initiation region functional in Brassica species cells;
(ii) a DNA sequence comprising a gene of interest encoding a protein product or a nucleic acid sequence complementary to an endogenous transcription product which when expressed will alter the phenotype of said transgenic cells; and
(iii) a transcription termination region functional in Brassica species cells;
wherein said expression cassette further comprises a structural gene capable of expression in said Brassica species cells and providing for selection of Brassica species cells comprising said structural gene, and a right border region of T-DNA capable of providing for integration of said expression cassette into the genome of said Brassica species cells; and
wherein said gene of interest or said nucleic acid sequence is expressed and imparts a detectable trait to said Brassica species cells.

Granted US 5463174 is a continuation of now granted US 5188958.

Method to transform Brassica leaf and hypocotyl explants with a disarmed A. tumefaciens having a cassette with a right T-DNA border. Media composition for callus and shoot regeneration are claimed. A claimed expression cassette contains npt II gene for conferring resistance to kanamycin; other elements of the cassette are also recited in the claims.
US 5750871

  • Earliest priority – 29 May 1986
  • Filed – 30 March 1995
  • Granted – 12 May 1998
  • Expected expiry – 22 February 2010
 Title – Transformation and foreign gene expression in Brassica species

Claim 1Transgenic Brassica species cells and progeny thereof comprising an expression cassette, wherein said cells are characterized as oncogene-free and capable of regeneration to morphologically normal whole plants, and wherein said expression cassette comprises, in the 5′ to 3′ direction of transcription:
(i) a transcription initiation region functional in Brassica species cells;
(ii) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product; and
(iii) a transcription termination region functional in Brassica species cells;
wherein at least one of said transcription initiation region and transcription termination region is not naturally associated with said DNA sequence or said nucleic acid sequence; and
wherein said expression cassette is integrated into the genome of said Brassica species cells and imparts a detectable trait when said Brassicaspecies cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 15A method for transforming Brassica species cells to produce morphologically normal whole Brassica plants having an altered phenotype as a result of said transformation, said method comprising:

A) co-cultivating a Brassica hypocotyl or leaf explant with disarmed A. tumefaciens comprising a plasmid containing an expression cassette joined to at least a right T-DNA border, whereby said expression cassette becomes integrated into the genome of cells in said Brassica explant to provide transformed oncogene-free cells;
B) transferring said explant to callus inducing medium to allow callus to form on said explant, wherein said callus inducing medium comprises about 1 mg/l of an auxin and from about 0 to 1 mg/l of a cytokinin;
C) transferring said callus to regeneration medium containing less than about 2% sucrose, or an organic caloric equivalent thereto, and comprising at least one milligram per liter of a cytokinin, and a means for selecting for transformed cells as a result of a marker carried on said plasmid, whereby shoots comprising transformed cells are produced from said callus; and
D) transferring said shoots to a growing medium to produce plants having an altered phenotype when grown under conditions whereby a DNA sequence in said expression cassette is expressed.

Granted US 5750871 is a continuation of now granted US 5463174.

The inventors claim elements of an expression cassette that is integrated into the genome of Brassica cells. Hypocotyl and leaf explants are used for transformation. Medium components for callus and shoot regeneration are also claimed.
EP 270615 B1

  • Earliest priority – 29 May 1986
  • Filed – 26 May 1987
  • Granted – 4 August 1993
  • Revoked (appeal of proprietor rejected) – 28 July 2000
 Title – Transformation and foreign gene expression in Brassica species

Claim 1Transgenic Brassica species cells having a DNA construct resulting from in vitro joining of at least two fragments, wherein said fragments comprise:
(i) a transcription initiation region functional in said Brassica;
(ii) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a sequence complementary to an endogenous transcription product;
(iii) a transcription termination region functional in said Brassica;
(iv) a right border of T-DNA; and
(v) a structural gene capable of expression in said Brassica providing for selection of transformed Brassica cells;
wherein said fragments provide an expression cassette capable of expression in said Brassica cells.
Claim 7A method for transforming Brassica cells to produce Brassica plants said method comprising:

A) co-cultivating Brassica cells with A. tumefaciens comprising a plasmid comprising an insertion sequence resulting from the in vitro joining of a transcription cassette to at least the right T-DNA border and a marker which provides for selection of cells containing said marker, whereby said Brassica cells are transformed with said insertion sequence which becomes integrated into the plant cell genome;
B) transferring said transformed Brassica cells to callus inducing media containing at least one auxin and selecting for cells comprising said marker to produce callus from said transformed cells;
C) transferring said callus to regeneration media containing less than about 2% sucrose or an organic caloric equivalent to produce shoots; and
D) transferring said shoots to a growing medium to produce plants.

Designated contracting States at the time of grant are: Austria, Belgium, Switzerland (patent lapsed as reported by INPADOC), Germany, France, United Kingdom, Italy, Liechtenstein, Luxembourg, Netherlands, Sweden (patent lapsed as reported by INPADOC)

Transgenic Brassica cells and progeny having an expression cassette that confers an altered phenotype. A method to transform those cells with a disarmed A. tumefaciens is also claimed. The method is the same as the one claimed in US 5188958.
CA 1341167 A1

  • Earliest priority – 29 May 1986
  • Filed – 28 May 1987
  • Granted – 16 January 2001
  • Expected expiry – 15 January 2018
 Title – Transformation and foreign gene expression in Brassica species

Claim 1
Transgenic Brassica species cells and cellular progeny thereof comprising an expression cassette, wherein said cells are characterized as oncogene-free and capable of regeneration to morphologically normal whole plants, and wherein said expression cassette comprises, in the 5′- 3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species cells;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product; and
(3) a transcription termination region functional in Brassica species cells; wherein at least one of said transcription initiation region and transcription termination region is not naturally associated with said DNA sequence or said nucleic acid sequence; and wherein said expression cassette imparts a detectable trait when said Brassica species cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 2
Transgenic Brassica species cells and cellular progeny thereof comprising an expression cassette, wherein said cells are characterized as oncogene-free and capable of regeneration to morphologically normal whole plants, and wherein said expression cassette comprises in the 5′ – 3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species cells;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product which when expressed will alter the phenotype of said transgenic cells;
(3) a transcription termination region functional in Brassica species cells; wherein said expression cassette further comprises a structural gene capable of expression in said Brassica species cells and providing for selection of transgenic Brassica species cells comprising said structural gene, and a right border region of T-DNA capable of providing for integration of said expression cassette into the genome of said Brassicaspecies cells; and wherein said expression cassette is capable of altering the phenotype of said Brassica species cells when said cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 6
A method for transforming Brassica species cells to produce morphologically normal whole Brassica plants capable of having an altered phenotype as a result of said transformation, said method comprising:

  • co-cultivating Brassica cells with A.tumefaciens comprising an oncogene-free plasmid containing an insertion sequence resulting from joining in vitro of a transcription cassette to at least the right T-DNA border of a Ti- or Ri-plasmid whereby said Brassica cells are transformed with said insertion sequence which becomes integrated into the plant cell genome to provide transformed oncogene-free cells;
  • transferring said transformed oncogene-free cells to callus inducing medium, wherein said callus inducing medium contains at least one auxin and a means for selecting for transformed cells as a result of a marker carried on said plasmid whereby callus comprising transformed cells is produced;
  • transferring said callus to regeneration medium containing less than about 2% sucrose or an organic caloric equivalent thereto to produce shoots; and
  • transferring said shoots to a growing medium to produce plants capable of having an altered phenotype when grown under conditions whereby a DNA sequence in said insertion sequence is expressed.
Claim 16
Transgenic Brassica species cells and cellular progeny thereof comprising an expression cassette, wherein said cells are derived from tissue explants and are characterized as oncogene-free, and wherein said expression cassette comprises, in the 5′- 3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species cells;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product; and
(3) a transcription termination region functional in Brassica species cells; wherein at least one of said transcription initiation region and transcription termination region is not naturally associated with said DNA sequence or said nucleic acid sequence; and wherein said expression cassette imparts a detectable trait when said Brassica species cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 17
Transgenic Brassica species cells and cellular progeny thereof comprising an expression cassette, wherein said cells are derived from tissue explants and are characterized as oncogene-free, and wherein said expression cassette comprises in the 5′ – 3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species cells;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product which when expressed will alter the phenotype of said transgenic cells;
(3) a transcription termination region functional in Brassica species cells; wherein said expression cassette further comprises a structural gene capable of expression in said Brassica species cells and providing for selection of transgenic Brassica species cells comprising said structural gene, and a right border region of T-DNA capable of providing for integration of said expression cassette into the genome of said Brassicaspecies cells; and wherein said expression cassette is capable of altering the phenotype of said Brassica species cells when said cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 18
A method for transforming Brassica species cells to produce morphologically normal whole Brassica plants capable of having an altered phenotype as a result of said transformation, said method comprising: transforming Brassica cells with a transcription cassette comprising, in the 5′-3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species cells;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product; and
(3) a transcription termination region functional in Brassica species cells; wherein at least one of said transcription initiation region and said transcription termination region is not naturally associated with said DNA sequence or said nucleic acid sequence; whereby said cassette becomes integrated into the plant cell genome to provide transformed cells which are not tumor cells; transferring said transformed cells to callus inducing medium, wherein said callus inducing medium contains at least one auxin and a means for selecting for transformed cells as a result of a marker carried on said plasmid whereby callus comprising transformed cells is produced; transferring said callus to regeneration medium; and transferring said shoots to a growing medium to produce plants capable of having an altered phenotype when grown under conditions whereby a DNA sequence in said insertion sequence is expressed.
CA 1341481 A1

  • Earliest priority – 29 May 1986
  • Filed – 28 May 1987
  • Granted – 26 April 2005
  • Expected expiry – 25 April 2025
 Title – Transformation and foreign gene expression in Brassica species

Claim 1
An expression or transcription cassette comprising in the 5′ – 3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species cells;
(2) a nucleic acid sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product which when expressed will alter the phenotype of Brassica species cells;
(3) a transcription termination region functional in Brassica species cells;
(4) a structural gene capable of expression in Brassica species cells and providing for selection of transgenic Brassica species cells comprising said structural gene; and
(5) a right border region of T-DNA capable of providing for integration of said expression or transcription cassette into the genome of Brassicaspecies cells; and wherein said expression or transcription cassette is capable of altering the phenotype of said transgenic Brassica species cells when said cells are grown under conditions whereby said nucleic acid sequence is expressed.
Claim 11
Transgenic Brassica species tissue consisting of a uniform cell type comprising an expression cassette, wherein said tissue is characterized as oncogene-free, and wherein said expression cassette comprises, in the 5′ – 3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species tissue;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product; and
(3) a transcription termination region functional in Brassica species tissue; wherein at least one of said transcription initiation region and transcription termination region is not naturally associated with said DNA sequence or said nucleic acid sequence; and wherein said expression cassette imparts a detectable trait when said Brassica species tissue is grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 12
Transgenic Brassica species tissue consisting of a uniform cell type comprising an expression cassette, wherein said tissue is characterized as oncogene-free, and wherein said expression cassette comprises in the 5′ – 3′ direction of transcription:
(1) a transcription initiation region functional in Brassica species tissue;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product which when expressed will alter the phenotype of said transgenic tissue; and
(3) a transcription termination region functional in Brassica species tissue; wherein said expression cassette further comprises a structural gene capable of expression in said Brassica species tissue and providing for selection of transgenic Brassica species tissue comprising said structural gene, and a right border region of T-DNA capable of providing for integration of said expression cassette into the genome of said Brassica species tissue; and wherein said expression cassette is capable of altering the phenotype of said Brassica species tissue when said tissue is grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 13
A transgenic Brassica species cell comprising an expression cassette, wherein said expression cassette comprises, in the 5′ – 3′ direction of transcription:
(1) a transcription initiation region functional in the Brassica species cell;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product; and
(3) a transcription termination region functional in the Brassica species cell; wherein at least one of said transcription initiation region and transcription termination region is not naturally associated with said DNA sequence or said nucleic acid sequence; and wherein said expression cassette imparts a detectable trait when said Brassica species cell is grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
Claim 14
A transgenic Brassica species cell comprising an expression cassette, wherein said expression cassette comprises in the 5′ – 3′ direction of transcription:
(1) a transcription initiation region functional in the Brassica species cell;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a nucleic acid sequence complementary to an endogenous transcription product which when expressed will alter the phenotype of said transgenic cell; and
(3) a transcription termination region functional in the Brassica species cell; wherein said expression cassette further comprises a structural gene capable of expression in said Brassica species cell and providing for selection of the transgenic Brassica species cell comprising said structural gene, and a right border region of T-DNA capable of providing for integration of said expression cassette into the genome of said Brassica species cell: and wherein said expression cassette is capable of altering the phenotype of said Brassica species cell when said cell is grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.
WO 1987/07299 A1

  • Earliest priority – 29 May 1986
  • Filed – 26 May 1987
  • OPI – 3 December 1987
 Title – Transformation and foreign gene expression in Brassica species

Claim 1
Transformed Brassica species cells having a DNA construct resulting from in vitro joining of at least two fragments, wherein said fragments comprise:(1) a transcription initiation region functional in said Brassica;
(2) a DNA sequence comprising an open reading frame having an initiation codon at its 5′ terminus or a sequence complementary to an endogenous transcription product;
(3) a transcription termination region func tional in said Brassica;
(4) a right border of T-DNA;
(5) a structural gene capable of expression in said Brassica providing for selection of transformed Brassica cells; wherein said fragments provide an expression cassette capable of expression in said Brassica cells.
Claim 7
A method for transforming Brassica cells to produce Brassica plants, said method comprising:

  • co-cultivating Brassica cells with A. tumefaciens comprising a plasmid comprising an insertion sequence resulting from the in vitrojoining of a transcription cassette to at least the right T-DNA border and a marker which provides for selection of cells containing said marker, whereby said Brassica cells are transformed with said insertion sequence which becomes integrated into the plant cell genome;
  • transferring said transformed Brassica cells to callus inducing media containing at least one auxin and selective for cells comprising said marker to produce callus from said transformed cells;
  • transferring said callus to regenerate media containing less than about 2% sucrose or organic caloric equivalent to produce shoots; and
  • transferring said shoots to a growing medium to produce plants.
Remarks
  1. Other national phase entries of WO 1987/07299 include: Finland (FI 880383; patent lapsed), Japan (JP H01/500718; appeal against rejection of application withdrawn by applicant).

Note: Patent information on this page was last updated on 13 March 2006.

Brassica – Patent Assigned to AgrEvo Canada Inc. (now Bayer Crop Science)

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 6316694

  • Earliest priority – 17 March 1995
  • Filed – 14 November 1997
  • Granted – 13 November 2001
  • Expected expiry – 14 March 2016
 Title – Transformed embryogenic microspores for the generation of fertile homozygous plants

Claim 1A method for producing a stably transformed Brassica embryogenic microspore, capable of leading to a non-chimeric transformed haploid or doubled haploid embryo which develops into a fertile homozygous Brassica plant within one generation, said process comprising the following steps:
a. infecting an embryogenic microspore with Agrobacteria, which contain a plasmid carrying a gene of interest under regulatory control of initiation and termination signals bordered by at least one T-DNA border, and
b. washing out and killing the Agrobacteria after co-cultivation using mucolytic enzymes, thereby producing a stably transformed Brassica embryogenic microspore.
Claim 2A method for producing a non-chimeric Brassica plant, containing a foreign DNA stably incorporated into its genome, said method comprising:
a. co-cultivating a Brassica embryogenic microspore with Agrobacteria which contains a plasmid carrying a gene of interest under regulatory control of initiation and termination signals;
b. washing out and killing the Agrobacteria after co-cultivation using mucolytic enzymes; and
c. regenerating a non-chimeric haploid or doubled haploid Brassica embryo from said microspore, wherein the embryo contains said gene of interest stably integrated into its genome, thereby producing a non-chimeric Brassica plant.

This patent discloses a method to generate transgenic plants from the Brassica family by using microspores as a target tissue forAgrobacterium carrying a gene of interest. After infection bacteria are eliminated by treatment with mucolytic enzymes. Microspores are further cultivated to produce haploid or doubled haploid embryos from which fully regenerated transgenic plants are obtained.

AgrEvo Canada Inc. (now Bayer Crop Science)
Remarks
  1. Claims of other granted national phase entries of the corresponding PCT application for US 6316694 (WO 1996/29419)  in Australia (AU 710201) and China (CN 1110562) are not limited to plants from the Brassica family.
  2. National phase entry of WO 1996/29419 in Canada (CA 2215763) is pending.
  3. Nationall phase entry of WO 1996/29419 n Europe (EP 737748) is deemed to be withdrawn on 6 September 2000.

Note: Patent information on this page was last updated on 13 March 2006.

Turnip rape (Brassica rapa) – Granted patents assigned to the University of Helsinki

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 6455761

  • Earliest priority – 18 September 1997
  • Filed – 10 March 2000
  • Granted – 24 September 2002
  • Expected expiry – 15 September 2018
 Title – Agrobacterium-mediated transformation of turnip rape

Claim 1
A method for transforming mature plants of turnip rape, comprising

(i) excising an internode section of the inflorescence-carrying stem of a mature turnip rape plant,
(ii) sterilizing said internode section, and cutting it in 4-8 mm segments to obtain an internode segment,
(iii) placing an internode segment in a horizontal position on an agar pre-cultivation medium supplemented with 15-90 μM of silver nitrate and 2,4-dichlorophenoxyacetic acid (2,4-D) hormone,
(iv) pre-cultivating the internode segment on said medium for 1 day,
(v) immersing the internode segment in a MS solution inoculated with Agrobacterium tumefaciens bacteria carrying at least one gene heterologous to said turnip rape,
(vi) placing the immersed internode segment in a horizontal position on an agar MS co-cultivation medium,
(vii) co-cultivating the internode segment with Agrobacteria for 2 days,
(viii) washing the internode segment to remove the Agrobacteria,
(ix) placing the internode segment in a vertical position with the basal side down on MS agar medium for selection with an antibiotic, the medium being supplemented with cytokinine hormones and silver nitrate, to obtain an internode segment with regenerated primordia or embryonic green nodules,
(x) placing the internode segment with regenerated primordia or embryogenic green nodules on a hormone-free regeneration medium, and
(xi) recovering the transgenic shoots regenerated.

The independent claim of United Stated patent US 6455761 discloses a quite detailed protocol of how to obtain transgenic turnip rape (Brassica rapa). This offers a number of opportunities to develop similar methods and avoid infringement with respect to this specific patent at the same time. Other patents have more general claims that cover the use of Agrobacterium to produce transgenic dicots for example.

University of Helsinki
AU 732372 B2

  • Earliest priority – 18 September 1997
  • Filed – 16 September 1998
  • Granted – 26 April 2001
  • Patent ceased – 22 April 2004
 Title – Agrobacterium-mediated transformation of turnip rape

Claim 1
A method for transformation of mature plants of turnip rape, comprising

– excising an internode section of the inflorescence carrying stem of a mature turnip rape plant,
– sterilizing said internode section, and cutting it in 4-8 mm segments,
– placing a segment in a horizontal position on an agar pre-cultivation medium supplemented with silver nitrate and 2, 4-dichlorophenoxyacetic acid (2, 4-D) hormone,
– pre-cultivating the segment on said medium for 1 day,
– immersing the segment in a MS solution inoculated with Agrobacterium tumefaciens bacteria carrying at least one gene heterologous to said turnip rape,
– placing the immersed segment in a horizontal position on an agar MS co-cultivation medium,
-co-cultivating the segment with Agrobacteria for 2 days,
– washing the segment from the Agrobacteria,
-placing the segment in a vertical position with the basal side down on MS agar medium for selection with an antibiotic, the medium being supplemented with cytokinine hormones and silver nitrate, and
-placing the segment with regenerated primordia or embryogenic green nodules on a hormone free regeneration medium, and
– recovering the transgenic shoots regenerated.
Remarks
  1. National phase entry of WO 1999/14349 in Canada (CA 2302835) is deemed dead on 16 September 2004.
  2. National phase entry of WO 1999/14349 in Europe (EP 1009845) is deemed to be withdrawn on 6 October 2004.
  3. Other national phase entry of WO 1999/14349 includes Finland (FI 973720).

Note: Patent information on this page was last updated on 13 March 2006.