Brassica - Patents granted to Calgene

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.

A 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.

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; 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.

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:

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.

A 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.

A cell culture of stably transformed Brassica species cells, wherein said cells are capable of regeneration into morphologically normal whole Brassica 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.

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 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.

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; 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 Brassica species cells are grown under conditions whereby said DNA sequence or said nucleic acid sequence is expressed.

A 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.

Transgenic 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.

A 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.

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 Brassica species 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 Brassica species 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.

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 Brassica species 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.

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.

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 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;
• 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.