A method for transforming a monocotyledon callus, comprising contacting a cultured tissue of a monocotyledon during dedifferentiation wherein said dedifferentiation is obtained by culturing an explant on a dedifferentiation-inducing medium for not less than 7 days or a dedifferentiated cultured tissue of a monocotyledon, with a bacterium belonging to the genus Agrobacterium containing a desired gene.

A method for transforming a monocotyledon with a desired gene, comprising: contacting a cultured tissue of said monocotyledon during dedifferentiation thereof, or a dedifferentiated cultured tissue of said monocotyledon, with a suspension of Agrobacterium tumefaciens having a cell population of 10⁶ to 10¹¹ cells/ml for 3-10 minutes and then culturing said cultured tissue of said monocotyledon during dedifferentiation thereof, or said dedifferentiated cultured tissue of said monocotyledon, on a solid medium for several days together with said Agrobacterium tumefaciens, or adding said Agrobacterium tumefaciens to culture medium in which said cultured tissue of said monocotyledon during dedifferentiation thereof or said dedifferentiated cultured tissue of said monocotyledon is cultured, and continuously culturing said cultured tissue of said monocotyledon during dedifferentiation or said dedifferentiated cultured tissue of said monocotyledon together with said Agrobacterium tumefaciens, wherein said dedifferentiated cultured tissue of said monocotyledon is selected from the group consisting of a tissue cultured during the process of callus formation which is cultured for not less than 7 days after an explant is placed on a dedifferentiation-inducing medium and a callus, and wherein said Agrobacterium tumefaciens contains plasmid pTOK162, and said desired gene is present between border sequences of the T region of said plasmid pTOK162, or wherein said desired gene is present in another plasmid contained in said Agrobacterium tumefaciens.

A method for transforming a monocotyledon comprising transforming a cultured tissue during dedifferentiation process or a dedifferentiated cultured tissue of said monocotyledon with a bacterium belonging to genus Agrobacterium containing a desired gene.

A method for transforming a monocotyledon comprising transforming a cultured tissue during dedifferentiation process or a dedifferentiated cultured tissue of said monocotyledon with a bacterium belonging to genus Agrobacterium containing a desired gene.

A method for transforming a monocotyledon, comprising contacting a cultured tissue of said monocotyledon during dedifferentiation thereof obtained by culturing an explant on a dedifferentiation-inducing medium for less than 7 days with a bacterium belonging to the genus Agrobacterium containing a super binary vector having the virulence region of Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281, left and right border sequences of T-DNA of a Ti plasmid or an Ri plasmid of a bacterium belonging to the genus Agrobacterium, and a desired gene located between said left and right border sequences.

A method for transforming a monocotyledon, comprising contacting a cultured tissue of said monocotyledon during dedifferentiation thereof obtained by culturing an explant derived from an immature tissue on a dedifferentiation-inducing medium for less than 7 days with a bacterium belonging to the genus Agrobacterium containing a desired gene and containing a vector having the virulence region of Ti plasmid contained in Agrobacterium tumefaciens.

A method for transforming monocotyledons comprising transforming scutellum of an immature embryo of a monocotyledon with a bacterium belonging to genus Agrobacterium containing a desired gene, which immature embryo has not been subjected to a dedifferentiation treatment, to obtain a transformant.

A method for transforming monocotyledons comprising transforming scutellum of an immature embryo of a monocotyledon with a bacterium belonging to genus Agrobacterium containing a desired gene, which immature embryo has not been subjected to a dedifferentiation treatment, to obtain a transformant.

A method for producing a transgenic plant comprising:

a. obtaining pollen from a first plant,
b. applying a lawn of Acrrobacteria to a solid pollen culture medium, the Agrobacteria comprising at least one heterologous gene sequence capable of being transferred to a plant cell,
c. applying the pollen to the solid medium,
d. allowing the pollen to germinate and grow on the medium, thereby producing transgenic pollen,
e. applying the transgenic pollen to the stigma of a second plant capable of being fertilized by the pollen of the first plant, thereby fertilizing the second plant,
f. obtaining transgenic seed from the second plant,
g. germinating the transgenic seed to obtain a transgenic plant.

A medium for pollen germination and pollen tube growth comprising agarose, sucrose, KNO₃, MnSO₄, H₃BO₃, MgSO₄, and gibberellic acid.

A method for producing a transgenic plant comprising:

a. obtaining pollen from a first plant,
b. applying a lawn of Agrobacteria to a solid pollen culture medium, the Agrobacteria comprising at least one heterologous gene sequence capable of being transferred to a plant cell,
c. applying the pollen to the solid medium,
d. allowing the pollen to germinate and grow on the medium, thereby producing transgenic pollen,
e. applying the transgenic pollen to the stigma of a second plant capable of being fertilized by the pollen of the first plant, thereby fertilizing the second plant,
f. obtaining transgenic seed from the second plant,
g. germinating the transgenic seed to obtain a transgenic plant.

A method for producing a transgenic plant comprising:

a. obtaining pollen from a first plant,

b. applying a lawn of Agrobacteria to a solid pollen culture medium, the Agrobacteria comprising at least one heterologous gene sequence capable of being transferred to a plant cell,

c. applying the pollen to the solid medium,

d. allowing the pollen to germinate and grow on the medium, thereby producing transgenic pollen,

e. applying the transgenic pollen to the stigma of a second plant capable of being fertilized by the pollen of the first plant, thereby fertilizing the second plant,

f. obtaining transgenic seed from the second plant,

g. germinating the transgenic seed to obtain a transgenic plant.

A method for producing a transgenic plant comprising:

a. obtaining pollen from a first plant,
b. applying a lawn of Acrrobacteria [sic] to a solid pollen culture medium, the Agrobacteria comprising at least one heterologous gene sequence capable of being transferred to a plant cell,
c. applying the pollen to the solid medium,
d. allowing the pollen to germinate and grow on the medium, thereby producing transgenic pollen,
e. applying the transgenic pollen to the stigma of a second plant capable of being fertilized by the pollen of the first plant, thereby fertilizing the second plant,
f. obtaining transgenic seed from the second plant,
g. germinating the transgenic seed to obtain a transgenic plant.

A medium for pollen germination and pollen tube growth comprising agarose, sucrose, KNO3, MnSO4, H3BO3, MgSO4, and gibberellic acid.

A method of transforming excised shoot apical tissue comprising:

a) excising shoot apical tissue consisting essential of the apical dome and two or more primordial leaves,

b) placing said excised tissue in a suitable growth medium,

c) inoculating said apical tissue with Agrobacterium tumefaciens to transform said tissue.

A method for transforming an excised shoot apex comprising:

a) excising a shoot apex,
b) placing said apex in a suitable growth medium,
c) inoculating said apical tissue with Agrobacterium tumefaciens to transform said tissue.

A method for producing a transformed plant comprising, injecting Agrobacterium cells harboring a vector, comprising a nucleic acid molecule capable of conferring a desired phenotypic trait to a plant, into a plant floral or meristematic tissue using a needleless injection device, which can be adapted for the injection of small volumes of material in a precise manner without causing massive tissue damage.

A method for producing a transgenic seed comprising injecting a Agrobacterium cells harboring a vector, comprising a gene capable of conferring a desired phenotypic trait, into the floral tissues of a plant before the division of the egg cell using a needleless-hypodermic injection device.

A method of producing a transgenic seed comprising injecting a recombinant Agrobacterium into the foloral tissues of a plant using a needleless-hypodermic injection device.

A method for producing a transgenic seed comprising injecting a Agrobacterium cells harboring a vector, comprising a gene capable of conferring a desired phenotypic trait, into the floral tissues of a plant before the division of the egg cell using a needleless-hypodermic injection device.

A method for producing a transformed plant including injecting a transforming agent comprising Agrobacterium cells harboring a vector having a nucleic acid molecule capable of conferring a desired phenotypic trait to a plant, into a plant floral or meristematic tissue using a needleless injection device which can be adapted for the injection of small volumes of material in a precise manner without causing massive tissue damage.

A method for producing a transgenic seed comprising injecting a recombinant Agrobacterium into the floral tissues of a plant using a needleless injection device which can be adapted for the injection of small volumes of material in a precise manner without causing massive tissue damage.

A method for producing a transgenic seed comprising injecting Agrobacterium cells harboring a vector, comprising a gene capable of conferring a desired phenotypic trait, into the floral tissues of a plant before the division of the egg cell using a needleless injection device.

An in planta germline transformation method including needleless injection of a transforming agent comprising an Agrobacterium vector, said injection being directly into a tissue including the floral or meristematic germline cells of said plant, said injection being selected as to pressure, volume and trajectory so as to limit penetration of said transforming agent to a region of said tissue whereby said germline cells are transformed without causing excessive tissue damage.

A method for producing a transformed plant or a transgenic seed substantially as herein described in the detailed description of the invention with reference to the drawings.

A method for producing a transformed plant comprising, injecting a transforming agent into a plant tissue using a needless injection device.

A method for producing a transgenic seed comprising injecting a recombinant Agrobacterium into the floral tissues of a plant using a needleless-hypodermic injection device.

A method for producing a transgenic seed comprising injecting a DNA molecule comprising a gene capable of conferring a desired phenotypic trait into the floral tissues of a plant using a needleless-hypodermic injection device.

A transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural plant environment, followed by generation of a transgenic plant via dedifferentiation and regeneration of the target tissue, wherein said inoculation is performed by injection of Agrobacterium into said tissue, with minimal damages to said tissue.

A transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural plant environment, followed by generation of dedifferentiated tissue from the target tissue, wherein said inoculation is performed by injection of Agrobacterium into said tissue, with minimal damages to said tissue.

Use of Agrobacterium in a transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural plant environment, followed by generation of transgenic plant material via dedifferentiation of the target tissue, wherein said inoculation is performed by injection of Agrobacterium into said tissue, with minimal damages to said tissue.

A transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural plant environment, followed by generation of a transgenic plant via dedifferentiation and regeneration of the target tissue, after isolation and culture of said target tissue, wherein said inolucation is performed by injection of Agrobacterium into said tissue, with minimal damage to said tissue.

A transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural plant environment, followed by generation of dedifferentiated tissue from the target tissue after isolation and culture of said target tissue, wherein said inoculation is performed by injection of Agrobacterium into said tissue, with minimal damage to said tissue.

Use of Agrobacterium in a transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural plant environment, followed by generation of transgenic plant material via dedifferentiation of the target tissue, after isolation and culture of said target tissue, wherein said inoculation is performed by injection of Agrobacterium into said tissue, with minimal damage to said tissue.

A transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural plant environment, followed by generation of a transgenic plant via dedifferentiation and regeneration of the target tissue.

A transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural environment, followed by generation of dedifferentiated tissue from the target tissue.

Use of Agrobacterium in a transformation method comprising inoculation and co-cultivation of a target tissue, from a target plant, with Agrobacterium, at a time when the target tissue is in its natural environment, followed by generation of transgenic plant material via dedifferentiation of the target tissue.

A method for preparing a transgenic plant or seed comprising:

a) growing a suspension of Agrobacterium cells until the optical density of the suspension is about 2 to about 2.4 at a wavelength of 600 nanometers, wherein said Agrobacterium cells contain at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said Agrobacterium cells of said suspension with an aqueous medium so that the optical density of the suspension is reduced to about 0.6 to about 1.5, wherein said diluting is not followed by pelleting via centrifugation with subsequent resuspension; and
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium cells in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.

A method for preparing a transgenic plant or seed comprising:

a) growing a suspension of Agrobacterium cells until growth of Agrobacterium cells in the suspension is substantially completed, wherein said Agrobacterium cells contain at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said suspension with an aqueous medium to reduce the concentration of Agrobacterium cells and any other components in the growth medium and allow the Agrobacterium cells to infect the plant without harming it, wherein said diluting is not followed by pelleting via centrifugation with subsequent resuspension;
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium cells in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.

A method for preparing a transgenic plant or seed comprising:

a) growing a suspension of Agrobacterium cells until growth of Agrobacterium cells in the suspension is substantially completed, wherein said Agrobacterium cells contains at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said Agrobacterium cells in said suspension with about 2 to about 10 volumes aqueous medium per volume of suspension, wherein said diluting is not followed by pelleting via centrifugation with subsequent resuspension;
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium cells in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.

A method for transforming plants, comprising the steps of:

1) selecting half a seed, which is germinated for one day as a target plant tissue;
2) wounding the target plant tissue using a bundle of needles;
3) inserting a target gene into the target plant tissue with Agrobacterium tumefaciens vector containing the target gene; and
4) regenerating a whole plant from the target plant tissue.

A plant transformation method mediated by Agrobacterium comprising:

(a) providing germinating seeds; and

(b) co-culturing the germinating seeds with an Agrobacterium strain containing Ti plasmids with an inserted nucleic acid sequence so that the foreign nucleic acid sequence carried by the Agrobacterium strain is transferred and integrated into the genome of the germinating seeds through Ti plasmids.

A plant transformation method mediated by Agrobacterium, characterized in that germinating seeds were used as the receptors, an Agrobacterium strain containing Ti plasmids with an inserted nucleic acid sequence was used as the donor, the germinating seeds are co-cultured with the Agrobacterium strain so that the foreign nucleic acid sequence carried by the Agrobacterium strain can be transferred and integrated into the genome of the receptor through Ti plasmids.

A process for the incorporation of foreign DNA into chromosomes of dicotyledonous plants, comprising infecting the plants or incubating plant protoplasts with Agrobacterium bacteria, which contain plasmids, said Agrobacterium bacteria containing at least one plasmid having the vir-region of a Ti plasmid but no T-region, and at least one other plasmid having a T-region with incorporated therein foreign DNA but no vir-region.

A process for the production of Agrobacterium bacteria which contains at least one plasmid which has the vir-region of a Ti-plasmid but no T-region and at least one other plasmid which has a T-region with foreign DNA incorporated in it but no vir-region, comprising:

(a) using Escherichia coli as a host and incorporating foreign DNA into a plasmid therein which contains a T-region and a replicator having a broad bacterial host range
(b) introducing the resulting plasmid into Agrobacterium bacteria which contain at least one plasmid which has the vir-region of a Ti-plasmid but no T-region.

A process for incorporating into the genome of dicotyledonous plants foreign DNA, comprising infecting the plants or plant cells or incubating plant protoplasts with Agrobacterium strains, which contain plasmids, said Agrobacterium strains containing at least one plasmid having the vir-region of a Ti plasmid but no T-region, and at least one other plasmid having a T-region but no vir-region, said T-region being composed of naturally occurring border sequences consisting of about 23 base pairs at the extremities of said T-region and only foreign DNA between said border sequences, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.

Agrobacterium strains, comprising at least one plasmid having the vir-region of a Ti plasmid but no T-region, and at least one other plasmid having a T-region but no vir-region, said T-region being composed of naturally occurring border sequences consisting of about 23 base pairs at the extremities of said T-region and only foreign DNA between said sequences, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.

A process for the production of Agrobacterium strains comprising at least one plasmid having the vir-region of a Ti plasmid but no T-region, and at least one other plasmid having a T-region but no vir-region, said T-region being composed of naturally occurring border sequences consisting of about 23 base pairs at the extremities of said T-region and only foreign DNA between said border sequences, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation, said process comprising:

(a) incorporating non-Agrobacterium foreign DNA into a plasmid having a T-region and a replicator having a broad bacterial host range, wherein said foreign DNA becomes part of the T-region,

(b) cloning the resulting plasmid in Escherichia coli; and

(c) introducing the resulting plasmid into Agrobacterium strains which contain at least one plasmid which has the vir-region of a Ti-plasmid but no T-region.

A process for incorporating in the genome of dicotyledonous plants foreign DNA, comprising infecting the plants or plant cells or incubating plant protoplasts with Agrobacterium strains, which contain plasmids, said Agrobacterium strains containing at least one plasmid having the vir-region of a Ti-plasmid but no T-region, and at least one other plasmid having a single T-region but no vir-region, said single T-region being composed of naturally occurring border sequences at the extremities of said single T-region, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.

A process for the incorporation of foreign DNA into chromosomes of dicotyledonous plants comprising infecting the plants or incubating plant protoplasts with Agrobacterium bacteria, which contain plasmids, said Agrobacterium bacteria containing at least one plasmid having the vir-region of a Ti-plasmid but no T-region, and at least one other plasmid having an artificial T-region with only foreign DNA between the 23 base pairs at the extremeties of the wild type T-region, but no vir-region, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.

Agrobacterium bacteria, comprising at least one plasmid having the vir-region of a Ti-plasmid but no T- region, and at least one other plasmid having an artificial T-region with only foreign DNA between the 23 base pairs at the extremeties of the wild type T-region, but no vir-region, the vir-region plasmid and the T-region plasmid containing no homology which could lead to cointegrate formation.

Claim 1

Agrobacteria comprising a T-region and a vir-region in their DNA, the T-region being stably integrated into the bacterial chromosome.

Claim 2

Agrobacteria comprising a T-region and a vir-region in their DNA, the vir-region being stably integrated into the bacterial chromosome.

Claim 3

A process for the transfer of recombinant DNA into the cells or protoplasts of plants selected from the group consisting of dicotyledonous plants and monocotyledonous plants of the families Liliaceae and Amaryllidaceae, said process comprising infecting the plants or plant cells, or incubating protoplasts from the plants, with Agrobacterium bacteria which contain in their genetic material a vir-region from the Ti plasmid of Agrobacterium and at least one T-region, wherein said T-region comprises said recombinant DNA flanked on both sides by border sequences as present in wild-type of Agrobacterium, and wherein said vir-region is integrated into the chromosome of said Agrobacterium bacteria prior to said infecting or incubating step.

Claim 5

A process for the transfer of recombinant DNA into the cells or protoplasts of plants selected from the group consisting of dicotyledonous plants and monocotyledonous plants of the families Liliaceae and Amaryllidaceae, said process comprising infecting the plants or plant cells, or incubating protoplasts from the plants, with Agrobacterium bacteria which contain in their genetic material a vir-region from the Ti plasmid of Agrobacterium and at least one T-region, wherein said T-region comprises said recombinant DNA flanked on both sides by border sequences as present in wild-type T-region of Agrobacterium, and wherein each of said T-region is integrated into the chromosome of said Agrobacterium bacteria prior to said infecting or incubating step.

Claim 1

A process for the incorporation of foreign DNA into the genome of plants by infecting the plants or by incubating plant protoplasts with Agrobacterium bacteria, characterized in that Agrobacterium bacteria are used, which contain in their chromosome at least one T-region or a vir-region or both, on the understanding that in the first two cases the bacteria contain a plasmid comprising a vir-region or a T-region resp.

Claim 4

Agrobacterium bacteria containing in their chromosomes at least one T-region or a vir-region or both.

A process for introducing a defined DNA sequence in a selected target locus of a nuclear plant cell genome by integrating at least a part of a recombinant DNA into said genome through homologous recombination at the target locus, comprising the steps of:

a) coincubating plant protoplasts or plant cells under transforming conditions, with a strain of the genus Agrobacterium capable of T-region transfer and containing a plant transformation vector which comprises recombinant DNA of the formula:

wherein boxes 1 and 7 are T-DNA border sequences, and wherein one of box 1 or 7 may be absent; and wherein box 3 comprises a DNA sequence sufficiently homologous to a DNA sequence inside the target locus and sufficiently long to promote homologous recombination; and wherein box 4 comprises a DNA sequence not homologous to sequences occurring in the target locus; and wherein the lines connecting the boxes shown may comprise any number of nucleotides or basepairs, and
b) identifying protoplasts or cells that have obtained the said defined DNA sequence in their genomes at said target locus.

A process for introducing a defined mutation in a selected target locus of a nuclear plant cell genome by integrating at least a part of a recombinant DNA into said genome through homologous recombination at the target locus, comprising the steps of:

a) coincubating plant protoplasts or plant cells under transforming conditions, with a strain of the genus Agrobacterium capable of T-region transfer and containing a plant transformation vector which comprises recombinant DNA of the formula:

wherein boxes 1 and 7 are T-DNA border sequences, and wherein one of box 1 or 7 may be absent; and wherein box 3 comprises a DNA sequence sufficiently homologous to a DNA sequence inside the target locus and sufficiently long to promote homologous recombination; and wherein box 4 comprises a DNA sequence sufficiently homologous to promote homologous recombination with a corresponding sequence in the target locus and wherein box (4) comprises the said defined mutation; and wherein the lines connecting the boxes shown may comprise any number of nucleotides or basepairs, and
b) identifying protoplasts or cells that have obtained the said defined mutation in their genome at the said target locus.

A recombinant DNA capable of introducing a defined mutation in a selected target locus of a nuclear plant cell genome through homologous recombination at the target locus, which recombinant DNA has the formula:

wherein box i comprises the said defined mutation; and wherein boxes 1 and 7 are T-DNA border sequences, and wherein one of box 1 or 7 may be absent; and wherein each of boxes 3 and 4 individually is a DNA sequence sufficiently homologous to a DNA sequence inside the target locus and sufficiently long to promote homologous recombination; and wherein the DNA sequences of the boxes 3 and 4 have the same 5'-to-3' orientation, but wherein said boxes 3 and 4 are rearranged with respect to the homologous sequences in the target locus.

Claim 1

A method for introducing a defined mutation in a selected target locus of a nuclear plant genome by integrating at least a part of a recombinant DNA into said genome through homologous recombination at the target locus, which recombinant DNA has the following general structure,

in which the boxes 1 and 7 and the connecting lines represent DNA sequences that are capable of functioning as a T-DNA border in the DNA transfer process, in which box 1 or 7 may be absent but not both, in which box 3 comprises a DNA sequence which is sufficiently homologous to a DNA sequence inside the target locus and sufficiently long to promote homologous recombination, in which box 4 represents a DNA sequence that is not homologous to sequences occurring in the target locus, and in which the lines connecting the boxes may represent any number of basepairs, or no basepairs, and identifying transformants having the defined mutation in said target locus using known methods.

Claim 8

A recombinant DNA capable of integrating a part of itself into the genome of a plant host via homologous recombination at the target locus,

in which the boxes 1 and 7 represent DNA sequences that are capable of functioning as a T-DNA border in the DNA transfer process, in which box 1 or 7 may be absent but not both, in which both box 3 and 4 represent DNA sequences which are sufficiently homologous and sufficiently long to promote homologous recombination with the target locus, in which the DNA sequences within the boxes have the same 5' to 3' orientation as in the target locus, but the order of the boxes themselves have been changed with respect to the situation in the target locus, resulting in insertion of box i in the target locus after homologous recombination, in which the lines connecting the boxes may represent any number of basepairs, or no basepairs.

A vector for transferring heterologous DNA into a plant cell, said vector comprising:

• a backbone which includes a first origin of replication for maintaining heterologous DNA as a single copy in an Escherichia coli host cell, and which further includes a second origin of replication for maintaining heterologous DNA as a single copy in an Agrobacterium tumefaciens host cell;

• a unique restriction endonuclease cleavage site for insertion of heterologous DNA; and

• left and right Agrobacterium T-DNA border sequences flanking said unique restriction endonuclease cleavage site, said left and right T-DNA border sequences allowing introduction of heterologous DNA located between said left and right T-DNA border sequences into a plant cell.

A method of making a transgenic plant transformed with a vector comprising:

(1) providing a vector comprising: a backbone which includes a first origin of replication capable of maintaining heterologous DNA as a single copy in an Escherichia coli host cell, and which further includes a second origin of replication capable of maintaining heterologous DNA as a single copy in an Agrobacterium tumefaciens host cell, a unique restriction endonuclease cleavage site for insertion of heterologous DNA, a heterologous DNA encoding a gene product inserted into the unique restriction site, and left and right Agrobacterium T-DNA border sequences flanking said unique restriction endonuclease cleavage site, said left and right T-DNA border sequences allowing introduction of heterologous DNA located between said left and right T-DNA border sequences into a plant cell;
(2) introducing said vector into plant cells; and
(3) propagating plants from said plant cells.

A method of introducing heterologous DNA into a plant cell comprising:

(1) providing a vector comprising: a backbone which includes a first origin of replication capable of maintaining heterologous DNA as a single copy in an Escherichia coli host cell, and which further includes a second origin of replication capable of maintaining heterologous DNA as a single copy in an Agrobacterium tumefaciens host cell, a unique restriction endonuclease cleavage site for insertion of heterologous DNA, a heterologous DNA encoding a gene product inserted into the unique restriction endonuclease cleavage site, and left and right Agrobacterium T-DNA border sequences flanking said unique restriction endonuclease cleavage site, said left and right T-DNA border sequences allowing introduction of heterologous DNA located between said left and right T-DNA border sequences into a plant cell; and
(2) introducing said vector into plant cells.

Claim 1

A vector for transferring heterologous DNA into a plant cell, said vector comprising:

• a backbone which includes a first origin of replication capable of maintaining heterologous DNA as a single copy in an Escherichia coli host cell, and which further includes a second origin of replication capable of maintaining heterologous DNA as a single copy in an Agrobacterium tumefaciens host cell;

• a unique restriction endonuclease cleavage site for insertion of heterologous DNA; and

• left and right Agrobacterium T-DNA border sequences flanking said unique restriction endonuclease cleavage site, said left and right T-DNA border sequences allowing introduction of heterologous DNA located between said left and right T-DNA border sequences into a plant cell.

Claim 1

A vector for transferring heterologous DNA into a plant cell, said vector comprising:

• a backbone which includes a first origin of replication capable of maintaining heterologous DNA as a single copy in an Escherichia coli host cell, and which further includes a second origin of replication capable of maintaining heterologous DNA as a single copy in an Agrobacterium tumefaciens host cell;

• a unique restriction endonuclease cleavage site for insertion of heterologous DNA; and

• left and right Agrobacterium T-DNA border sequences flanking said unique restriction endonuclease cleavage site, said left and right T-DNA border sequences allowing introduction of heterologous DNA located between said left and right T-DNA border sequences into a plant cell.

An Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids that are stably inherited, wherein each of said binary vector plasmids comprises at least one T-DNA region comprising a heterologous nucleotide sequence of interest.

A method for co-transformation of a plant with at least two unique heterologous nucleotide sequences of interest, said method comprising the steps of

a) contacting a tissue from said plant with an Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids that are stably inherited, wherein each of said binary vector plasmids comprises at least one T-DNA region, wherein each of said T-DNA regions comprises one of said unique nucleotide sequences, wherein at least one of said nucleotide sequences comprises at least an expression cassette comprising a plant scorable marker gene;
b) co-cultivating said tissue with said Agrobacterium;
c) culturing the tissue in a culture medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium;
d) screening the tissue for expression of said plants scorable marker gene; and
e) regenerating a transformed plant from tissue that expresses said plant scorable marker gene.

An Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids, wherein each of said binary vector plasmids comprises at least one T-DNA region comprising a heterologous nucleotide sequence of interest.

A method for co-transformation of a plant with at least two unique heterologous nucleotide sequences of interest, said method comprising the steps of:

a) contacting a tissue from said plant with an Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids, wherein each of said binary vector plasmids comprises at least one T-DNA region, wherein each of said T-DNA regions comprises one of said unique nucleotide sequences, wherein at least one of said nucleotide sequences comprises at least an expression cassette comprising a plant scorable marker gene;
b) co-cultivating said tissue with said Agrobacterium;
c) culturing the tissue in a culture medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium;
d) screening the tissue for expression of said plant scorable marker gene; and
e) regenerating a transformed plant from tissue that expresses said plant scorable marker gene.

An Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids, wherein each of said binary vector plasmids comprises at least one T-DNA region comprising a heterologous nucleotide sequence of interest.

A method for co-transformation of a plant with at least two unique heterologous nucleotide sequences of interest, said method comprising the steps of:

a) contacting a tissue from said plant with an Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids, wherein each of said binary vector plasmids comprises at least one T-DNA region, wherein each of said T-DNA regions comprises one of said unique nucleotide sequences, wherein at least one of said nucleotide sequences comprises at least an expression cassette comprising a plant scorable marker gene;
b) co-cultivating said tissue with said Agrobacterium;
c) culturing the tissue in a culture medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium;
d) screening the tissue for expression of said plant scorable marker gene; and
e) regenerating a transformed plant from tissue that expresses said plant scorable marker gene.

An Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids, wherein each of said binary vector plasmids comprises at least one T-DNA region comprising a heterologous nucleotide sequence of interest.

A methid for co-transformation of a plant with at least two unique heterologous nucleotide sequences of interest, said method comprising the steps of:

a) contacting a tissue from said plant with an Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids, wherein each of said binary vector plasmids comprises at least one T-DNA region, wherein each of said T-DNA regions comprises one of said unique nucleotide sequences, wherein at least one of said nucleotide sequences comprises at least an expression cassette comprising a plant scorable marker gene;
b) co-cultivating said tissue with said Agrobacterium;
c) culturing the tissue in a culture medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium;
d) screening the tissue for expression of said plants scorable marker gene; and
e) regenerating a transformed plant from tissue that expresses said plant scorable marker gene.

An Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids that are stably inherited, wherein each of said binary vector plasmids comprises at least one T-DNA region comprising a heterologous nucleotide sequence of interest.

A method for co-transformation of a plant with at least two unique heterologous nucleotide sequences of interest, said method comprising the steps of:

a) contacting a tissue from said plant with an Agrobacterium strain comprising a helper plasmid and at least two binary vector plasmids that are stably inherited, wherein each of said binary vector plasmids comprises at least one T-DNA region, wherein each of said T-DNA regions comprises one of said unique nucleotide sequences, wherein at least one of said nucleotide sequences comprises at least an expression cassette comprising a plant scorable marker gene;
b) co-cultivating said tissue with said Agrobacterium;
c) culturing the tissue in a culture medium comprising an antibiotic capable of inhibiting the growth of Agrobacterium;
d) screening the tissue for expression of said plants scorable marker gene; and
e) regenerating a transformed plant from tissue that expresses said plant scorable marker gene.

A vector combination consisting of:

A) an acceptor Ti plasmid which is substantially free of internal T-DNA sequences of wild-type Ti plasmid and incapable of inducing tumors in plants comprising:
(i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) a DNA sequence derived from a cloning vehicle, located between the two border sequences; and
(iii) a DNA segment of a wild type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes, and

B) an intermediate cloning vector, said cloning vector comprising:
(i) at least one gene of interest under the control of a promoter capable of directing gene expression in plants; and
(ii) a cloning vehicle segment containing a DNA sequence which is homologous with the DNA sequence (ii) in said acceptor Ti plasmid permitting a single crossover event.

A vector combination consisting of:

A) an acceptor Ti plasmid which is incapable of inducing tumors in plants being free of border sequences and internal T-DNA sequences of a wild-type Ti plasmid, comprising:
(i) a DNA segment of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(ii) a DNA sequence derived from a cloning vehicle, and

B) an intermediate cloning vector comprising:
(i) a cloning vehicle segment containing the two border sequences of the T-region of a wild-type Ti plasmid; and
(ii) a DNA sequence located between said two border sequences which is homologous with the DNA sequence (ii) in said acceptor Ti plasmid permitting a single crossover event, wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and
(iii) at least one gene of interest under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome.

A hybrid Ti plasmid vector obtained by cointegration either between

A) an acceptor Ti plasmid which is incapable of inducing tumors in plants comprising:
(i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) a DNA sequence devoid of oncogenic internal T-DNA regions of the wild-type T-DNA, derived from a cloning vehicle, located between the two border sequences, and containing a DNA sequence which is homologous with at least a part of a DNA sequence in an intermediate cloning vector permitting a single crossover event; and
(iii) a DNA segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes; and

B) an intermediate cloning vector comprising
(i) at least one gene of interest under the control of a promoter capable of directing gene expression in plants; and
(ii) a cloning vehicle segment containing a DNA sequence which is homologous with the above DNA sequence (ii) in the acceptor Ti plasmid; or between
I) an acceptor Ti plasmid comprising:
(a) a DNA segment of the wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence derived from a cloning vehicle; and II) an intermediate cloning vector comprising:
(a) a cloning vehicle segment containing the two border sequences of the T-region of a wild-type Ti plasmid and a DNA sequence located between said two border sequences which is homologous with the above DNA sequence (b) in the acceptor Ti plasmid permitting a single crossover event, wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and
(b) at least one gene of interest under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome; III) said hybrid Ti plasmid vector comprising at least:
(1) the two border sequences in the T-region of a wild-type Ti plasmid;
(2) non-oncogenic DNA sequences derived from a cloning vehicle;
(3) a DNA segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer of the T-region of wild type Ti plasmid by Agrobacterium into plant cell genomes; and
(4) at least one gene of interest under the control of a promoter capable of directing gene expression in plants which is located between the two border sequences.

Acceptor Ti plasmid comprising: (i) two border sequences of the T-region of the wild-type Ti plasmid; and
(ii) a non-oncogenic DNA segment derived from a cloning vehicle located between the two border sequences containing a DNA sequence which is homologous with at least a part of a DNA in an intermediate cloning vector permitting a single crossover event; and
(iii) a segment of the wild type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes.

Acceptor Ti plasmid comprising: (i) a DNA segment of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(ii) a DNA sequence derived from a cloning vehicle which is homologous with a DNA sequence of an intermediate cloning vector which contains the two border sequences of the T-region of the wild-type Ti plasmid.

Hybrid Ti plasmid vector comprising: (i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) non-oncogenic DNA segments derived from a cloning vehicle;
(iii) a segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes; and
(iv) at least one gene of interest which is located between the two border sequences.

A cell of a dicotyledonous plant, obtainable by Agrobacterium transformation, which contains stably integrated into its genome a foreign DNA which is characterised in that:

(a) it does not contain T-DNA genes that control neoplastic growth and it is substantially free of internal T-DNA sequences of a wild-type Ti-plasmid; and
(b) it comprises at least one gene of interest containing:
(i) a coding sequence; and
(ii) a promoter region that contains a promoter sequence other than the natural promoter sequence of said coding sequence, and wherein said promoter sequence regulates transcription of downstream sequences containing said coding sequence to produce an RNA in said cell.

A cloning vector which comprises:

(a) a cloning vehicle segment (3') containing a left border sequence (1) and a right border sequence (2) of a T-region of a wild-type Ti-plasmid, and
(b) a DNA segment which is located between said border sequences in a manner allowing its integration into a plant genome, wherein said DNA segment does not contain T-DNA genes that control neoplastic growth and wherein said DNA segment is substantially free of internal T-DNA sequences of a wild-type Ti-plasmid, except for promoter sequences, and which contains at least one gene of interest which comprises:
(i) a coding sequence, and
(ii) a promoter region that contains a promoter sequence other than the natural promoter sequence of said coding sequence, and wherein said promoter sequence regulates transcription of downstream sequences containing said coding sequence to produce an RNA in a cell of a plant.

Claim 9

A vector combination consisting of
(i) an acceptor Ti plasmid, which is incapable of inducing tumors in plants being free of border sequences and intemal T-DNA sequences of a wild-type Ti plasmid, comprising:
(a) a DNA segment (4) of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence (3) derived from a cloning vehicle, and
(ii) an intermediate cloning vector comprising:
(c) a cloning vehicle segment (3') containing the two border sequences of the T-region of a wild-type Ti plasmid (1; 2) and a DNA sequence located outside of said two border sequences which is homologous with the DNA sequence (b) in said acceptor Ti plasmid permitting a single cross-over event wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and
(d) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome.

Claim 16

A hybrid Ti plasmid vector obtained by co-integration between an acceptor Ti plasmid comprising:
(a) a DNA segment (4) of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence (3) derived from a cloning vehicle; and an intermediate cloning vector comprising:
(a') a cloning vehicle segment (3') containing the two border sequences of the T-region of a wild-type Ti plasmid (1; 2) and a DNA sequence located outside of said two border sequences which is homologous with the above DNA sequence (b) in the acceptor Ti plasmid permitting a single crossover event, wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and

(b') at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome;

said hybrid Ti plasmid vector comprising at least:

(1) the two border sequences (1; 2) of the T-region of a wild-type Ti plasmid;

(2) non-oncogenic DNA sequences (3; 3') derived from a cloning vehicle;

(3) a DNA segment (4) of the wild-type Ti plasmid containing DNA sequences essential for the transfer of the T-region of wild-type Ti plasmids by Agrobacterium into plant cell genomes; and

(4) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants which is located between the two border sequences (1; 2).

Recombinant plant DNA genome being free of oncogenic internal T-DNA regions of the wild-type Ti plasmid containing an integrated gene of interest foreign to said plant DNA obtainable by infecting a plant cell with an Agrobacterium harboring a hybrid Ti plasmid vector created by homologous recombination between:

A) an acceptor Ti plasmid which is incapable of inducing tumors in plants and comprises:
(i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) a DNA segment devoid of oncogenic internal T-DNA regions of the wild-type T-DNA, derived from a cloning vehicle, located between the two border sequences, and containing a DNA sequence "A" which is homologous with at least a part of a DNA sequence in an intermediate cloning vector permitting a single crossover event; and
(iii) a segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmid into plant cell genomes, and

B) an intermediate cloning vector which comprises:
(i) at least one gene of interest; and
(ii) a cloning vehicle segment containing a DNA sequence which is homologous to the DNA sequence "A" in said acceptor Ti plasmid,
or a hybrid Ti plasmid vector created by homologous recombination between:
I) an acceptor Ti plasmid which is incapable of inducing tumors in plants and which comprises:
(a) a DNA segment of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence derived from a cloning vehicle which is homologous with at least a part of a DNA sequence of an intermediate cloning vector which contains the two border sequences of the T-region of the wild-type Ti plasmid, and II) an intermediate cloning vector which comprises:
(a) a cloning vehicle segment containing the two border sequences of the T-region of the wild-type Ti plasmid and a DNA sequence which is homologous with the DNA sequence (b) in said acceptor Ti plasmid; and
(b) at least one gene of interest located between the two border sequences in a manner allowing its integration into the plant genome; said hybrid Ti plasmid comprising at least:
1) the two border sequences of the T-region of the wild-type Ti plasmid;
2) non-oncogenic DNA segments derived from a cloning vehicle;
3) a segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmid into plant cell genomes, and
4) at least one gene of interest which is located between the two border sequences.

A non-oncogenic Ti plasmid vector system free of T-DNA genes controlling neoplastic growth of transformed plants which comprises:

(i) DNA sequences coding for functions which are essential for the transfer of the T-region of a wild-type Ti plasmid by Agrobacterium into a plant cell genome; and
(ii) at least one gene of interest which has been inserted into said vector system and which is capable of being expressed in plants which are susceptible to infection by Agrobacterium,
said gene being under the control of at least one promoter capable of directing the expression of said gene in the plant and associated with at least the right border sequence of the T-region of a wild-type Ti plasmid, said border sequence allowing the integration of said gene into the plant genome.

An intermediate cloning vector comprising:

(i) a cloning vehicle segment containing the right border sequence of the T-region of a wild-type Ti plasmid and a DNA sequence which is homologous with a DNA sequence in an acceptor Ti plasmid; and
(ii) at least one gene of interest under the control of a promoter capable of directing gene expression in plants which is associated with said border sequence in a manner allowing its integration into the plant genome.

A method for transforming plant cell which comprises

• contacting plant cells, which are susceptible to genetic transformation by Agrobacterium cells, with Agrobacterium tumefaciens cells containing a co-integrated Ti plasmid comprising a disarmed T-DNA region which comprises in sequence:

(i) a left Agrobacterium T-DNA border sequence,
(ii) a chimeric selectable marker gene which functions in plant cells comprising: (a) a promoter which functions in plant cells;
(b) a structural coding sequence encoding a neomycin phosphotransferase; and
(c) a 3í non-translated region encoding a polyadenylation signal, and (iii) a right Agrobacterium T-DNA border sequence.

A method of creating transformed plant cells, comprising the following steps:

(i) culturing a microorganism containing

• a first plasmid containing:

(a) at least one first gene that is expressed in plant cells and
(b) at least one second gene which serves as a marker in a selected microorganism, and

• a Ti second plasmid, under conditions which allow the first plasmid to recombine with the Ti second plasmid in the microorganism, thereby creating a third plasmid having at least one T-DNA border on each side of the first gene, said gene being located in said first plasmid between a region of homology from the T-region of the Ti plasmid and a T-DNA border;

(ii) selecting those microorganisms containing the third plasmid;
(iii) inserting the third plasmid, or a portion thereof, into plant cells; and
(iv) culturing the plant cells under conditions which allow a segment of DNA from the third plasmid to be inserted into the genome of the plant cells.

A method of transforming plant cells, comprising

• inserting into the plant cells a co-integrated plasmid, or a portion thereof, which was formed by a single crossover event between a first plasmid and a Ti second plasmid, wherein the co-integrated plasmid contains a region comprising:

(i) a first T-DNA border;
(ii) a gene which expressed in plant cells; and
(iii) a second T-DNA border which is complementary to the first T-DNA border,
wherein the region does not contain any sequences which would render the plant cells incapable of being regenerated into morphologically normal plants.

A co-integrated plasmid for use in transforming plant cells, and produced by recombination by a single crossover event of:

A) a chimeric plasmid comprising a gene which functions in plants to express an encoded polypeptide, said plasmid comprising in sequence:

(i) a region of DNA which is homologous to T-DNA located near the left T-DNA border of a tumor-inducing plasmid of Agrobacterium and which is capable of causing in vivo recombination of the chimeric plasmid with said tumor-inducing plasmid of Agrobacterium by a single crossover event;
(ii) a gene comprising a promoter which functions in plant cells, a structural coding sequence and a 3í non-translated region encoding a polyadenylation signal, said promoter and polyadenylation signal being operably linked to said structural coding sequence; and
(iii) an Agrobacterium plasmid T-DNA right border sequence which enables the transfer and incorporation of T-DNA into the genome of a plant cell;
said chimeric plasmid containing no plant tumorigenic genes between and including the region (i), the gene (ii) and the border sequence (iii), and

B) a Ti plasmid capable of transferring the T-DNA region into the genome of a plant cell,

such that the co-integrated plasmid comprises a T-DNA region having the following elements in sequence:
1) a left T-DNA border sequence;
2) the gene (ii) of the chimeric plasmid;
3) at least one right T-DNA border sequence;
wherein said T-DNA border sequences enable the transfer of the T-DNA region into the genome of a plant cell, and wherein, between and including the left T-DNA border sequence, the gene (ii) and the right T-DNA border sequence or (if there is more than one right T-DNA border sequence) between the gene (ii) and the right T-DNA border sequence which is nearest to the left T-DNA border sequence, there are no genes which would render a transformed plant cell tumorous or incapable of regeneration into a morphologically normal plant, and there is no duplication of DNA sequences, which duplication could, through homologous recombination, result in loss of the gene (ii).

A process for the incorporation of foreign DNA into the genome of dicotyledonous plants comprising

• infecting the plants or incubating plant protoplasts with A. tumefaciens bacteria, which contain one or more Ti (tumor inducing) plasmids, characterized in that as Ti plasmid a stable co-integrated plasmid composed of the plasmid R772 and the plasmid pTiB6 with foreign DNA incorporated in the T-region of the Ti component of the co-integrated plasmid is applied.

A process for the production of Agrobacterium tumefaciens bacteria, which contain one or more Ti plasmids, comprising

• combining in Escherichia coli a vector known per se for use in E. coli, provided with T-DNA region in which foreign DNA has been incorporated, with co-integrated plasmid pAL969 and the co-integrated plasmid with foreign DNA incorporated by double crossing-over in the T-region of the Ti component of transferring the co-integrated plasmid to A. tumefaciens.

A process for the incorporation of foreign DNA into the genome of dicotyledonous plants comprising

• infecting the plants or incubating plant protoplasts with Agrobacterium tumefaciens bacteria containing at least one plasmid which is derived from the co-integrated plasmid pAL969 by the incorporation of foreign DNA in the T-region of the component pTiB6 characterized in that the co-integrated plasmid with foreign DNA contains only foreign DNA between the 23 base pairs of the wild-type T-region.

An Agrobacterium strain comprising: (i) a structural DNA sequence encoding a site-specific recombinase that is Cre recombinase; and

(ii) a DNA sequence linked thereto which operationally controls expression of said Cre recombinase, said strain further comprising
(iii) a first recombination site.

An Agrobacterium strain capable of producing a site-specific recombinase capable of effecting site-specific recombination of a first and second recombination site in said Agrobacterium strain, when present therein, comprising a structural DNA sequence encoding said recombinase and a DNA sequence capable of controlling expression in said Agrobacterium strain.

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.

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.

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.

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.

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 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 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;

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.

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.

A hybrid vector comprising a first T-DNA containing: 1) a gene giving a drug resistance, which functions in plant, and

2) 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.

A method for transferring genetic material, which is not a typical T-DNA surrounded by Agrobacterium T-borders from an Agrobacterium virulence system, to a eukaryotic host cell, said method comprising:

• providing said genetic material on a mobilizable plasmid capable of forming a relaxosome;
• bringing said mobilizable plasmid into an Agrobacterium having at least the activity of the transfer genes of Agrobacterium not present on said mobilizable plasmid so that the necessary gene products providing the same or similar activity as a functional virB operon are also present; and
• co-cultivating said Agrobacterium with the eukaryotic host cell.

A mobilizable plasmid comprising:

• genetic material to be transferred into a eukaryotic cell by Agrobacterium transfer;
• a functional oriT;
• sequences encoding functional virD-like mobilization products;
• a virD4-like coupling factor; and
• sequences encoding functional virB-like activity.

Claim 1
A method for transforming a plant cell or tissue with a gene construct, comprising heat shocking said plant cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said plant cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.

A method for producing a fertile transgenic plant comprising a gene construct, which method comprises: (a) transforming a plant cell or tissue comprising heat shocking said plant cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said plant cell or tissue and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed plant cell or tissue to produce said fertile transgenic plant.

Claim 12
A method for transforming a maize cell or tissue with a gene construct, comprising heat shocking said maize cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.

Claim 13
A method for producing a fertile transgenic maize plant comprising a gene construct, which method comprises: (a) transforming a maize cell or tissue comprising heat shocking said maize cell or tissue before co-cultivating with Agrobacterium, wherein said heat shock treatment inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed maize cell or tissue to produce said fertile transgenic maize plant.

A method for transforming a plant cell or tissue with a gene construct, comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said plant cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said plant cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.

A method for producing a fertile transgenic plant comprising a gene construct, which method comprises: (a) transforming a plant cell or tissue comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said plant cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said plant cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed plant cell or tissue to produce said fertile transgenic plant.

Claim 24
A method for transforming a maize cell or tissue with a gene construct, comprising exposing said maize cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said maize cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct.

Claim 25
A method for producing a fertile transgenic maize plant comprising a gene construct, which method comprises: (a) transforming a maize cell or tissue comprising exposing said maize cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise delivering to or expressing in said maize cell or tissue a nucleotide sequence comprising a coding sequence of a p35, iap or dad-1 gene, said delivery or expression of said nucleotide sequence inhibits Agrobacterium induced necrosis in said maize cell or tissue, and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed maize cell or tissue to produce said fertile transgenic maize plant.

Claim 26
A method for transforming a gramineceous plant cell or tissue with a gene construct, comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise culturing said plant cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and said Agrobacterium comprises a vector comprising said gene construct.

Claim 27
A method for producing a fertile transgenic gramineceous plant comprising, a gene construct, which method comprises: (a) transforming a gramineceous plant cell or tissue comprising exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise culturing said plant cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and said Agrobacterium comprises a vector comprising said gene construct; and (b) regenerating the transformed plant cell or tissue to produce said fertile transgenic gramineceous plant.

Claim 33
A method for transforming a maize cell or tissue with a gene construct, comprising exposing said maize cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise culturing said maize cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and said Agrobacterium comprises a vector comprising said gene construct.

Claim 34
A method for producing a fertile transgenic maize plant comprising a gene construct, which method comprises: (a) transforming a maize cell or tissue comprising exposing said maize cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis, wherein said conditions comprise culturing said maize cell or tissue in a necrosis inhibiting medium, said necrosis inhibiting medium comprises (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor, and said Agrobacterium comprises a vector comprising said gene construct.; and (b) regenerating the transformed maize cell or tissue to produce said fertile transgenic maize plant.

A transgenic plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which comprises a coding sequence of a p35, iap or dad-1 gag.

A transgenic plant, plant tissue or plant cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin which comprises a coding sequence of a p35, iap or dad-1 gene.

Claim 40
A transgenic maize plant, tissue or cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin comprising a coding sequence of a p35 gene.

Claim 41
A transgenic maize plant, tissue or cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin which comprises a coding sequence of an iap gene.

Claim 42
A transgenic maize plant, tissue or cell comprising a genome having a stably integrated nucleotide sequence of heterologous origin which comprises a coding sequence of a dad-1 gene.

Claim 44
A gramineceous plant cell or tissue culture medium, comprising (a) (i) an ethylene inhibitor other than silver nitrate, or (ii) an ethylene synthesis inhibitor; and (b) an Agrobacterium comprising a plasmid comprising a gene construct.

Claim 1

A method of transforming a plant cell with a gene of interest, comprising

• exposing said plant cell to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN), wherein said Agrobacterium comprises a vector comprising said gene of interest.

A method of making a fertile, transgenic plant comprising

• transforming plant tissue by exposing the tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN) and regenerating tissue thus transformed, wherein said Agrobacterium comprises a vector comprising a gene of interest.

A plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which inhibits AIN.

A plant cell or tissue culture medium, comprising

a) a chemical inhibitor of AIN,
b) an Agrobacterium comprising a plasmid comprising a gene of interest, and
c) water and essential salts.

Claim 11
A method of transforming a totipotent cell of a plant of the family Gramineae, comprising exposing a population of said totipotent cells to Agrobacterium comprising a plasmid comprising a gene of interest, wherein the Agrobacterium is of a strain which does not induce significant levels of necrosis in said population at an exposure duration and concentration sufficient to achieve transformation of said cell.

Claim 12
A method for determining the suitability of an Agrobacterium strain for use in the transformation of a regenerable cell of a plant of the family Gramineae comprising exposing a population of said regenerable cells of the plant to the Agrobacterium strain and observing the necrosis in said cell population.

An Agrobacterium strain which has been genetically modified to reduce or eliminate expression of the Agrobacterium necrosis factor or a derivative of such a modified strain.

Claim 1

A method of transforming a plant cell or tissue with a gene of interest, comprising:

• exposing said plant cell or tissue to Agrobacterium under conditions which inhibit Agrobacterium-induced necrosis (AIN), wherein
  said Agrobacterium comprises a vector comprising said gene of interest, wherein
  said conditions which inhibit AIN comprise:
  • exposing said plant cell or tissue to Agrobacterium after heat shock treatment; or
  • exposing said plant cell or tissue to Agrobacterium in the presence of an agent inhibiting AIN, wherein said agent comprises:
    • a chemical inhibitor, wherein said chemical inhibitor is a compound selected from the group consisting of ethylene inhibitors other than silver nitrate, ethylene synthesis inhibitors, gibberellin antagonists, and phosphatase inhibitors; or
    • a nucleotide sequence encoding mRNA or protein inhibiting AIN.

A method of making a fertile, transgenic plant comprising: A) transforming plant tissue by exposing the tissue to Agrobacterium under conditions which inhibit Agrobacterium -induced necrosis (AIN); and
B) regenerating tissue thus transformed, wherein
said Agrobacterium comprises a vector comprising said gene of interest, wherein
said conditions which inhibit AIN comprise:

• exposing said plant cell or tissue to Agrobacterium after heat shock treatment; or
• exposing said plant cell or tissue to Agrobacterium in the presence of an agent inhibiting AIN, wherein said agent comprises:
  • a chemical inhibitor, wherein said chemical inhibitor is a compound selected from the group consisting of ethylene inhibitors other than silver nitrate, ethylene synthesis inhibitors, gibberellin antagonists, and phosphatase inhibitors; or
  • a nucleotide sequence encoding mRNA or protein inhibiting AIN.

A plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which inhibits AIN.

A plant cell or tissue culture medium, comprising:

1. a chemical inhibitor, wherein said chemical inhibitor is a compound selected from the group consisting of ethylene inhibitors other than silver nitrate, ethylene synthesis inhibitors, gibberellin antagonists, and phosphatase inhibitors;
2. an Agrobacterium comprising a plasmid comprising a gene of interest; and
3. water and essentials salts.

Claim 1

A method of transforming a plant cell with a gene of interest, comprising

• exposing said plant cell to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN), wherein said Agrobacterium comprises a vector comprising said gene of interest.

A method of making a fertile, transgenic plant comprising

• transforming plant tissue by exposing the tissue to Agrobacterium under conditions which inhibit Agrobacterium induced necrosis (AIN) and regenerating tissue thus transformed, wherein said Agrobacterium comprises a vector comprising a gene of interest.

A plant, plant tissue or plant cell comprising a nucleotide sequence of heterologous origin which inhibits AIN.

A plant cell or tissue culture medium, comprising

a) a chemical inhibitor of AIN,
b) an Agrobacterium comprising a plasmid comprising a gene of interest, and
c) water and essential salts.

Claim 11
A method of transforming a totipotent cell of a plant of the family Gramineae, comprising exposing a population of said totipotent cells to Agrobacterium comprising a plasmid comprising a gene of interest, wherein the Agrobacterium is of a strain which does not induce significant levels of necrosis in said population at an exposure duration and concentration sufficient to achieve transformation of said cell.

Claim 12
A method for determining the suitability of an Agrobacterium strain for use in the transformation of a regenerable cell of a plant of the family Gramineae comprising exposing a population of said regenerable cells of the plant to the Agrobacterium strain and observing the necrosis in said cell population.

An Agrobacterium strain which has been genetically modified to reduce or eliminate expression of the Agrobacterium necrosis factor or a derivative of such a modified strain.

Claim 1
A method for transforming plant explant tissue, comprising:

a) contacting a cotyledon explant from a plant seedling infected with an Agrobacterium containing DNA to be introduced into the explant with an agent that inhibits enzymatic browning of a wounded plant, plant tissue or plant cell so as to yield transformed explant tissue; and

b) identifying transformed explant tissue.

Claim 22
A method to identify an agent that enhances Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part, comprising:

a) contacting the plant cell, plant tissue or plant part with Agrobacterium containing DNA to be introduced into the plant cell, plant tissue or plant part and the agent so as to yield a transformed plant cell, plant tissue or plant, wherein the agent is not a phenolic compound; and

b) detecting or determining whether the agent enhances Agrobacterium-mediated transformation of the plant cell, plant tissue or plant part relative to Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part in the absence of the agent.

Claim 37
A method for the stable transformation of plant tissue or cells, comprising:

a) contacting plant tissue or cells with an Agrobacterium containing DNA and an agent selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidase; and

b) identifying stably transformed plant tissue or cells.

Claim 44
A plant medium comprising: an amount of an agent effective to inhibit the enzymatic browning of a plant organ, tissue or cell, wherein the agent is selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of polyphenol oxidase and an inhibitor of peroxidase.

Claim 57
A method for the stable transformation of monocot plant tissue or cells, comprising:

a) contacting monocot plant tissue or cells with an Agrobacterium containing a recombinant DNA and one or more agents selected from the group consisting of a sulfhydryl-containing agent, methionine, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidases, which one or more agents are present in solid media in an amount effective to enhance the stable transformation of the monocot plant tissue or cells relative to corresponding monocot plant tissue or cells contacted with Agrobacterium in the absence of the one or more agents; and

b) identifying stably transformed plant tissue or cells.

Claim 62
A method for the stable transformation of plant tissue or cells, comprising:

a) contacting plant tissue or cells with an Agrobacterium containing a recombinant DNA and one or more agents selected from the group consisting of a sulfhydryl-containing agent, methionine, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidases, which one or more agents are present in solid media in an amount effective to enhance the stable transformation of the tissue or cells relative to corresponding tissue or cells contacted with Agrobacterium in the absence of the one or more agents; and

b) identifying stably transformed plant tissue or cells.

A method for stable transformation of leguminous plant tissue or cells, comprising:

a) contacting leguminous plant tissue or cells with an Agrobacterium comprising a recombinant DNA and one or more sulfhydryl-containing agents which one or more agents are present in solid media in an amount effective to enhance the stable transformation of the leguminous plant tissue or cells relative to corresponding plant tissue or cells contacted with the Agrobacterium in the absence of the one or more agents, wherein the plant tissue or cells are embryogenic somatic cells, immature embryo, meristem, or a cotyledon explant, and wherein the stable transformation is enhanced by at least 0.5%; and
b) identifying stably transformed plant tissue or cells.

Claim 1

A method for transforming plant explant tissue, comprising:

a) contacting a cotyledon explant from a plant seedling infected with an Agrobacterium containing DNA to be introduced into the explant with an agent that inhibits enzymatic browning of a wounded plant, plant tissue or plant cell so as to yield transformed explant tissue; and
b) identifying transformed explant tissue.

A method to identify an agent that enhances Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part, comprising:

a) contacting the plant cell, plant tissue or plant part with Agrobacterium containing DNA to be introduced into the plant cell, plant tissue or plant part and the agent so as to yield a transformed plant cell, plant tissue or plant, wherein the agent is not a phenolic compound; and
b) detecting or determining whether the agent enhances Agrobacterium-mediated transformation of the plant cell, plant tissue or plant part relative to Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part in the absence of the agent.

A method for the stable transformation of plant tissue or cells, comprising:

a) contacting plant tissue or cells with an Agrobacterium containing DNA and an agent selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidase; and
b) identifying stably transformed plant tissue or cells.

A plant medium comprising: an amount of an agent effective to inhibit the enzymatic browning of a plant organ, tissue or cell, wherein the agent is selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of polyphenol oxidase and an inhibitor of peroxidase.

Claim 1

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:
A) inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;
B) co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media not containing a growth inhibiting agent;
C) selecting transformed plant cells or tissue; and
D) regenerating a transformed plant expressing the genetic component from the selected transformed plant cells or tissue.

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of: A) inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue;
B) co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
C) selecting transformed plant cells or tissue; and
D) regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:
A) inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;
B) co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of the plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
C) selecting transformed plant cells or tissue; and
D) regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.

Claim 1

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:

• inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;

• co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media not containing a growth inhibiting agent;

• selecting transformed plant cells or tissue; and

• regenerating a transformed plant expressing the genetic component from the selected transformed plant cells or tissue.

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:

• inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue;
• co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
• selecting transformed plant cells or tissue; and
• regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.

A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of:

• inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;
• co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of the plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;
• selecting transformed plant cells or tissue; and
• regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.

A process to produce a dicotyledonous transgenic plant, said plant comprising

• a foreign DNA fragment integrated into the genome of at least some of its cells, said process comprising the following steps:

1) providing a plant which is systemically infected with an Agrobacterium strain auxotrophic for methionine or cysteine, or adenine and histidine, harboring a DNA of interest which is operably linked to at least one T-DNA border sequence; and
2) generating a transgenic plant from a single cell or a group of cells isolated from said systemically infected plant.

Bacterial strain LBA4404metHV, deposited as LMG P-18486.

Bacterial strain ATHV ade,his, deposited as LMG P-18485.

A method for producing a transgenic plant, said plant comprising

• a foreign DNA fragment integrated into the genome of at least some of its cells, said process comprising:

a. cocultivating a plant cell, plant tissue, explant or plant with an Agrobacterium strain auxotrophic for methionine or cysteine, or for adenine and histidine, to generate a transgenic cell; and
b. regenerating a transgenic plant from said transgenic cell.

A process to produce a transgenic plant comprising

• a foreign DNA fragment integrated into the genome of at least some of its cells, said process comprising the following steps:

1) providing a plant which is systemically infected with an auxotrophic Agrobacterium strains harbouring a DNA of interest which is operably linked to at least one T-DNA border sequence; and
2) generating a transgenic plant from a single cell or a group of cells isolated from said systemically infected plant.

Bacterial strain LBA4404met.

Bacterial strain ATHVade, his.

The use of an Agrobacterium strain auxotrophic for methionine or cysteine or for adenine and histidine in Agrobacterium-mediated transformation of plants.

Claim 15
The use of Agrobacterium strain auxotrophic for methionine or cysteine or for adenine and histidine in Agrobacterium mediated transformation of callus from sugarbeet.

Claim 16
The use of Agrobacterium strain auxotrophic for methionine or cysteine or for adenine and histidine in Agrobacterium mediated transformation of corn protoplasts.

A process to produce a transgenic plant comprising

• a foreign DNA fragment integrated into the genome of at least some of its cells, said process comprising the following steps:

1) providing a plant which is systemically infected with an auxotrophic Agrobacterium strain harbouring a DNA of interest which is operably linked to at least one T-DNA border sequence; and
2) generating a transgenic plant from a single cell or a group of cells isolated from said systemically infected plant.

Bacterial strain LBA4404metHV, deposited as BCCM/LMG P-18486.

Bacterial strain ATHV ade,his, deposited as BCCM/LMG P-18485.

The use of an Agrobacterium strain auxotrophic for methionine or cysteine, or for adenine and histidine, in Agrobacterium-mediated transformation of plants.

Claim 15
The use of an Agrobacterium strain auxotrophic for methionine or cysteine, or for adenine and histidine, in Agrobacterium mediated transformation of callus from sugarbeet.

Claim 16
The use of an Agrobacterium strain auxotrophic for methionine or cysteine, or for adenine and histidine, in Agrobacterium mediated transformation of corn protoplasts.

Claim 1

A process to produce a transgenic piant comprising a foreign DNA fragment integrated into the genome of at least some of its cells, said process comprising the following steps:
1) providing a plant which is systemically infected with an auxotrophic Agrobacterium strain harbouring a DNA of interest which is operably linked to at least one T-DNA border sequence; and
2) generating a transgenic plant from a single cell or a group of cells isolated from said systemically infected plant.

Claim 12

Bacterial strain LBA44O4met.

Claim 13

Bacterial strain ATHVade, his.

Claim 14

The use of an Agrobacterium strain auxotrophic for methionine or cysteine or for adenine and histidine in Agrobacterium-mediated transformation of plants.

Claim 15
The use of Agrobacterium strain auxotrophic for methionine or cysteine or for adenine and histidine in Agrobacterium mediated transformation of calius from sugarbeet.

Claim 16
The use of Agrobacterium strain auxotrophic for methionine or cysteine or for adenine and histidine in Agrobacterium mediated transformation of corn protoplasts.

A method for producing a fertile transgenic plant, comprising the steps of:

(a) introducing one or more genetic component(s) one desires to introduce into the genome of a plant by co-culturing a regenerable plant cell or tissue with Agrobacterium containing said genetic component(s);
(b) co-culturing said Agrobacterium and regenerable plant cells or tissues of step (a) under conditions that decrease the weight of said Arobacterium-inoculated explant;
(c) identifying or selecting a transformed cell line; and
(d) regenerating a fertile transgenic plant therefrom.

Claim 1

A method for producing a fertile transgenic plant, comprising the steps of:

(a) introducing one or more genetic component(s) into the genome of a plant by co-culturing a regenerable plant cell or tissue with Agrobacterium containing said genetic component(s);
(b) co-culturing said Agrobacterium and regenerable plant cells or tissues of step (a) in a manner controlling a reduction in the weight of said Agrobacterium-inoculated explant of from about 20% to 35% of the weight of the plant cell or tissue prior to co-culture, wherein the manner for controlling a reduction in the weight of the Agrobacterium-inoculted explant comprises limitation or removal of water from the explant;
(c) identifying or selecting a transformed cell line; and
(d) regenerating a fertile transgenic plant therefrom.

A method for transforming a plant sample comprising the following steps:

a. providing a non-tumor inducing vector containing nucleic acid to be transferred to the plant sample, wherein the vector is a non-tumor inducing Agrobacterium;
b. combining the plant sample with said vector;
c. sonicating the plant sample; wherein the vector is combined with the plant sample before, during, or after sonication;
d. lastly growing the plant sample and selecting for the transformed plant sample.

A method for transforming a plant sample comprising the following steps:

a. providing a non-tumor inducing vector containing nucleic acid to be transferred to the plant sample, wherein the vector is a non-tumor inducing Agrobacterium;
b. combining the plant sample with said vector;
c. sonicating the plant sample, wherein the vector is combined with the plant sample before, during or after sonication;
d. lastly growing the plant sample.

A method for transforming a plant sample comprising the following steps:

a. providing a non-tumor inducing vector containing nucleic acid to be transferred to the plant sample;
b. combining the plant sample with said vector;
c. sonicating the plant sample;
d. lastly growing the plant sample.

A method for direct plant transformation using seedlings and Agrobacterium comprising:

A) contacting at least one seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said seedling;
B) applying a vacuum to said seedling in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said seedling such that said Agrobacterium cells transfer said T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different.

A method for direct plant transformation using seedlings and Agrobacterium comprising:

A) contacting at least one seedling with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said seedling;
B) applying a vacuum to said seedling in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said seedling such that said Agrobacterium cells transfer T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different.

A method for direct plant transformation using seedlings and Agrobacterium comprising:

A) contacting at least one seedling with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said seedling;
B) applying a vacuum to said seedling in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said seedling such that said Agrobacterium cells transfer said T-DNA to cells of said seedling at a second time, wherein said first and second time are the same or different;
C) allowing said transformed seedling to grow to maturity and set seed;
D) germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression;
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

A method for direct plant transformation using plants and Agrobacterium comprising:

A) contacting the aerial portions of at least one plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.

A method for direct transformation of a plant comprising:

A) vernalizing and germinating initial seed to form said plant contacting the aerial portions of said plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.

A method for direct plant transformation using plants at the time of flowering and Agrobacterium comprising:
A) contacting aerial portions of at least one plant at the time of flowering with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector with a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.

A method for direct transformation of a plant at the time of flowering comprising:

A) vernalizing and germinating initial seed to form said plant contacting aerial portions of said plant at the time of flowering with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector with a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.

A method for direct plant transformation using plants at the time of flowering and Agrobacterium

comprising:A) contacting aerial portions of at least one plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said plant;
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different;
C) allowing said transformed plant to grow to maturity and set seed;
D) germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression; and
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

A method for direct transformation of a plant at the time of flowering comprising:

A) vernalizing and germinating initial seed to form said plant;
B) contacting aerial portions of said plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said plant;
C) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different;
D) allowing said transformed plant to grow to maturity and set seed; germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression; and
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.

A method for transforming a monocotyledon callus, comprising contacting a cultured tissue of a monocotyledon during dedifferentiation wherein said dedifferentiation is obtained by culturing an explant on a dedifferentiation-inducing medium for not less than 7 days or a dedifferentiated cultured tissue of a monocotyledon, with a bacterium belonging to the genus Agrobacterium containing a desired gene.

A method for transforming a monocotyledon with a desired gene, comprising:

A) contacting a cultured tissue of said monocotyledon during dedifferentiation thereof, or a dedifferentiated cultured tissue of said monocotyledon, with a suspension of Agrobacterium tumefaciens having a cell population of 10⁶ to 10¹¹ cells/ml for 3-10 minutes, and then
B) culturing said cultured tissue of said monocotyledon during dedifferentiation thereof, or said dedifferentiated cultured tissue of said monocotyledon, on a solid medium for several days together with said Agrobacterium tumefaciens, or
C) adding said Agrobacterium tumefaciens to culture medium in which said cultured tissue of said monocotyledon during dedifferentiation thereof or said dedifferentiated cultured tissue of said monocotyledon is cultured, and continuously culturing said cultured tissue of said monocotyledon during dedifferentiation or said dedifferentiated cultured tissue of said monocotyledon together with said Agrobacterium tumefaciens,

wherein said dedifferentiated cultured tissue of said monocotyledon is selected from the group consisting of a tissue cultured during the process of callus formation which is cultured for not less than 7 days after an explant is placed on a dedifferentiation-inducing medium and a callus, and
wherein said Agrobacterium tumefaciens contains plasmid pTOK162, and said desired gene is present between border sequences of the T region of said plasmid pTOK162, or wherein said desired gene is present in another plasmid contained in said Agrobacterium tumefaciens.

A method for transforming a monocot plant comprising:

i) culturing an explant of said monocot plant, or a tissue isolated from said explant, for one to six days in a medium comprising at least one auxin to obtain a cultured tissue;
ii) co-culturing the cultured tissue from step i) with an Agrobacterium bacterium comprising a polynucleotide of interest;
iii) selecting cultured tissue into which the polynucleotide of interest has been introduced; and
iv) culturing the selected tissue on a regeneration medium to obtain a transformed monocot plant.

A method for transforming a tissue of a monocot plant comprising:

i) culturing an explant of an immature tissue of a monocot plant for one to six days on a medium comprising at least one auxin and that induces dedifferentiation of the cells of the explanted tissue to obtain a dedifferentiating or dedifferentiated cultured immature tissue;
ii) contacting the dedifferentiating or dedifferentiated cultured immature tissue with cells of Agrobacterium bacteria that comprise a vector comprising at least one virulence gene of a Ti plasmid, a left T-DNA border, a right T-DNA border and a polynucleotide of interest located between the left T-DNA border and the right T-DNA border;
thereby obtaining a transformed plant tissue.

A method for obtaining a transformed monocot plant comprising

i) culturing an explant of an immature tissue of a monocot plant for one to six days on a medium comprising at least one auxin and that induces dedifferentiation of the cells of the explanted tissue to obtain a dedifferentiating or dedifferentiated cultured immature tissue;
ii) contacting the dedifferentiating or dedifferentiated cultured immature tissue with cells of Agrobacterium bacteria that comprise a vector comprising at least one virulence gene of a Ti plasmid, a left T-DNA border, a right T-DNA border and a polynucleotide of interest located between the left T-DNA border and the right T-DNA border; thereby obtaining a transformed plant tissue; and
iii) culturing the transformed plant tissue on at least one regeneration medium, thereby obtaining a transformed monocot plant.

A method for transforming a monocotyledon, comprising contacting a cultured tissue of said monocotyledon during dedifferentiation thereof obtained by culturing an explant on a dedifferentiation-inducing medium for less than 7 days with a bacterium belonging to the genus Agrobacterium containing a super binary vector having the virulence region of Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281, left and right border sequences of T-DNA of a Ti plasmid or an Ri plasmid of a bacterium belonging to the genus Agrobacterium, and a desired gene located between said left and right border sequences.

A method for transforming a monocotyledon, comprising contacting a cultured tissue of said monocotyledon during dedifferentiation thereof obtained by culturing an explant derived from an immature tissue on a dedifferentiation-inducing medium for less than 7 days with a bacterium belonging to the genus Agrobacterium containing a desired gene and containing a vector having the virulence region of Ti plasmid contained in Agrobacterium tumefaciens.

A method for transforming a monocotyledon comprising transforming a cultured tissue during dedifferentiation process or a dedifferentiated cultured tissue of said monocotyledon with a bacteria belonging to genus Agrobacterium containing a desired gene.

A method for transforming monocotyledons comprising transforming scutellum of an immature embryo of a monocotyledon with a bacterium belonging to genus Agrobacterium containing a desired gene, which immature embryo has not been subjected to a dedifferentiation treatment, to obtain a transformant.

A method for transforming monocotyledons comprising transforming scutellum of an immature embryo of a monocotyledon with a bacterium belonging to genus Agrobacterium containing a desired gene, which immature embryo has not been subjected to a dedifferentiation treatment, to obtain a transformant.

A method of transforming a monocot comprising co-cultivating a monocot inflorescence with Agrobacterium containing a plasmid comprising a heterologous nucleic acid.

A method of transforming a monocot comprising:

A) dissecting an inflorescence from a monocot;
B) initiating a callus from the inflorescence to generate an inflorescence-derived callus; and
C) co-cultivating the inflorescence-derived callus with Agrobacterium containing a plasmid comprising a heterologous nucleic acid.

A method of making a transgenic monocot comprising:

A) dissecting an inflorescence from a monocot;
B) initiating a callus from the inflorescence to generate an inflorescence-derived callus;
C) co-cultivating the inflorescence-derived callus with Agrobacterium containing a plasmid comprising a heterologous nucleic acid; and
D) regenerating a transgenic monocot from the callus.

A method of making a transgenic monocot comprising:

A) dissecting an inflorescence from a monocot;
B) co-cultivating the inflorescence with Agrobacterium containing a plasmid comprising a heterologous nucleic acid;
C) initiating a callus from the inflorescence; and
D) regenerating a transgenic monocot from the callus.

An in planta method of transforming a monocotyledonous plant comprising:

A) contacting at least one flower of the monocotyledonous plant with a solution or suspension comprising an Agrobacterium clone; and
B) subjecting said plant to a vacuum effective to cause entry of the Agrobacterium clone into at least one flower of the plant.

An apparatus for the transformation of a monocotyledonous plant, comprising:

1. a vacuum chamber of sufficient size to contain at least one monocotyledonous plant;
2. means for generating a vacuum;
3. a connector that connects the means for generating a vacuum with the vacuum chamber; and
4. means for affixing the monocotyledonous plant inside the vacuum chamber.

A method of transforming cells of a monocotyledonous plant with genetic material, said method comprising:

A) obtaining an explant from said plant;
B) co-cultivating the explant with Agrobacterium species having a T-DNA or T-DNA region comprising the genetic material to be transformed into the plant cells for a time and under conditions sufficient for the genetic material to transfer into the plant cells without said Agrobacterium overgrowing the plant cells; and
C) selecting for the transformed plant cells and permitting the cells to form organogenic callus.

A method for producing a genetically modified monocotyledonous plant, said method comprising:

A) obtaining explant from a plant to be genetically modified;
B) co-cultivating the explant with Agrobacterium species having a T-DNA or T-DNA region comprising genetic material to be transformed into said plant cells for a time and under conditions sufficient for the genetic material to transfer to plant cells without said Agrobacterium overgrowing the plant cells;
C) selecting transformed plant cells and permitting the cells to form organogenic callus; and then
D) regenerating a plant from selected transformed plant cells.

A method for producing a genetically modified monocotyledonous plant, said method comprising:

A) obtaining an explant from said plant to be genetically modified;
B) co-cultivating the explant with Agrobacterium species having a T-DNA or T-DNA region comprising the genetic material to be transformed into the plant cells for a time and under conditions sufficient for the genetic material to transfer into the plant cells without the Agrobacterium overgrowing the plant cells;
C) selecting for the transformed plant cells and permitting the cells to form organogenic callus; and
D) regenerating a plant from said transformed organogenic callus.

Claim 1

A method of transforming cells of a pineapple plant with genetic material, said method comprising:

A) obtaining an explant from said plant;
B) co-cultivating the explant with Agrobacterium species having a T-DNA or T-DNA region comprising the genetic material to be transformed into the plant cells for a time and under conditions sufficient for the genetic material to transfer into the plant cells without said Agrobacterium overgrowing the plant cells; and
C) selecting for the transformed plant cells and permitting the cells to form organogenic callus.

Claim 19

A method for producing a genetically modified pineapple plant, said method comprising:

A) obtaining explant from a plant to be genetically modified;
B) co-cultivating the explant with Agrobacterium species having a T-DNA or T-DNA region comprising genetic material to be transformed into said plant cells for a time and under conditions sufficient for the genetic material to transfer to plant cells without said Agrobacterium overgrowing the plant cells;
C) selecting transformed plant cells and permitting the cells to form organogenic callus; and then
D) regenerating a plant from selected transformed plant cells.

Claim 37

A method for producing a genetically modified pineapple plant, said method comprising:

A) obtaining an explant from said plant to be genetically modified;
B) co-cultivating the explant with Agrobacterium species having a T-DNA or T-DNA region comprising the genetic material to be transformed into the plant cells for a time and under conditions sufficient for the genetic material to transfer into the plant cells without the Agrobacterium overgrowing the plant cells;
C) selecting for the transformed plant cells and permitting the cells to form organogenic callus; and
D) regenerating a plant from said transformed organogenic callus.

A method for the transformation of a monocot plant comprising,
i) exposing explant tissue of said monocot plant to an Agrobacterium strain under vacuum in the presence of a phenolic compound, said Agrobacterium strain comprising a heterologous gene of interest within a vector;
ii) removing said Agrobacterium from said explant tissue;
iii) adding an antibiotic against said Agrobacterium; and
iv) selecting explant tissue for occurrence of said heterologous gene of interest.

A method for the transformation of a monocot plant comprising,
i) placing explant tissue of said monocot plant into media comprising a suspension of Agrobacterium to obtain a mixture, said Agrobacterium strain comprising a heterologous gene of interest within a vector;
ii) maintaining said mixture under vacuum in the presence of acetosyringone;
iii) releasing said vacuum and further incubating said explant tissue in the presence of said Agrobacterium;
iv) transferring said explant tissue to fresh media comprising acetosyringone and incubating said explant tissue in the dark
v) washing said explant tissue with an antibiotic against said Agrobacterium,
vi) transferring said explant tissue to fresh media and allowing said explant tissue to differentiate, thereby producing differentiated calli;
vii) placing said differentiated calli onto media containing a selection agent, and maintaining said differentiated calli in the light; and
viii) obtaining calli that grow in the presence of the selection agent.

A method for the transformation of a monocot plant comprising,
i) placing explant tissue of said monocot plant into media comprising a phenolic compound, and a suspension of Agrobacterium to obtain a mixture, said Agrobacterium strain comprising a heterologous gene of interest within a vector;
ii) washing said explant tissue with an antibiotic against said Agrobacterium and transferring said explant tissue to fresh media comprising acetosyringone and incubating said explant tissue in the dark;
iii) transferring said explant tissue to fresh media and allowing said explant tissue to differentiate, thereby producing differentiated calli;
iv) placing said differentiated calli to media containing a selection agent, and maintaining said differentiated calli in the light; and
v) obtaining calli that grow in the presence of the selection agent.

A method for transforming a monocotyledon, comprising a step of infecting an intact seed with an Agrobacterium which contains a desired recombinant gene.

A method for transforming a monocotyledon, comprising a step of infecting an intact seed with an Agrobacterium which contains a desired recombinant gene wherein the seed is a germinated seed which is germinated by preculturing four to five days after sowing on a medium comprising 2,4-D.

A method of producing transformed Gramineae, said method comprising:

A) making a wound in a graminaceous seedling with newly emerging radicle and stem, said wound being made in an area of the seedling containing rapidly dividing cells, wherein said area extends from the base of the scutellar node to slightly beyond the coleoptile node; and
B) inoculating the wound with vir⁺ A. tumefaciens.

A transformed pollen grain of a Gramineae produced by a plant grown from a seedling infected with vir+ Agrobacterium tumefaciens containing a vector comprising genetically-engineered T-DNA.

A transformed Gramineae plant derived from a seedling infected with vir+ Agrobacterium tumefaciens which contains a vector comprising genetically-engineered T-DNA.

A transformed Gramineae produced by making a wound in a graminaceous seedling with newly emerged radicle and stem, the wound being made in an area of the seedling containing rapidly dividing cells, wherein said area extends from the base of the scutellar node to slightly beyond the coleoptile node; and inoculating the wound with vir+ Agrobacterium tumefaciens.

A transformed Gramineae produced by making a wound in a graminaceous seedling with newly emerged radicle and stem, the wound being made in an area of the seedling containing rapidly dividing cells, wherein said area extends from the base of the scutellar node to slightly beyond the coleoptile node; and inoculating the wound with vir+ Agrobacterium tumefaciens; the transformed Gramineae containing a foreign gene which is an opine synthesis gene that is a nopaline synthase gene or an octopine synthase gene.

An Agrobacterium--mediated transformed Gramineae.

A transformed Gramineae plant comprising a genetically-engineered T-DNA further comprising a heterologous gene and a transcription unit in operable order.

A method of producing transformed Gramineae, as hereinbefore defined, comprising:
A) making a wound in a seedling in an area of the seedling containing rapidly dividing cells that give rise to germ line cells; and
B) inoculating the wound with vir⁺ A. tumefaciens.

A method of producing transformed Gramineae comprising:
A) making a wound in a seedling in an area of the seedling containing rapidly dividing cells; and
B) inoculating the wound with vir⁺ A. tumefaciens.

A transformed pollen grain of a Gramineae.

A transformed pollen grain of a Gramineae produced by a plant grown from a seedling infected with vir⁺ Agrobacterium tumefaciens.

A transformed pollen grain of a Gramineae produced by a plant grown from a seedling infected with vir⁺ Agrobacterium tumefaciens which contains a vector comprising genetically-engineered T-DNA.

A transformed pollen grain of a Gramineae produced by a plant grown from a seedling infected with vir⁺ Agrobacterium tumefaciens which contains a vector comprising genetically-engineered T-DNA.

A process for the stable integration of a DNA, comprising a gene that is functional in a cell of a cereal plant, wherein said DNA is integrated into the nuclear genome of said cereal plant, said process comprising the steps of:

A) providing a compact embryogenic callus of said cereal plant;
B) wounding said compact embryogenic callus or treating said compact embryogenic callus with a cell wall degrading enzyme for a period of time so as not to cause a complete disruption of tissues, and transferring said DNA into the nuclear genome of a cell in said compact embryogenic callus by means of Agrobacterium-mediated transformation to generate a transformed cell; and
C) regenerating a transformed cereal plant from said transformed cell.

A method for the stable integration of a DNA comprising a gene that is functional in a cell of a cereal plant, into the nuclear genome of a cereal plant, said method comprising:

A) providing a compact embryogenic callus of a corn plant;
B) wounding and/or degrading said compact embryogenic callus and transferring said DNA in the nuclear genome of a cell in said compact embryogenic callus by means of electroporation, bombardment with DNA-coated microprojectiles or Agrobacterium-mediated transformation to generate a transformed cell; and optionally
C) regenerating a transformed cereal plant from said transformed cell.

The use of compact embryogenic callus of a cereal plant as starting material for transferring a DNA comprising a gene that is functional in a cell of a cereal plant, by means of electroporation, bombardment with DNA-coated microprojectiles or Agrobacterium-mediated transformation, into the nuclear genome of said cereal plant.

A method for the stable integration of a DNA comprising a gene that is functional in a cell of a cereal plant, into the nuclear genome of a cereal plant, said method comprising:

a) providing a compact embryogenic callus of a cereal plant;
b) wounding and/or degrading said compact embryogenic callus and transferring said DNA in the nuclear genome of a cell in said compact embryogenic callus by means of electroporation, bombardment with DNA-coated microprojectiles or Agrobacterium-mediated transformation to generate a transformed cell; and optionally
c) regenerating a transformed cereal plant from said transformed cell.

The use of compact embryogenic callus of a cereal plant as starting material for transferring a DNA comprising a gene that is functional in a cell of a cereal plant, by means of electroporation, bombardment with DNA-coated microprojectiles or Agrobacterium-mediated transformation, into the nuclear genome of said cereal plant.