Genetically transformed plant cells comprising

    a desired nucleotide sequence and

    a co-introduced nucleotide sequence

wherein expression or transcription of the co-introduced nucleotide sequence in the transformed cells gives said transformed cells a competitive advantage when a population of cells including the transformed and the non-transformed cells is supplied with a compound that only the transformed cells are able to utilize, and
the desired nucleotide sequence codes for a gene other than a toxin, antibiotic or herbicide resistance gene.

Genetically transformed maize cells comprising

    a desired nucleotide sequence and

    a co-introduced nucleotide sequence

wherein the co-introduced nucleotide sequence gives the transformed cells a competitive advantage when a population of cells including the transformed cells and nontransformed cells is supplied with a compound,

wherein the co-introduced nucleotide sequence codes for a phosphomanno-isomerase or a mannophosphatase and the compound is mannose, a mannose derivative or a mannose precursor.

A method of selecting genetically transformed cells from a population of cells comprising the steps of:

a) introducing into the genome of a plant cell a desired nucleotide sequence and a co-introduced nucleotide sequence wherein said desired nucleotide sequence or co-introduced nucleotide sequence codes for a sequence other than a toxin, antibiotic or herbicide resistance gene;

b) obtaining transformed cells;

c) supplying to the population of cells a compound that only transformed cells are able to utilize wherein said transformed cells have a competitive advantage over non-transformed cells due to the expression or transcription of the desired nucleotide sequence or co-introduced nucleotide sequence in the presence of the compound; and

d) selecting said transformed cells from the population of cells.

A method of selecting genetically transformed maize cells from a population of cells comprising the steps of:

a) introducing into the genome of a maize cell a desired nucleotide sequence and a co-introduced nucleotide;

b) obtaining transformed cells;

c) supplying to the population of cells a compound wherein said transformed cells have a competitive advantage over non-transformed cells due to the expression or transcription of the desired nucleotide sequence or co-introduced nucleotide sequence in the presence of the compound; and

d) selecting said transformed cells from the population of cells wherein said co-introduced nucleotide sequence comprises a sequence encoding a phosphomanno-isomerase or a manno-phosphatase and the compound is mannose, a mannose derivative or a mannose precursor.

A method for selecting from a population of cells genetically transformed cells into which a desired nucleotide sequence has been introduced, wherein in the transformed cells the desired nucleotide sequence or a co-introduced nucleotide sequence induces or increases a positive effect of a compound or nutrient supplied to the population of cells, thereby allowing the transformed cells to be identified or selected from non-transformed cells.

Genetically transformed cells whose genome does not contain as a selection marker a non-native nucleotide sequence coding for toxin, antibiotic or herbicide resistance.

A compound of the general formula I

wherein

R² is H, CH₃, S-CH₃, SO₂-CH₃, SCH₂-phenyl, SH, OH, Cl or a group -S-R¹⁰, -NH-R¹⁰ or -O-R¹⁰, where R¹⁰ is a β-D-glucopyranuronosyl group or a salt thereof or an ester or amide derivative thereof at the carboxylic acid function,

R⁶ is benzyl which may be substituted on the phenyl ring with OH, C_^1-6-alkoxy, halogen, C_1-4-alkyl, NH₂ or CF₃, or with -O-R¹⁰, -S-R¹⁰ or -NH-R¹⁰, where R¹⁰ is as defined above; C_1-8-alkyl or C_2-8-alkenyl which may be substituted with from 1 to 3 hydroxy, glucosyloxy or C_1-6-alkoxy groups, with phenyl, and/or with -O-R¹⁰, -S-R¹⁰ or -NH-R¹⁰, where R¹⁰ is as defined above; esterified C_1-6-alkyl or C_2-6-alkenyl; furfuryl; or cyclohexylureido, phenylureido or tolylureido;

either

    i) R⁷ and Y are half-bonds which together form a bond,
    ii) one of R³ and R⁹ is H or a group R¹⁰ as defined above and the other is a half-bond which together with a half-bond X forms a bond, or R⁹ is ribosyl, 5 min -phosphoribosyl, glucosyl or -CH2CH(NH₂)COOH and R³ is a half-bond which together with the half-bond X forms a bond, and
    iii) R⁸ is H, CH₃, S-CH₃, SO₂-CH₃, SCH₂-phenyl, SH, OH, Cl or a group -S-R¹⁰, -NH-R¹⁰ or -O-R¹⁰, where R¹⁰ is as defined above,

or

    iv) R⁷ is ribosyl, 5 min -phosphoribosyl or glucosyl, R⁸ is H, R⁹ and Y are half-bonds which together form a bond, and R³ is a half-bond which together with the half-bond X forms a bond; with the proviso that one of R², R³, R⁶, R⁸ and R⁹ is or comprises a β-D-glucopyranuronosyl group or a salt thereof or an ester or amide derivative thereof at the carboxylic acid function.

A method of selecting genetically transformed plant cells from a population of cells which comprises

supplying the said population with a compound which can be metabolized by the expression product of a nucleotide sequence which has been introduced into the said transformed cells, so as to provide the transformed cells with a physiological advantage when compared to the non-transformed cells, wherein the compound is not an antibiotic or herbicide and has no direct adverse effect on the non-transformed cells.

31 May 2000

Opposition file by CAMBIA, BASF AG and Unilever PLC

28 Aug 2002

Decision under appeal: Interloculory decision of the Opposition Division of the EPO posted concerning     maintenance of EP 601092 in amended form

21 Jan 2006

Legal effect of interlocutory decision A.106(3)

16 Sep 2006

Fee for printing new specification R.58(5) paid

A plant cell whose genome does not contain as a selection marker an introduced, non-native nucleotide sequence coding for a resistance to a compound having a direct adverse effect on the nontransformed cells such as an antibiotic or herbicide and also not a nucleotide sequence coding for a β-glucuronidase but comprises at least an introduced, non-native nucleotide sequence the expression product of which is capable of providing the transformed plant cell with a physiological advantage when compared to the non-transformed plant cells such that upon supplying a population of plant cells comprising transformed and non-transformed cells with a compound which can be metabolized by the expression product of said introduced, non-native nucleotide sequence, genetically transformed cells can be selected from said population of plant cells.

A plant whose genome does not contain as a selection marker an introduced, non-native nucleotide sequence coding for a resistance to a compound having a direct adverse effect on the non-transformed cells such as an antibiotic or herbicide and also not a nucleotide sequence coding for a β-glucuronidase but comprises at least an introduced, non-native nucleotide sequence the expression product of which is capable of providing transformed plant cells derived therefrom with a physiological advantage when compared to the non-transformed plant cells such that upon supplying a population of plant cells comprising transformed and non-transformed cells with a compound which can be metabolized by the expression product of said introduced, non-native nucleotide sequence, genetically transformed cells can be selected from said population of plant cells.

A method for selecting genetically transformed plant cells from a population of cells comprising:

    a) introducing into plant cells a desired nucleotide sequence and a co-introduced nucleotide sequence to obtain transformed plant cells;

    b) supplying to a population of plant cells including the transformed cells and non-transformed cells a compound selected from the group consisting of mannose and a derivative or precursor of mannose, wherein said transformed cells have a competitive advantage over the non-transformed plant cells in the population due to the expression or transcription of the co-introduced nucleotide sequence in the transformed cells; and

    c) selecting the transformed plant cells based on said competitive advantage wherein said co-introduced nucleotide sequence is a gene encoding an enzyme involved in mannose metabolism selected from the group comprising a phosphomanno isomerase, a phosphomanno mutase, a mannose epimerase, a phosphatase, and a permease.

Genetically transformed corn cells comprising,

an introduced desired nucleotide sequence and a co-introduced nucleotide sequence, wherein the expression or transcription of said co-introduced nucleotide sequence gives the transformed cells a competitive advantage over non-transformed cells when both transformed and non-transformed cells in a population of cells are supplied with a mannose compound and said transformed cells are selected based on said competitive advantage, wherein the co-introduced nucleotide sequence codes for a phosphosugar isomerase.

Genetically transformed plant cells comprising an introduced nucleotide sequence and a co-introduced nucleotide sequence wherein the expression or transcription of the co-introduced nucleotide sequence gives the transformed cells a competitive advantage over non-transformed cells when both transformed and non-transformed cells in a population of cells are supplied with a compound and said transformed cells are selected based on said competitive advantage, wherein the co-introduced nucleotide sequence is a gene encoding an enzyme involved in mannose metabolism selected from the group comprising a phosphomanno isomerase, a phosphomanno mutase, a mannose epimerase, a phosphatase, and a permease.

Claim 1
A method for identifying or selecting from a population of plant cells selected from the group consisting of fruits, small grain cereals, vegetables, canola, sunflower, tobacco, sugar beet corn and cotton cultivated or in a medium containing at least one compound, cells which have a metabolic advantage as a result of having being transformed, wherein:

    i) the cells are transformed with a nucleotide sequence or a co-introduced nucleotide sequence one of which comprises a region which encodes an enzyme protein which is involved in the metabolism of the compound selected from the group consisting of phosphosugar-isomerases, phosphosugar-mutases, phosphatases, and sugar-epimerases;

    ii) the compound is mannose or xylose or a derivative or a precursor of these, or a substrate of the enzyme protein involved, either directly or indirectly, in the metabolism of mannose or xylose.

An isolated nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:14) or a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.

An isolated nucleic acid molecule that encodes one of the amino acid sequences of SEQ ID NOs.: 19-21 (19, 20, 21), or a variant thereof wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.: 19-21 and which encodes a functional β-glucuronidase.

An expression vector comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the β-glucuronidase is encoded by a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.

An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises one of the amino acid sequences of SEQ ID NOs.: 19-21 (19. 20, 21), or variant thereof, wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.: 19-21, and which encodes a functional β-glucuronidase.

A method for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO: 14) and which encodes functional β-glucuronidase and a nucleic acid molecule encoding a product of the gene of interest; wherein the β-glucuronidase and the gene of interest are co-expressed; (b) detecting the presence of the microbial β-glucuronidase, thereby monitoring expression of the gene of interest.

A method for transforming a host cell with a gene of interest or portion thereof, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase, and a nucleic acid sequence encoding a product of the gene of interest, such that the vector construct integrates into the genome of the host cell; wherein the β-glucuronidase and the gene of interest are co-expressed; (b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

A method for positive selection for a transformed cell, comprising: (a) introducing into a host cell a vector construct, the vector construct comprising a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO: 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase; (b) exposing the host cell to a sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that an aglycone is released, wherein the aglycone is required for growth of the host cell; wherein a host cell that expresses the β-glucuronidase grows, thereby positively selecting a transformed cell.

Claim 17

An isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 22, or a variant thereof wherein the variant has at least 90% amino acid identity to SEQ ID NO: 22 and which encodes a functional β-glucuronidase.

An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises the amino acid sequence of SEQ ID NO: 22, or variant thereof, wherein the variant has at least 90% amino acid identity to SEQ ID NO: 22, and which encodes a functional β-glucuronidase.

An isolated nucleic acid molecule consisting essentially of a nucleotide sequence that encodes a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

An isolated nucleic acid molecule encoding a thermostable β-glucuronidase, wherein the β-glucuronidase has a half-life of at least 10 min at 65° C.

An isolated nucleic acid molecule encoding a microbial β-glucuronidase, wherein the β-glucuronidase converts at least 50 nmoles of p-nitrophenyl-glucuronide to p-nitrophenyl per minute per μg of protein at 37° C.

An isolated nucleic acid molecule encoding a microbial β-glucuronidase, wherein the β-glucuronidase retains at least 80% of its activity in 10 mM glucuronic acid.

An isolated nucleic acid molecule encoding a fusion protein of a microbial β-glucuronidase or an enzymatically active portion thereof and a second protein.

An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

An isolated form of recombinant microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

A method for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising:

    (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule according to claim 1 and a nucleic acid molecule encoding a product of the gene of interest or a portion thereof;    

    (b) detecting the presence of the microbial glucuronidase, thereby monitoring expression of the gene of interest.

A method for transforming a host cell with a gene of interest or portion thereof, comprising:

    (a) introducing into the host cell a vector construct, the vector construct comprising

        a nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase, and

        a nucleic acid sequence encoding a product of the gene of interest or a portion thereof, such that the vector construct integrates into the genome of the host cell;

    (b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

A method for positive selection for a transformed cell, comprising:

(a) introducing into a host cell a vector construct, the vector construct comprising-nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli p-glucuronidase;

(b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth.

A method of producing a transgenic plant that expresses a microbial β-glucuronidase, comprising:

    (a) introducing an expression vector comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase, into an embryogenic plant cell; and

    (b) producing a plant from the embryogenic plant cell, wherein the plant expresses the β-glucuronidase.

A method for positive selection for a transformed cell, comprising:

    (a) introducing into a host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase;

    (b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth.

A transgenic plant cell comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

A transgenic plant comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, provided that the microbial β-glucuronidase is not E. coli β-glucuronidase.

A transgenic aquatic animal cell comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter.

A transgenic aquatic animal comprising an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter.

A method for identifying a microorganism that secretes β-glucuronidase, comprising:

    (a) culturing the microorganism in a medium containing a substrate for β-glucuronidase, wherein the cleaved substrate is detectable, and wherein the microorganism is an isolate of a naturally occurring microorganism or a transgenic microorganism; and
    (b) detecting the cleaved substrate in the medium; therefrom identifying an organism that secretes β-glucuronidase.

A method for providing an effector compound to a cell in a transgenic plant, comprising:

    (a) growing a transgenic plant that comprises an expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter and a nucleic acid sequence comprising a gene encoding a cell surface receptor for an effector compound.
    (b) exposing the transgenic plant to a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the effector compound is released.

Claim 1

An isolated nucleic acid molecule that encodes a microbial β-glucuronidase, comprising nucleotides 1-1689 of Figures 4I-J (SEQ ID NO.14) or a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of Figure 4I-J (SEQ ID NO.14) and which encodes a functional β-glucuronidase.

Claim 16

An isolated form of recombinant microbial β-glucuronidase, wherein the microbial β-glucuronidase comprises the amino acid sequence presented in Figure 3B panel E (SEQ ID NO: 6) or a variant thereof having at least 75% amino acid sequence identity thereto.

Claim 17
A method for monitoring expression of a gene of interest or a portion thereof in a host cell, comprising:

(a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule according to claims 1 or 2 and a nucleic acid sequence encoding a product of the gene of interest or a portion thereof;
(b) detecting the presence of the microbial β-glucuronidase, thereby monitoring expression of the gene of interest.

Claim 18
A method for transforming with a host cell a gene of interest or a portion thereof, comprising:

(a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid sequence according to claims 1 or 2 and a nucleic acid sequence encoding a product of the gene of interest or a portion thereof such that the vector construct integrates into the genome of the host cell; and
(b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.

Claim 19
A method for positive selection for a transformed cell, comprising:

(a) introducing into a host cell a vector construct, the vector construct comprising nucleic acid sequence according to claims 1 or 2; and
(b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth.

Claim 22
A method of producing a transgenic plant that expresses a microbial β-glucuronidase, comprising:
(a) introducing into an embryogenic plant cell an expression vector comprising a nucleic acid sequence according to claims 1 or 2 in operative linkage with a heterologous promoter; and
(b) producing a plant from the embryogenic plant cell, wherein the plant expresses the β-glucuronidase.

Claim 28
A method for providing an effector compound to a cell of a transgenic plant, comprising:
(a) growing a transgenic plant that comprises an expression vector, comprising nucleic acid sequence encoding a microbial β-glucuronidase according to claims 1 or 2 in operative linkage with a heterologous promoter and a nucleic acid sequence comprising a gene encoding a cell surface receptor for an effector compound; and
(b) exposing the transgenic plant to a glucuronide, wherein the glucuronide in cleaved by the β-glucuronidase, such that the effector compound is released.

A method of preparing cellobiuronic acid comprising: exposing gellan gum to partially hydrolyzing conditions to produce a hydrolysate comprising anionic saccharides, the anionic saccharides comprising at least 50 wt. % cellobiuronic acid; and isolating the cellobiuronic acid by anion exchange chromatography.

A method of hydrolyzing gellan gum comprising (a) contacting gellan gum with a hydrolyzing agent selected from the group consisting of acid, base and hydrolytic enzyme, under conditions that provide a hydrolysate comprising anionic saccharides, the anionic saccharides comprising at least 50 wt. % cellobiuronic acid, where the cellobiuronic acid is present in the hydrolysate at a concentration of at least 5 wt. % based on the total weight of gellan gum, and wherein the gellan gum has not previously been subjected to oxidizing conditions; and (b) isolating an anionic fraction comprising cellobiuronic acid from a neutral fraction comprising one or more monosaccharides using anion exchange chromatography.

A method of hydrolyzing gellan gum comprising contacting gellan gum with an aqueous composition having a pH between 2 and 7, under conditions effective to partially hydrolyze the gellan to form a hydrolysate comprising anionic saccharides, the anionic saccharides comprising at least 50 wt. % cellobiuronic acid, isolating the cellobiuronic acid from neutral saccharides by anion exchange chromatography, and separating the cellobiuronic acid from water.

A method of preparing cellobiuronic acid comprising:
    exposing gellan to partially hydrolyzing conditions to produce a hydrolysate comprising saccharides, the saccharides comprising cellobiuronic acid; and
    isolating the cellobiuronic acid.

A method of hydrolyzing gellan comprising contacting gellan with a hydrolyzing agent selected from the group consisting of acid, base and hydrolytic enzyme, under conditions that provide a hydrolysate comprising disaccharides and monosaccharides, wherein the disaccharid comprises cellobiuronic acid, and the cellobiuronic acid is present in the hydrolysate at a concentration of at least 5 wt. % based on the total weight of gellan, and wherein the gellan has not previously been subjected to oxidizing conditions.

A method of hydrolyzing gellan comprising contacting gellan with an aqueous composition having a pH between 2 and 7, under conditions effective to partially hydrolyze the gellan to cellobiuronic acid and separate the cellobiuronic acid from water.

An isolated polynucleotide molecule comprising at least one gene of interest and at least one selectable marker gene, wherein said at least one selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 2, or a plant optimized version thereof, wherein said nucleotide sequence encodes for a protein possessing ribitol dehydrogenase enzymatic activity and a protein possessing ribitol kinase enzymatic activity.

A method of selecting transformed cells from a population of cells comprising;

a) introducing into the genome of a cell a gene of interest and a selectable marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed cells have a selective advantage over non-transformed cells due to expression or transcription of the the selectable marker gene in the presence of the marker compound; and
d) selecting said transformed cells from the population of cells; wherein said selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 2, or a plant optimized version thereof, wherein said nucleotide sequence encodes a protein that possesses ribitol dehydrogenase enzymatic activity and a protein that possesses ribitol kinase enzymatic activity; and said marker compound comprises arabitol, ribitol, or mannitol.

Claim 12
An isolated polynucleotide molecule comprising a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a plant optimized version of the nucleotide sequences shown in SEQ ID NO: 2, and wherein said nucleotide sequence encodes for a protein possessing ribitol dehydrogenase activity and a protein possessing ribitol kinase activity.

Claim 13
An isolated polynucleotide molecule comprising at least one gene of interest, and at least one selectable marker gene, wherein said at least one selectable marker gene comprises a nucleotide sequence encoding SEQ ID NOS.: 3 and 4.

Claim 14
An isolated polynucleotide molecule comprising at least one gene of interest, and at least one selectable marker gene, wherein said at least one selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 1, or a plant optimized version thereof, wherein said at least one selectable marker gene encodes for a protein possessing arabitol dehydrogenase enzymatic activity.

Claim 15
A method of selecting transformed cells from a population of cells comprising

a) introducing into the genome of a cell a gene of interest and a selectable marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed cells have a selective advantage over non-transformed cells due to expression or transcription of the selectable marker gene in the presence of the marker compound; and
d) selecting said transformed cells from the population of cells; wherein said selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 1, or a plant optimized version thereof, and encodes a protein having arabitol dehydrogenase enzymatic activity; and wherein said marker compound is arabitol.

Claim 16
A method of selecting transformed cells from a population of cells comprising

a) introducing into the genome of a cell a gene of interest and a selectable marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed cells have a selective advantage over non-transformed cells due to expression or transcription of the selectable marker gene in the presence of the marker compound; and
d) selecting said transformed cells from the population of cells; wherein said selectable marker gene comprises a nucleotide sequence encoding SEQ ID NO.: 3, and a nucleotide sequence encoding SEQ ID NO.: 4; and wherein said marker compound is ribitol.

Claim 1

A process for selectively increasing the number of transformed plant cells regenerated from a mixture of transformed and non-transformed plant cells cultured under heterotrophic culture conditions, the method comprising the steps of:

    (a) culturing a mixture of transformed and non-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by non-transformed plant cells except for a source of carbon that supports growth and proliferation and about 1.5 to 3 times the standard amount of phosphorus, said source of carbon being replaced by an encrypted carbon source that does not support growth and proliferation of said non-transformed cells and that is selected from the group of: mannose, mannitol, sorbitol, lactose, trehalose and salicin, said transformed cells having a heterologous genomic DNA segment that contains at least one expression cassette,
        the one expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a heterologous gene that encodes a heterologous enzyme that is selected from the group of: phosphomannose isomerase , mannitol-1-oxidoreductase , L-iditoldehydrogenase, D-sorbitol-1-oxidoreductase, lactase, β-gatactosidase and α,α-trehalase and that on expression converts said encrypted carbon source into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a promoter DNA segment that controls expression of said heterologous gene, and
            (iii) a termination DNA segment;
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express said heterologous enzyme, grow and proliferate; and
    (c) recovering said transgenic proliferating cells.

Claim 10

A process for selectively increasing the number of transformed plants regenerated from a mixture of transformed and non-transformed plant cells cultured under heterotrophic culture conditions, the method comprising the steps of

    (a) culturing a mixture of transformed and non-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by non-transformed plant cells except for a source of carbon that supports growth and proliferation and about 1.5 to 3 times the standard amount of phosphorus; said source of carbon being replaced by an encrypted carbon source that does not support growth and proliferation of said non-transformed cells and that is selected from the group of: mannose, mannitol, sorbitol, lactose, trehalose and salicin, said transformed cells containing a heterologous genomic DNA segment that contains at least one expression cassette,
        the one expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first heterologous gene that encodes a heterologous enzyme that is selected from the group of: phospho-mannose isomerase, mannitol-1-oxidoreductase, L-iditol dehydrogenase, D-sorbitol-1-oxidoreductase, lactase, β-galactosidase and , α,α-trehalase and that on expression converts said encrypted carbon source into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of said heterologous gene, and
            (iii) a termination DNA segment;
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express said heterologous enzyme, grow and proliferate;
    (c) recovering said proliferating cells; and
    (d) regenerating plant meristematic tissues or plant embryos from said proliferating cells.

Claim 19

A process for selectively increasing the number of transformed plant cells regenerated from a mixture of transformed and non-transformed plant cells placed under selective heterotrophic culture conditions, the method comprising the steps of:

(a) culturing a mixture of transformed and non-transformed plant cells for up to two weeks in a first culture medium that contains the minimal nutrients required for proliferation and growth by non-transformed plant cells including a source of carbon that supports growth and proliferation, said transformed plants cells containing a genomic heterologous DNA segment that contains at least one expression cassette,
        the one expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a heterologous gene that encodes a heterologous enzyme that is selected from the group of: phosphomannose isomerase , mannitol-1-oxidoreductase , L-iditoldehydrogenase, D-sorbitol-1-oxidoreductase, lactase, β-gatactosidase and α,α-trehalase and that on expression converts an encrypted carbon source into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a promoter DNA segment that controls expression of said heterologous gene, and
            (iii) a termination DNA segment;
    (b) removing the mixture of transformed and non-transformed plant cells from the first culture medium;
    (c) placing the transformed and non-transformed plant cells under heterotrophic culture conditions in a second culture medium that contains the minimal nutrients required for proliferation and growth of the non-transformed plant cells except for an encrypted carbon source that does not support growth and proliferation of said non-transformed plant cells and that is selected from the group of: mannose, mannitol, sorbitol, lactose, trehalose and salicin, and 1.5 to 3 times the standard amount of phosphorous;
    (d) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express said heterologous enzyme, grow and proliferate; and
    (e) recovering said proliferating cells.

Claim 28

A process for selectively growing twice-transformed plant cells from a mixture of twice- and once-transformed plant cells comprising the steps of:

    (a) culturing a mixture of twice- and once-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by said once-transformed plant cells except for a source of carbon that supports growth and proliferation of said once-transformed cells and about 1.5 to 3 times the standard amount of phosphorus, said source of carbon being replaced by a second encrypted carbon source that does not support growth and proliferation of said once-transformed plant cells and that is selected from the group of: mannose and mannitol; said twice-transformed cells containing first and a second heterologous DNA segments that contains at least two expression cassettes, wherein at least one expression cassette is in the first heterologous DNA segment and at least one expression cassette is in the second heterologous DNA segment;
        the expression cassette in the first heterologous DNA segment containing a heterologous DNA selectable marker segment that includes
            (i) a first heterologous gene that encodes a heterologous enzyme that is selected from the group of: phosphomannose isomerase and mannitol-1-oxidoreductase and that on expression converts a first encrypted carbon source into a carbon source that supports growth and proliferation of said once- and twice-transformed plant cells under heterotrophic culture conditions but does not support growth and proliferation of non-transformed plant cells, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of said heterologous gene, and
            (iii) a termination DNA segment;
        the expression cassette in the second heterologous DNA segment containing a second heterologous DNA selectable marker segment that includes
            (i) a second heterologous gene that encodes a second heterologous enzyme that is selected from the group of: phosphomannose isomerase, and mannitol-1-oxidoreductase and that on expression during heterotrophic culture of said twice-transformed cells converts said second
encrypted carbon source that does not support growth and proliferation of once-transformed and non-transformed plant cells of the same type into said first encrypted carbon source that supports growth and proliferation of said twice- and once-transformed cells, said second gene being operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second heterologous gene and
            (iii) a termination DNA segment;
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said twice-transformed plant cells to express said first and second heterologous enzymes, grow and proliferate; and
    (c) recovering said proliferating cells.

A process for selectively growing twice-transformed plants from a mixture of twice- and once-transformed plant cells comprising the steps of:

    (a) culturing a mixture of twice- and once-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by said once-transformed plant cells except for a source of carbon that supports growth and proliferation of said once-transformed cells and about 1.5 to 3 times the standard amount of phosphorus, said source of carbon being replaced by a second encrypted carbon source that does not support growth and proliferation of said once-transformed plant cells and that is selected from the group of: mannose and mannitol; said twice-transformed cells containing first and a second heterologous DNA segments that contains at least two expression cassettes, wherein at least one expression cassette is in the first heterologous DNA segment and at least one expression cassette is in the second heterologous DNA segment;
        the expression cassette in the first heterologous DNA segment containing a heterologous DNA selectable marker segment that includes
            (i) a first heterologous gene that encodes a heterologous enzyme that is selected from the group of: phosphomannose isomerase and mannitol-1-oxidoreductase and that on expression converts a first encrypted carbon source into a carbon source that supports growth and proliferation of said once- and twice-transformed plant cells under heterotrophic culture conditions but does not support growth and proliferation of non-transformed plant cells, said first heterologous gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of said first heterologous gene, and
            (iii) a termination DNA segment;
        the expression cassette in the second heterologous DNA segment containing a second heterologous DNA selectable marker segment that includes
            (i) a second heterologous gene that encodes a second heterologous enzyme that is selected from the group of: phosphomannose isomerase and mannitol-1-oxidoreductase and that on expression during heterotrophic culture of said twice-transformed cells converts said second encrypted carbon source that does not support growth and proliferation of once-transformed and non-transformed plant cells of the same type into said first encrypted carbon source that supports growth and proliferation of said twice- and once-transformed cells, said second heterologous gene being operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second heterologous gene and
            (iii) a termination DNA segment;
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said twice- transformed plant cells to express said first and second heterologous enzymes, grow and proliferate;
    (c) recovering said proliferating cells; and
    (d) regenerating plant meristematic tissues or plant embryos from said proliferating cells.

A kit for forming transformed plant cells comprising:

    (a) a first package containing a DNA segment for transforming plant cells that contains an expression cassette operatively linked to a linker segment containing at least one restriction endonuclease site, said expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first heterologous gene that encodes a heterologous enzyme that that is selected from the group of: phosphomannose isomerase, mannitol-1-oxidoreductase, L-iditol dehydrogenase, D-sorbitol-1-oxidoreductase, lactase, β-galactosidase and , α,α-trehalase and on expression during heterotrophic culture of transformed plant cells converts an encrypted carbon source that does not support growth and proliferation of non-transformed plant cells into a carbon source that supports growth and proliferation of said transformed cells and that is selected from the group of: mannose, mannitol, sorbitol, lactose, trehalose and salicin, said first gene being operatively linked to
            (ii) a promoter DNA segment that controls expression of said first heterologous gene and
            (iii) a termination DNA segment; and
    (b) a second package that contains minimal nutrients required for proliferation and growth of non-transformed plant cells during heterotrophic culture except for a source of carbon and about 1.5 to 3 times the standard amount of phosphorus, said source of carbon being replaced by an encrypted carbon source that does not support growth and proliferation of non-transformed plant cells but supports growth and proliferation of transformed plant cells whose genome contains the DNA segment of said first package and that is selected from the group of: mannose, mannitol, sorbitol, lactose, trehalose and salicin.

A process for selectively growing transformed plant cells in a mixture of transformed and non-transformed plant cells comprising the steps of:

    (a) culturing a mixture of transformed and non-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by non-transformed plant cells except for a source of carbon that supports growth and proliferation and at least about 58.1 mg/L of phosphorus in the culture medium, said source of carbon being replaced by mannose that does not support growth and proliferation of said non-transformed cells, said transformed cells containing a genomic heterologous DNA segment that contains two expression cassettes,
        the first expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first gene that encodes phosphomannose isomerase that on expression allows mannose to be converted into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of the phosphomannose isomerase, and
            (iii) a termination DNA segment;
        the second expression cassette containing
            (i) a second gene that is expressed in a transformed plant and that is operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second gene and
            (iii) a termination DNA segment; and
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express the phosphomannose isomerase, to grow and proliferate.

A process for selectively growing transformed plants from a mixture of transformed and non-transformed plant cells comprising the steps of

    (a) culturing a mixture of transformed and non-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by non-transformed plant cells except for a source of carbon that supports growth and proliferation and at least about 58.1 mg/L of phosphorus in the culture medium, said source of carbon being replaced by mannose that does not support growth and proliferation of said non-transformed cells, said transformed cells containing a genomic heterologous DNA segment that contains two expression cassettes,
        the first expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first gene that encodes phosphomannose isomerase that on expression allows mannose to be converted into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of said heterologous enzyme, and
            (iii) a termination DNA segment,
        the second expression cassette containing
            (i) a second gene that is expressed in a transformed plant and that is operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second gene and
            (iii) a termination DNA segment;  
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express the phosphomannose isomerase, grow and proliferate;
    (c) recovering said proliferating cells; and
    (d) forming plant meristematic tissues or plant embryos from said proliferating cells.

A process for selectively growing twice-transformed plant cells in a mixture of twice- and once-transformed plant cells comprising the steps of:

    (a) culturing a mixture of twice- and once-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by said once-transformed plant cells except for a source of carbon that supports growth and proliferation of said once-transformed cells and at least about 58.1 mg/L of phosphorus in the culture medium, said source of carbon being replaced by mannitol that does not support growth and proliferation of said once-transformed plant cells; said twice-transformed cells containing first and second heterologous DNA segments that contain four expression cassettes, wherein the first and second expression cassettes are in the first heterologous DNA segment and the third and fourth expression cassettes are in the second heterologous DNA segment;
        the first expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first gene that encodes phosphomannose isomerase that on expression allows mannose to be converted into a carbon source that supports growth and proliferation of said once- and twice-transformed plant cells under heterotrophic culture conditions but does not support growth and proliferation of non-transformed plant cells, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of the phosphomannose isomerase, and
            (iii) a termination DNA segment,
        the second expression cassette containing
            (i) a second gene that is expressed in a transformed plant and that is operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second gene and
            (iii) a termination DNA segment;
        the third expression cassette containing a second heterologous DNA selectable marker segment that includes    
            (i) a second gene that encodes mannitol 1-oxidoreductase that on expression during heterotrophic culture of said twice-transformed cells, converts the mannitol that does not support growth and proliferation of once-transformed and non-transformed cells, of the same type into mannose that supports growth and proliferation of said twice-and once-transformed cells, said second gene being operatively linked to
            (ii) a third promoter DNA segment that controls expression of said second heterologous enzyme, and
            (iii) a termination DNA segment;
        the fourth expression cassette containing
            (i) a fourth gene that is expressed in said transformed plant and that is operatively linked to
            (ii) a fourth promoter that controls expression of said forth gene and
            (iii) a termination DNA segment; and
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said twice-transformed plant cells to express said first and second heterologous enzymes, grow and proliferate.

A process for selectively growing transformed plant cells in a mixture of transformed and non-transformed plant cells comprising the steps of:

    (a) culturing a mixture of transformed and non-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by non-transformed plant cells except for a source of carbon that supports growth and proliferation and at least about 178.5 mg/L of phosphate in the culture medium, said source of carbon being replaced by mannose that does not support growth and proliferation of said non-transformed cells, said transformed cells containing a genomic heterologous DNA segment that contains two expression cassettes,
        the first expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first gene that encodes phosphomannose isomerase that on expression allows mannose to be converted into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of the phosphomannose isomerase, and  
            (iii) a termination DNA segment;
        the second expression cassette containing
            (i) a second gene that is expressed in a transformed plant and that is operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second gene and
            (iii) a termination DNA segment; and
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express the phosphomannose isomerase, to grow and proliferate.

A process for selectively growing transformed plants from a mixture of transformed and non-transformed plant cells comprising the steps of

    (a) culturing a mixture of transformed and non-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by non-transformed plant cells except for a source of carbon that supports growth and proliferation and at least about 178.5 mg/L of phosphate in the culture medium, said source of carbon being replaced by mannose that does not support growth and proliferation of said non-transformed cells, said transformed cells containing a genomic heterologous DNA segment that contains two expression cassettes,
        the first expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first gene that encodes phosphomannose isomerase that on expression allows mannose to be converted into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of said heterologous enzyme, and
            (iii) a termination DNA segment,
        the second expression cassette containing
            (i) a second gene that is expressed in a transformed plant and that is operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second gene and
            (iii) a termination DNA segment;
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express the phosphomannose isomerase, grow and proliferate;
    (c) recovering said proliferating cells; and
    (d) forming plant meristematic tissues or plant embryos from said proliferating cells.

A process for selectively growing twice-transformed plant cells in a mixture of twice- and once-transformed plant cells comprising the steps of:

    (a) culturing a mixture of twice- and once-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by said once-transformed plant cells except for a source of carbon that supports growth and proliferation of said once-transformed cells and at least about 178.5 mg/L of phosphate in the culture medium, said source of carbon being replaced by mannitol that does not support growth and proliferation of said once-transformed plant cells; said twice-transformed cells containing first and second heterologous DNA segments that contain four expression cassettes, wherein the first and second expression cassettes are in the first heterologous DNA segment and the third and fourth expression cassettes are in the second heterologous DNA segment;
        the first expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a first gene that encodes phosphomannose isomerase that on expression allows mannose to be converted into a carbon source that supports growth and proliferation of said once-transformed plant cells under heterotrophic culture conditions but does not support growth and proliferation of non-transformed plant cells, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of the phosphomannose isomerase, and
            (iii) a termination DNA segment,
        the second expression cassette containing
            (i) a second gene that is expressed in a transformed plant and that is operatively linked to  
            (ii) a second promoter DNA segment that controls expression of said second gene and
            (iii) a termination DNA segment;
        the third expression cassette containing a second heterologous DNA selectable marker segment that includes
            (i) a second gene that encodes mannitol 1-oxidoreductase that on expression during heterotrophic culture of said twice-transformed cells converts the mannitol that does not support growth and proliferation of once-transformed and non-transformed cells, of the same type into mannose that supports growth and proliferation of said twice- and once-transformed cells, said second gene being operatively linked to
            (ii) a third promoter DNA segment that controls expression of said second heterologous enzyme, and             (iii) a termination DNA segment;
        the fourth expression cassette containing
            (i) a fourth gene that is expressed in said transformed plant and that is operatively linked to
            (ii) a fourth promoter that controls expression of said fourth gene and
            (iii) a termination DNA segment; and
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said twice-transformed plant cells to express said first and second heterologous enzymes, grow and proliferate.

Claim 1

A process for selectively increasing the number of transformed plants regenerated from a mixture of transformed and non-transformed plant cells cultured under heterotrophic culture conditions, the method comprising the steps of:

    (a) culturing a mixture of transformed and non-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by non-transformed plant cells, except for a source of carbon,and at least about 58.1 mg/L of phosphorus, said source of carbon being replaced by an encrypted carbon source that does not support growth and proliferation of said non-transformed cells, said transformed cells having a heterologous genomic DNA segment that contains at least one expression cassette,
        the at least one expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a heterologous gene that encodes a heterologous enzyme that on expression converts said encrypted carbon source into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a promoter DNA segment that controls expression of said heterologous gene, and
            (iii) a termination DNA segment;
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express said heterologous enzyme, grow and proliferate; and
    (c) recovering said transgenic proliferating cells.

Claim 2

A process for selectively increasing the number of transformed plants regenerated from a mixture of transformed and non-transformed plant cells placed under selective heterotrophic culture conditions, the method comprising the steps of:

(a) culturing a mixture of transformed and non-transformed plant cells for up to two weeks in a first culture medium that contains the minimal nutrients required for proliferation and growth by both transformed and non-transformed plant cells including a source of carbon that supports growth and proliferation of both transformed and non-transformed plant cells, said transformed plants cells containing a genomic heterologous DNA segment that contains at least one expression cassette,
        the at least one expression cassette containing a heterologous DNA selectable marker segment that includes
            (i) a heterologous gene that encodes a heterologous enzyme that on expression converts an encrypted carbon source into a carbon source that supports growth and proliferation of said transformed plant cells under heterotrophic culture conditions, said first gene being operatively linked to
            (ii) a promoter DNA segment that controls expression of said heterologous gene, and
            (iii) a termination DNA segment;
    (b) removing the mixture of transformed and non-transformed plant cells from the first culture medium;
    (c) placing the transformed and non-transformed plant cells under heterotrophic culture conditions in a second culture medium that contains the minimal nutrients required for proliferation and growth of the non-transformed plant cells, except for an encrypted carbon source that does not support growth and proliferation of said non-transformed plant cells and at least about 58.1 mg/L of phosphorous;
    (d) maintaining said heterotrophic culture conditions for a time period sufficient for said transformed plant cells to express said heterologous enzyme, grow and proliferate; and
    (e) recovering said proliferating cells.

Claim 13
A process for selectively increasing the number of transformed plants regenerated from a mixture of transformed and non-transformed plant cells placed under heterotrophic culture conditions, said process including at least a step comprising culrturing the transformed and non-transformed plant cells in the presence of an encrypted carbon source which does not support growth or proliferation of the non-transformed cells, and at least about 58,1 mg/L P or 178.5 mg/L PO₄, said process being substantially as hereinbefore described with reference to any one of the examples.

A process for selectively growing twice transformed plants from a mixture of twice- and once-transformed plant cells comprising the steps of:

    (a) culturing a mixture of twice- and once-transformed plant cells under heterotrophic culture conditions in a culture medium that contains minimal nutrients required for proliferation and growth by said once-transformed plant cells, except for a source of carbon that supports growth and proliferation of said once-transformed cells and at least about 58.1 mg/L of phosphorus, said source of carbon being replaced by a second encrypted carbon source that does not support growth and proliferation of said once-transformed plant cells; said twice-transformed cells containing a first and a second heterologous DNA segments and containing at least two expression cassettes, wherein at least one expression cassette is in the first heterologous DNA segment and at least one expression cassette is in the second heterologous DNA segment;
        at least one expression cassette in the first heterologous DNA segment containing a heterologous DNA selectable marker segment that includes
            (i) a first heterologous gene that encodes a heterologous enzyme that on expression converts a first encrypted carbon source into a carbon source that supports growth and proliferation of said once- and twice-transformed plant cells under heterotrophic culture conditions but does not support growth and proliferation of non-transformed plant cells, said first gene being operatively linked to
            (ii) a first promoter DNA segment that controls expression of said heterologous gene, and
            (iii) a termination DNA segment;
        at least one expression cassette in the second heterologous DNA segment containing a second heterologous DNA selectable marker segment that includes
            (i) a second heterologous gene that encodes a second heterologous enzyme that on expression during heterotrophic culture of said twice-transformed cells converts said second encrypted carbon source that does not support growth and proliferation of once-transformed and non-transformed plant cells of the same type into said first encrypted carbon source that supports growth and proliferation of said twice- and once-transformed cells, said second gene being operatively linked to
            (ii) a second promoter DNA segment that controls expression of said second heterologous gene and
            (iii) a termination DNA segment;
    (b) maintaining said heterotrophic culture conditions for a time period sufficient for said twice-transformed plant cells to express said first and second heterologous enzymes, grow and proliferate; and
    (c) recovering said proliferating cells.

Claim 25
A process for selectively growing twice transformed plants from a mixture of twice- and once-transformed plant cells, said process including at least a step comprising culrturing the twice-transformed and once-transformed cells in the presence of an encrypted carbon source that only support growth or proliferation of twice-transformed plant cells, and at least about 58,1 mg/L P or 178.5 mg/L PO₄, said process being substantially as hereinbefore described with reference to any one of the examples.

Method for selecting genetically transformed cells from a population of cells, comprising of

    a) introducing into a cell at least one desired nucleotide sequence and at least one selection-nucleotide sequence to obtain a genetically transformed cell, wherein the selection-nucleotide sequence comprises a region which codes for a protein involved in the metabolizing of trehalose;
    b) placing a population with transformed and non-transformed cells into contact with trehalose and/or derivative thereof; and
    c) selecting the transformed cells from the population on the basis of the capacity of the transformed cells to metabolize the trehalose and/or derivative.

Use of trehalose and/or derivative thereof for the selection of transformed cells from a population of transformed and non-transformed cells, wherein the genome of the transformed cells comprises at least one selection-nucleotide sequence comprising a region which codes for a protein involved in the metabolizing of trehalose.

The same as  Claim 1 in EP 1442126 B1.

Method for using trehalose and/or a derivative thereof for the selection of transformed cells from a population of transformed and non-transformed cells, wherein the genome of the transformed cells comprises at least one selection-nucleotide sequence comprising a region which codes for a protein involved in the metabolizing of trehalose.

An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide which has a D-amino acid metabolizing activity, wherein the nucleotide sequence is operably linked to a heterologous plant specific regulatory element.

Claim 14
A composition having herbicide activity and comprising one or more D-amino acids.

Claim 16
A method of stimulating stress tolerance of a plant comprising: expressing in said plant a polypeptide which oxidizes a D-amino acid substrate; and treating said plant with said D-amino acid substrate.

Claim 17
A method of inhibiting growth of a transgenic plant that expresses a polypeptide which oxidizes a D-amino acid substrate, the method comprising: allowing the polypeptide to accumulate in cytosol of the plant; and treating the plant with the D-amino acid substrate.

Claim 19
A method for selective growth of plant cells that have a D-amino acid metabolizing activity comprising treating said plant cells with a fertilizer that contains D-amino acids.

An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide which has a D-amino acid metabolising activity, the sequence being operably linked to a plant specific regulatory element heterologous to the gene encoding the D-amino acid metabolizing enzyme.

A method for selecting a cell transformed with one or more nucleic acid coding sequences comprising:

    (a) transforming a population of cells with a foreign DNA construct comprising at least one first nucleic acid coding sequence and a second nucleic acid coding sequence encoding a temperature sensitive marker protein selected from the group consisting of heat shock proteins, heat shock transcription factors, cold regulated proteins, and cold regulated protein transcription factors, each of said first and second nucleic acid coding sequences being operatively linked to gene expression control sequences, said transforming generating a second population of cells comprising cells which have been successfully transformed with said foreign DNA construct and non-transformed cells,

    (b) selecting said cells which have been successfully transformed from said non-transformed cells in said second population of cells from said transforming in (a), said selecting consisting essentially of culturing said second population of cells under temperature conditions wherein growth of said non-transformed cells is suppressed or prevented while growth of said cells which have been successfully transformed is supported, and

    (c) recovering cells from (b) exhibiting significant growth, wherein the recovered cells exhibiting significant growth are said cells which have been successfully transformed.

A method for selecting a cell transformed with one or more nucleic acid coding sequences comprising:

    (a) transforming a population of cells with a foreign DNA construct comprising at least one first nucleic acid coding sequence and a second nucleic acid coding sequence encoding a temperature sensitive marker protein selected from the group consisting of heat shock proteins, heat shock transcription factors, cold regulated proteins, and cold regulated protein transcription factors, each of said first and second nucleic acid coding sequences being operatively linked to gene expression control sequences, said transforming generating a second population of cells comprising cells which have been successfully transformed with said foreign DNA construct and non-transformed cells,

    (b) selecting said cells which have been successfully transformed from said non-transformed cells in said second population from said transforming in (a), wherein said selecting comprises culturing said second population of cells under temperature conditions wherein growth of said non-transformed cells is suppressed or prevented while growth of said cells which have been successfully transformed is supported, and said selecting does not comprise any of: culturing said second population of cells transformed in (a) on culture media comprising an antibiotic or pesticide, or detection of toxins, enzyme products, or pigments produced by said cells, and

    (c) recovering cells from (b) exhibiting significant growth, wherein the recovered cells exhibiting significant growth are said cells which have been successfully transformed.

A method for selecting a cell transformed with one or more nucleic acid coding sequences comprising:

    (a) transforming a population of cells with a foreign DNA construct comprising at least one first nucleic acid coding sequence and a second nucleic acid coding sequence encoding a temperature sensitive marker protein selected from the group consisting of heat shock proteins, heat shock transcription factors, cold regulated proteins, and cold regulated protein transcription factors, each of said first and second nucleic acid coding sequences being operatively linked to gene expression contrcl sequences,

    (b) culturing said populatior. of cells transformed in (a) under temperature conditions wherein growth of non-transformed cells is suppressed or prevented while growth of cells transformed with said DNA construct is supported, and

    (c) recovering cells from (b) exhibiting significant growth as presumptive positive transformants,

wherein said DNA construct does not comprise an antibiotic resistance marker or a pesticide resistance marker.

A selection method for selecting genetically transformed plant cells from a population of plant cells, comprising:

    transforming a population of plant cells with an expressible nucleotide sequence having at least 75% identity to SEQ ID NO: 1 and which encodes an enzyme having glucosamine-6-phosphate deaminase activity that converts a component or a metabolic derivative thereof when present in a high concentration in a medium into a nutrient, thereby producing a population of genetically transformed and non-transformed cells;

    introducing said population of plant cells to a medium comprising said component or metabolic derivative thereof, wherein the component or the metabolic derivative thereof is present in said medium in a high concentration and is toxic to said non-transformed plant cells and is a source of both carbohydrate and nitrogen for the selectable, genetically transformed plant cells or if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatized substrate, then that derivatized substrate provides an allosteric effect on the gene product; and

    culturing said plant cells thereby selecting at least a portion of said transformed plant cells over said non-transformed plant cells; and, wherein said enzyme having glucosamine-6-phosphate deaminase activity converts glucosamine-6-phosphate to fructose-6-phosphate.

A composition comprising a population of transformed and non-transformed plant cells, said transformed plant cells being transformed with an expressible nucleotide sequence having at least 75% identity to SEQ ID NO: 1 and which encodes an enzyme having glucosamine-6-phosphate deaminase activity which converts a component or a metabolic derivative thereof into a nutrient, and a medium comprising said component or metabolic derivative thereof, wherein the component or the metabolic derivative thereof is present in said medium in a high concentration and is toxic to said non-transformed plant cells; and, wherein said enzyme having glucosamine-6-phosphate deaminase activity converts glucosamine-6-phosphate to fructose-6-phosphate.

A construct for genetically transforming a non-transformed plant cell to produce a selectable genetically transformed plant cell, said construct comprising: a plant promoter operably linked to an expressible nucleotide sequence encoding a glucosamine-6-phosphate deaminase gene product and having at least 75% identity to SEQ ID NO: 1; and, wherein said glucosamine-6-phosphate deaminase gene product converts glucosamine-6-phosphate to fructose-6-phosphate.

A transgenic plant produced from a cell transformed with an expressible nucleotide sequence having at least 75% identity to SEQ ID NO: 1 and encoding an enzyme having glucosamine-6-phosphate deaminase activity.

An E. coli strain selected from the group consisting of NCIMB 40852, NCIMB 40853, and NCIMB 40854.

Claim 1

A selection method for selecting from a population of cells one or more selectable genetically transformed cells;
    wherein the population of cells comprises selectable genetically transformed cells and possible non-transformed cells;
    wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence;
    wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells;
    wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells;
    wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells;
    the method comprising the step of introducing the population of cells to a medium;
    wherein the medium optionally comprises a high concentration of the component or the metabolic derivative thereof; and
    either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells;
    or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.

Use of glucosanmine-6-phosphate deaminase as a selection means for selecting a genetically transformed cell over a non-transformed cell.

Claim 22

Use of a gene coding for glucosamine-6-phosphate deaminase for providing a selection means for selecting a genetically transformed cell over a non-transformed cell.

Claim 23

Use of a gene coding for glucosamine-6-phosphate deaminase obtained from NCIMvB 40852 or NCIMB 40853 or NCIMB 40854 for providing a selection means for selecting a genetically transformed cell over a non-transformed cell.

Use of a DNA sequence coding for a linear polyol carrier, in plants and fungi, such as polyols having main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, in particular 6 carbon atoms, these polyols being advantageously chosen from mannitol, sorbitol, dulcitol, galactitol, inositol, myo-inositol, ribitol and xylitol, and being in particular mannitol, for the preparation of transgenic plants.

Protein characterized in that it comprises or is constituted by: sequence SEQ ID NO: 2, or any sequence derived from SEQ ID NO: 2, in particular by substitution, suppression or addition of one or more amino acids, having the property of transporting linear polyols in plants and fungi, such as polyols having a main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, in particular 6 carbon atoms, these polyols being advantageously chosen from mannitol, sorbitol, dulcitol, galactitol, inositol, ribitol and xylitol, and being in particular mannitol, any homologous sequence of SEQ ID NO: 2, preferably having a homology of at least approximately 50% with sequence SEQ ID NO: 2 and possessing the property of transporting, in plants and fungi, polyols as defined above, or any fragment of one of the sequences defined above, on the condition that it possesses the property of transporting, in plants and fungi, polyols as defined above, in particular any fragment being constituted by at least approximately 10 amino acids adjacent in the sequence SEQ ID NO: 2.

DNA sequence which comprises or is constituted by: nucleotide sequence SEQ ID NO: 1, or any nucleotide sequence derived, by degeneration of the genetic code, from the sequence SEQ ID NO: 1 coding for a protein represented by SEQ ID NO: 2, or any nucleotide sequence derived, in particular by substitution, suppression or addition of one or more nucleotides, from the sequence SEQ ID NO: 1 coding for a protein derived from SEQ ID NO: 2, as defined in claim 3, or any homologous nucleotide sequence of SEQ ID NO: 1, preferably having a homology of at least approximately 35% with the sequence SEQ ID NO: 1 coding for a homologous protein of SEQ ID NO: 2, as defined in claim 3, or any fragment of the nucleotide sequence SEQ ID NO: 1 or of the nucleotide sequences defined above, said fragment being preferably constituted of at least approximately 30 nucleotides adjacent in said sequence, or any complementary nucleotide sequence of the above-mentioned sequences or fragments, or any nucleotide sequence capable of hybridizing in stringent conditions with the complementary sequence of one of the abovementioned sequences fragments.

Claim 1
A protein, comprising: sequence SEQ ID NO: 2.

A vector for introducing a gene into a plant, which comprises:

    a desired gene,

    a cytokinin signal transduction gene which comprises the CKI1 gene from Arabidopsis thaliana and a cytokinin synthesis gene together as selectable marker genes, and

    a removable DNA element,

wherein the selectable marker genes are positioned such that they behave integrally with the removable DNA element, and wherein the desired gene is positioned such that it does not behave integrally with the removable DNA element.

A vector for introducing a gene into a plant, which comprises

    a desired gene, and
    a plant hormone signal transduction gene as a selectable marker gene.

Claim 1

A method of increasing transformation efficiency of a monocotyledonous plant upon obtaining transformed monocotyledonous plant tissue or body, characterised by introducing into said monocotyledonous plant cell
    a desired gene, and
    a cytokinin-related gene.

A method for producing a transgenic plant, which comprises:

    (A) introducing a vector into a plant cell, wherein the vector is a vector for gene introduction into a plant and comprises: a desired gene, and a selectable marker gene comprising a gene encoding an enzyme which synthesizes auxin from an auxin precursor;
    (B) culturing the plant cell into which the genes are introduced by the vector, in the presence of an auxin precursor and/or an analogue thereof to thereby prepare a redifferentiated tissue, and detecting and selecting the redifferentiated tissues; and
    (C) culturing the redifferentiated tissue selected in (B) to redifferentiate a plant individual.

A vector for gene introduction into a plant, which comprises:

    a desired gene, and

    a selectable marker gene comprising

            an indoleacetamide hydrolase, iaaH, gene and

            an isopentenyl transferase, ipt, gene

            and being free of an tryptophan monooxygenase, iaaM, gene.