The Seminis Vegetable Seeds' patent family

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.