Antisense gene systems of pollination control for hybrid seed production

Claim 1

A method of producting hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a genetically transformed plant by: (i) inserting into the genome of a plant cell of said pollen producing plant which is capable of being regenerated into a differentiated whole plant, one or more recombinant DNA sequences comprising antisense DNA which block the production of functional pollen grains or render the developing pollen grains susceptible to a chemical agent or physiological stress which blocks the production of functional pollen grains; (ii) obtaining a transformed plant cell of said plant; and (iii) regenerating from said plant cell a plant which is genetically transformed with said DNA sequences described in (a)(i) above and which is male sterile or carries the male sterile trait; (b) increasing the number of genetically transformed plants by: (i) fertilizing the genetically transformed plant described in step (a) with pollen produced by a suitable male fertile plant, in a manner to effect a seed increase of genetically transformed plants; or (ii) clonal propogation of said genetically transformed plant described in step (a) using tissue explants thereof, or other in vitro propagation techniques (c) effecting a hybrid cross by pollinating said genetically transformed plants with pollen from suitable male fertile plant donors.

Claim 2

A method of producting hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a genetically transformed plant by: (i) inserting into the genome of a plant cell of said pollen producing plant which is capable of being regenerated into a differentiated whole plant, one or more recombinant DNA sequences comprising antisense DNA which block the production of functional pollen grains or render the developing pollen grains susceptible to a chemical agent or physiological stress which blocks the production of functional pollen grains; (ii) obtaining a transformed plant cell of said plant; and (iii) regenerating from said plant cell a plant which is genetically transformed with said DNA sequences described in (a)(i) above and which is male sterile or carries the male sterile trait; (b) increasing the number of genetically transformed plants by: (i) fertilizing the genetically transformed plant described in step (a) with pollen produced by a suitable male fertile plant, in a manner to effect a seed increase of genetically transformed plants; or (ii) clonal propogation of said genetically transformed plant described in step (a) using tissue explants thereof, or other in vitro propagation techniques (c) effecting a hybrid cross by pollinating said genetically transformed plants with pollen from suitable male fertile plant donors.

Claim 6

A method of producing a male sterile plant from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) identifying and isolating, preferably from said plant, a sense gene or coding sequence, that is critical to pollen formation or function in said plant; (b) inserting into the genome of a plant cell of said plant that is capable of regeneration into a differentiated whole plant, a recombinant double stranded DNA molecule comprising: (i) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (ii) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene or coding sequence; and preferably (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (c) obtaining a transformed plant cell in said plant; and (d) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 7

A method of producing a male sterile plant from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) identifying and isolating, preferably from said plant, a sense gene or coding sequence, that is critical to pollen formation or function in said plant; (b) inserting into the genome of a plant cell of said plant that is capable of regeneration into a differentiated whole plant, a recombinant double stranded DNA molecule comprising: (i) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (ii) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene or coding sequence; and preferably (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (c) obtaining a transformed plant cell in said plant; and (d) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 10

The method any one of claims 2, 4, 5 or 6 wherein said promotor is the promotor of the sense gene.

Claim 12

A method of producing a male sterile plant from a plant selected from those species of pollen bearing plants that are capable of being genetically transformed which method comprises the steps of: (a) identifying and isolating preferably from said pollen producing plant, a sense gene or coding sequence that is critical to cellular function or development and expressed in metabolically competent cell types; (b) inserting into the genome of a plant cell a recombinant double stranded DNA molecule comprising: i) a DNA sequence that codes for RNA that is complimentary to the RNA encoded by said sense gene or coding sequence; ii) a pollen specific promoter which functions in said plant cell to cause transcription of said DNA sequence into RNA in a time frame which enables said RNA to block the function of the RNA encoded by said sense gene or coding sequence; and preferably iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence; (c) obtaining a plant cell that has been genetically transformed with said DNA sequence; (d) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 13

A method of producing a male sterile plant from a plant selected from those species of pollen bearing plants that are capable of being genetically transformed which method comprises the steps of: (a) identifying and isolating preferably from said pollen producing plant, a sense gene or coding sequence that is critical to cellular function or development and expressed in metabolically competent cell types; (b) inserting into the genome of a plant cell a recombinant double stranded DNA molecule comprising: i) a DNA sequence that codes for RNA that is complimentary to the RNA encoded by said sense gene or coding sequence; ii) a pollen specific promoter which functions in said plant cell to cause transcription of said DNA sequence into RNA in a time frame which enables said RNA to block the function of the RNA encoded by said sense gene or coding sequence; and preferably iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence; (c) obtaining a plant cell that has been genetically transformed with said DNA sequence; (d) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 14

A method of producing hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: i) identifying and isolating preferably from said pollen producing plant, a sense gene or a coding sequence that is critical to celular function or development and expressed in metabolically competent cell types. ii) inserting into the genome of a plant cell of said plant that is capable of regeneration into a differentiated whole plant, a gene which confers said plant on resistance to a chemical agent or physiological stress and, linked to said gene, a recombinant DNA sequence comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (B) a promoter which functions in said plant cell to cause transcription of said DNA sequence into RNA in a time frame which enables said RNA to block the function of the RNA encoded by said sense gene or coding sequence; and preferably; (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence; iii) obtaining a transformed plant cell of said plant; and iv) regenerating from said transformed plant cell a plant which is genetically transformed with the genes described in step (a)(ii) above and which is male sterile; and (b) increasing the number of genetically transformed plants by: i) crossing the genetically transformed plant described in step (a)(iv) above with a suitable male fertile plant; ii) using a chemical agent or physiological stress to eliminate plants which do not contain the genes described in step (a)(ii) above among plants grown from seed produced by such cross; and iii) repeating such a cross over several generations with the plants obtained as in step (b)(ii) above in the presence of said chemical agent or physiological stress to increase the numbers of male sterile plants; (c) effecting a hybrid cross by pollinating said male sterile plants with pollen from a suitable male fertile donors.

Claim 15

A method of producing hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: i) identifying and isolating preferably from said pollen producing plant, a sense gene or a coding sequence that is critical to celular function or development and expressed in metabolically competent cell types. ii) inserting into the genome of a plant cell of said plant that is capable of regeneration into a differentiated whole plant, a gene which confers said plant on resistance to a chemical agent or physiological stress and, linked to said gene, a recombinant DNA sequence comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (B) a promoter which functions in said plant cell to cause transcription of said DNA sequence into RNA in a time frame which enables said RNA to block the function of the RNA encoded by said sense gene or coding sequence; and preferably; (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence; iii) obtaining a transformed plant cell of said plant; and iv) regenerating from said transformed plant cell a plant which is genetically transformed with the genes described in step (a)(ii) above and which is male sterile; and (b) increasing the number of genetically transformed plants by: i) crossing the genetically transformed plant described in step (a)(iv) above with a suitable male fertile plant; ii) using a chemical agent or physiological stress to eliminate plants which do not contain the genes described in step (a)(ii) above among plants grown from seed produced by such cross; and iii) repeating such a cross over several generations with the plants obtained as in step (b)(ii) above in the presence of said chemical agent or physiological stress to increase the numbers of male sterile plants; (c) effecting a hybrid cross by pollinating said male sterile plants with pollen from a suitable male fertile donors.

Claim 16

A method of producing a male sterile plant from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) identifying and isolating preferably from said pollen producing producing plant a sense gene or coding sequence that is critical to pollen formation or function in said plant; (b) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant; a gene which confers on said plant resistance to a selective agent and, linked to said gene, a gene or coding sequence comprising: (i) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence ; (ii) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene; and preferably (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (c) obtaining a transformed plant cell in said plant; and (d) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 17

A method of producing a male sterile plant from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) identifying and isolating preferably from said pollen producing producing plant a sense gene or coding sequence that is critical to pollen formation or function in said plant; (b) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant; a gene which confers on said plant resistance to a selective agent and, linked to said gene, a gene or coding sequence comprising: (i) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence ; (ii) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene; and preferably (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (c) obtaining a transformed plant cell in said plant; and (d) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 18

A method of producing hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: (i) identifying and isolating, preferably from said pollen producing plant a sense gene or coding sequence that is critical to pollen formation or function in said plant; (ii) inserting into the genome of a plant cell of said plant which is capable of being regenerated into a differentiated whole plant, a gene which confers on said plant resistance to a chemical agent or physiological stress and, linked to said gene, a gene comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (B) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene or coding sequence; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence (iii) obtaining a transformed plant cell of said plant; and (iv) regenerating from said transformed plant cell a plant which is genetically transformed with the genes described in step (a)(ii) above and is male sterile; and (b) increasing the number of genetically transformed plants by: (i) crossing the genetically transformed plant described in step (a)(iv) above with a suitable male fertile plant; (ii) using said chemical agent or physiological stress to eliminate plants which do not contain the genes described in step (a)(ii) above among plants grown from seed produced by such cross; and (iii) repeating such a cross over several generations with the plants selected for as in step (b)(ii) above in the presence of said chemical agent or physiological stress to increase the numbers of male sterile plants; (c) effecting a hybrid cross by pollinating said male sterile plants with pollen from suitable male fertile donors.

Claim 19

A method of producing hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: (i) identifying and isolating, preferably from said pollen producing plant a sense gene or coding sequence that is critical to pollen formation or function in said plant; (ii) inserting into the genome of a plant cell of said plant which is capable of being regenerated into a differentiated whole plant, a gene which confers on said plant resistance to a chemical agent or physiological stress and, linked to said gene, a gene comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (B) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene or coding sequence; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence (iii) obtaining a transformed plant cell of said plant; and (iv) regenerating from said transformed plant cell a plant which is genetically transformed with the genes described in step (a)(ii) above and is male sterile; and (b) increasing the number of genetically transformed plants by: (i) crossing the genetically transformed plant described in step (a)(iv) above with a suitable male fertile plant; (ii) using said chemical agent or physiological stress to eliminate plants which do not contain the genes described in step (a)(ii) above among plants grown from seed produced by such cross; and (iii) repeating such a cross over several generations with the plants selected for as in step (b)(ii) above in the presence of said chemical agent or physiological stress to increase the numbers of male sterile plants; (c) effecting a hybrid cross by pollinating said male sterile plants with pollen from suitable male fertile donors.

Claim 22

The method of any of one of claims 2, 10, 11, 12, 13, 14 or 15 wherein the selective agent is glyphosate or chlorsulfuron.

Claim 23

A method of producing hybrid seed with restored male fertility among plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: (i) identifying and isolating preferably from said pollen producing plant, a sense gene or coding sequence that is critical to pollen formation or function in said plant; (ii) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant, a recombinant double stranded DNA molecule comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (B) an inducible promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA during the time of transcription of said sense gene; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence (iii) obtaining a transformed plant cell of said plant; and (iv) regenerating from said transformed plant cell a plant which is genetically transformed with said double stranded DNA molecule described in (ii) above and is capable of being rendered male sterile by said inducer; (b) increasing the number of genetically transformed plants by: (i) growing the genetically transformed plant described in step (a)(iv) above in the absence of the relevant inducer to produce a male fertile plant; and (ii) permitting self-fertilzation; and (iii) growing seed of such a male fertile plant, over a number of generations, in the absence of the relevant inducer to increase the number of genetically transformed plants; (c) pollinating said genetically transformed plants in the presence of the relevant inducer during pollen formation with pollen from a suitable line of male fertile donors.

Claim 24

A method of producing hybrid seed with restored male fertility among plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: (i) identifying and isolating preferably from said pollen producing plant, a sense gene or coding sequence that is critical to pollen formation or function in said plant; (ii) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant, a recombinant double stranded DNA molecule comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene or coding sequence; (B) an inducible promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA during the time of transcription of said sense gene; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence (iii) obtaining a transformed plant cell of said plant; and (iv) regenerating from said transformed plant cell a plant which is genetically transformed with said double stranded DNA molecule described in (ii) above and is capable of being rendered male sterile by said inducer; (b) increasing the number of genetically transformed plants by: (i) growing the genetically transformed plant described in step (a)(iv) above in the absence of the relevant inducer to produce a male fertile plant; and (ii) permitting self-fertilzation; and (iii) growing seed of such a male fertile plant, over a number of generations, in the absence of the relevant inducer to increase the number of genetically transformed plants; (c) pollinating said genetically transformed plants in the presence of the relevant inducer during pollen formation with pollen from a suitable line of male fertile donors.

Claim 26

A method of producing a male sterile plant from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant, a recombinant double stranded DNA molecule comprising: (i) a pollen specific promotor; (ii) a DNA sequence that codes for a cytotoxic molecule; and (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (b) obtaining a transformed plant cell; and (c) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 27

A method of producing a male sterile plant from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant, a recombinant double stranded DNA molecule comprising: (i) a pollen specific promotor; (ii) a DNA sequence that codes for a cytotoxic molecule; and (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (b) obtaining a transformed plant cell; and (c) regenerating from said transformed plant cell a genetically transformed plant which is male sterile.

Claim 28

A method to produce hybrid seed with restored male fertility from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) inserting into the genome of a plant cell of said pollen producing plant that is capable of being regenerated into a differentiated whole plant, a gene which confers on said plant resistance to a chemical agent or physiological stress and linked to said gene a recombinant double stranded DNA molecule comprising: (i) a DNA sequence which codes for cytotoxic molecule; (ii) a pollen specific promoter which functions in said plant cell to cause transcription of said DNA sequence; and (iii) a terminator sequence which defines a termination signal during transcription of such DNA sequence. (b) obtaining a transformed plant cell; (c) regenerating from said plant cell a genetically transformed plant which is male sterile; (d) increasing the number genetically transformed plants by: i) crossing the geneticaly transformed plant described in step (c) above with a suitable male fertile plant; ii) using a chemical agent or physiological stress to eliminate plants which do not contain the genes described in step (a) above among plants grown from seed produced by such cross; and iii) repeating such a cross over several generations with the plants obtained as in step (d) ii) above in the presence of said chemical agent or physiological stress to increase the numbers of male sterile plants; (e) inserting into a plant cell of suitable male fertile plant selected from the same species a recombinant double stranded DNA molecule comprising: (i) a DNA sequence which codes for RNA that is complimentary to the RNA sequence coding for said cytotoxic molecule; (ii) a promoter which causes transcription of the DNA sequence defined in step (d)(i) above at about the time of transcription of the DNA sequence defined in step (a)(i); (iii) a terminator sequence which defines a termination signal during transcription of the DNA sequence described in step (e)(i) above; (f) obtaining a transformed plant cell from step (d); (g) regenerating from said transformed plant cell described in step (d) above a genetically transformed male fertile plant. (h) producing a restorer line by permitting said genetically transformed male fertile plant to self fertilize and growing seed of such plant, over a number of generations to increase the mumbers of genetically transformed male fertile plants; (i) effecting a hybrid cross by pollinating said male sterile plants with pollen from said genetically transformed male fertile plants.

Claim 29

A method to produce hybrid seed with restored male fertility from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) inserting into the genome of a plant cell of said pollen producing plant that is capable of being regenerated into a differentiated whole plant, a gene which confers on said plant resistance to a chemical agent or physiological stress and linked to said gene a recombinant double stranded DNA molecule comprising: (i) a DNA sequence which codes for cytotoxic molecule; (ii) a pollen specific promoter which functions in said plant cell to cause transcription of said DNA sequence; and (iii) a terminator sequence which defines a termination signal during transcription of such DNA sequence. (b) obtaining a transformed plant cell; (c) regenerating from said plant cell a genetically transformed plant which is male sterile; (d) increasing the number genetically transformed plants by: i) crossing the geneticaly transformed plant described in step (c) above with a suitable male fertile plant; ii) using a chemical agent or physiological stress to eliminate plants which do not contain the genes described in step (a) above among plants grown from seed produced by such cross; and iii) repeating such a cross over several generations with the plants obtained as in step (d) ii) above in the presence of said chemical agent or physiological stress to increase the numbers of male sterile plants; (e) inserting into a plant cell of suitable male fertile plant selected from the same species a recombinant double stranded DNA molecule comprising: (i) a DNA sequence which codes for RNA that is complimentary to the RNA sequence coding for said cytotoxic molecule; (ii) a promoter which causes transcription of the DNA sequence defined in step (d)(i) above at about the time of transcription of the DNA sequence defined in step (a)(i); (iii) a terminator sequence which defines a termination signal during transcription of the DNA sequence described in step (e)(i) above; (f) obtaining a transformed plant cell from step (d); (g) regenerating from said transformed plant cell described in step (d) above a genetically transformed male fertile plant. (h) producing a restorer line by permitting said genetically transformed male fertile plant to self fertilize and growing seed of such plant, over a number of generations to increase the mumbers of genetically transformed male fertile plants; (i) effecting a hybrid cross by pollinating said male sterile plants with pollen from said genetically transformed male fertile plants.

Claim 30

The method of claims 20 or 24 wherein said pollen specific promotor consists of a DNA sequence which comprises the sequence of nucleotides, 1-595, in figure 2a or a functional fragment thereof.

Claim 31

A method of producing a plant which carries the male sterile trait from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) transforming a plant cell of said plant which is capable of being regenerated into a differentiated whole plant, with a sense gene which confers on said plant resistance to achemical agent or naturally occurring or artificially induced physiological stress; (b) regenerating from said transformed plant cell a genetically transformed plant which is resistant to the same stress; (c) inserting into the genome of a plant cell of said stress resistant plant which is capable of being regenerated into a differentiated whole plant a recombinant double stranded DNA molecule comprising: (i) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by the said sense gene; (ii) a pollen specific promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA; and preferably (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (d) obtaining a transformed plant cell; and (e) regenerating from said transformed plant cell a plant which has been genetically transformed with the genes described in step (a) and step (c) above and which can be rendered male sterile by said chemical agent or stress.

Claim 32

A method of producing a plant which carries the male sterile trait from a plant selected from those species of pollen producing plants which are capable of being genetically transformed, which method comprises the steps of: (a) transforming a plant cell of said plant which is capable of being regenerated into a differentiated whole plant, with a sense gene which confers on said plant resistance to achemical agent or naturally occurring or artificially induced physiological stress; (b) regenerating from said transformed plant cell a genetically transformed plant which is resistant to the same stress; (c) inserting into the genome of a plant cell of said stress resistant plant which is capable of being regenerated into a differentiated whole plant a recombinant double stranded DNA molecule comprising: (i) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by the said sense gene; (ii) a pollen specific promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA; and preferably (iii) a terminator sequence which defines a termination signal during transcription of said DNA sequence. (d) obtaining a transformed plant cell; and (e) regenerating from said transformed plant cell a plant which has been genetically transformed with the genes described in step (a) and step (c) above and which can be rendered male sterile by said chemical agent or stress.

Claim 33

A method of producing hybrid seed with restored male fertility from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a plant which carries a male sterile trait by: i) inserting concomitantly or independently into the genome of a plant cell of said pollen producing plant which is capable of being regenerated into a differentiated whole plant, a sense gene which confers on said plant resistance to a chemical agent or a naturally occurring or artificially induced physiological stress and a recombinant double stranded DNA molecule comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene; (B) a pollen specific promoter which functions in said plant cell to cause transcription of said DNA sequence into RNA; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence; ii) obtaining a plant cell of a plant which has been transformed with the genes described in step (i) above; iii) regenerating from said transformed plant cell a plant which is genetically transformed with the genes described in step (i) above and can be rendered male sterile by said chemical agent or stress; (b) increasing the number of genetically transformed plants: i) growing the genetically transformed plant described in step (a)(iii) above in isolation from the same stress or chemical agent to produce a self-fertile plant; iii) permitting self-fertilization; and iii) growing seed of such self-fertile plant, over a number of generations in isolation from the same stress or chemical agent to increase the number of genetically transformed plants; (c) effecting a hybrid crossing growing said genetically transformed plants alongside plants of a suitable line of male fertile donors in the presence of the same stress or chemical agent during pollen formation to produce male sterile plant and to permit pollination of the male sterile plants.

Claim 34

A method of producing hybrid seed with restored male fertility from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a plant which carries a male sterile trait by: i) inserting concomitantly or independently into the genome of a plant cell of said pollen producing plant which is capable of being regenerated into a differentiated whole plant, a sense gene which confers on said plant resistance to a chemical agent or a naturally occurring or artificially induced physiological stress and a recombinant double stranded DNA molecule comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by said sense gene; (B) a pollen specific promoter which functions in said plant cell to cause transcription of said DNA sequence into RNA; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence; ii) obtaining a plant cell of a plant which has been transformed with the genes described in step (i) above; iii) regenerating from said transformed plant cell a plant which is genetically transformed with the genes described in step (i) above and can be rendered male sterile by said chemical agent or stress; (b) increasing the number of genetically transformed plants: i) growing the genetically transformed plant described in step (a)(iii) above in isolation from the same stress or chemical agent to produce a self-fertile plant; iii) permitting self-fertilization; and iii) growing seed of such self-fertile plant, over a number of generations in isolation from the same stress or chemical agent to increase the number of genetically transformed plants; (c) effecting a hybrid crossing growing said genetically transformed plants alongside plants of a suitable line of male fertile donors in the presence of the same stress or chemical agent during pollen formation to produce male sterile plant and to permit pollination of the male sterile plants.

Claim 35

The method of claims 4, 22, or 23 herein said pollen specific promotor consists of DNA sequence which comprises the sequence of nucleotides, 1-595 in figure 2a, or a functional fragment thereof.

Claim 36

The method of any one of claims 4, 22, 23 or 24 wherein said physiological stress is a herbicide.

Claim 38

A method of producing hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: (i) identifying and isolating, preferably from said pollen producing plant, a sense gene or coding sequence that is critical to pollen formation or function in said plant; (ii) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant a recombinant double stranded DNA molecule comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by the said sense gene or coding sequence; (B) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene or coding sequence; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence (iii) obtaining a transformed plant cell of said plant and (iv) regenerating from said transformed plant cell a genetically transformed plant which is male sterile; (b) producing a male sterile line by clonal propagation of said genetically transformed plant using cells derived from tissue explants of said genetically transformed plant; (c) growing plants of the male sterile line alongside plants of a suitable line of male fertile donors to permit pollination of the male sterile plant.

Claim 39

A method of producing hybrid seed from plants selected from those species of pollen producing plants which are capable of being genetically transformed comprising the steps of: (a) producing a male sterile plant by: (i) identifying and isolating, preferably from said pollen producing plant, a sense gene or coding sequence that is critical to pollen formation or function in said plant; (ii) inserting into the genome of a plant cell of said plant that is capable of being regenerated into a differentiated whole plant a recombinant double stranded DNA molecule comprising: (A) a DNA sequence that codes for RNA that is complimentary to the RNA sequence encoded by the said sense gene or coding sequence; (B) a promotor which functions in said plant cell to cause transcription of said DNA sequence into RNA at about the time of transcription of said sense gene or coding sequence; and preferably (C) a terminator sequence which defines a termination signal during transcription of said DNA sequence (iii) obtaining a transformed plant cell of said plant and (iv) regenerating from said transformed plant cell a genetically transformed plant which is male sterile; (b) producing a male sterile line by clonal propagation of said genetically transformed plant using cells derived from tissue explants of said genetically transformed plant; (c) growing plants of the male sterile line alongside plants of a suitable line of male fertile donors to permit pollination of the male sterile plant.

Claim 40

The method of any of one of claims 1 to 27, wherein the selected pollen producing plant is a plant of the species Brassica napus or Brassica campestris .

Claim 41

The method of any one of claims 1 to 27 wherein the selected pollen producing plant is of the genus Brassica .

Claim 42

The method of any one of claims 1 to 27 wherein the selected pollen producing plant is of the family Cruciferae .

Claim 43

The method of any one of claims 1 to 27 wherein the selected pollen producing plant is of the family Solanacae