Claims for Patent: 6,008,437

Summary for Patent: 6,008,437

Title:	Use of anthocyanin genes to maintain male sterile plants
Abstract:	A plant consisting essentially of cells which comprise in their genome a homozygous male-sterility genotype at a first genetic locus; and a color-linked restorer genotype at a second genetic locus, which is heterozygous (Rf/-) for a foreign DNA Rf. The foreign DNA Rf comprises: a) a fertility-restorer gene capable of preventing the phenotypic expression of the male-sterility genotype, and b) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in cells of seeds of the plant which is capable of producing anthocyanin at least in the seeds of the plant, so that anthocyanin production in the seeds is visible externally. Preferably, the anthocyanin regulatory gene is a shortened R, B or C1 gene or a continuation thereof. The invention also relates to DNA sequences encoding shortened R, B or C1 anthocyanin regulatory genes and to a process for maintaining a line of male-sterile plants which comprises crossing a male-sterile parent plant and a maintainer parent plant comprising homozygous male-sterility genotype and a restore genotype comprising fertility-restorer gene and an anthocyanin regulatory gene.
Inventor(s):	Krebbers; Enno (Houston, TX), Williams; Mark (Ghent, BE), Leemans; Jan (Deurle, BE)
Assignee:	Plant Genetic Systems (Brussels, BE)
Application Number:	08/750,357
Patent Claims:	1. A process for maintaining a line of male-sterile plants or of maintainer plants, said process comprising: (i) crossing: (a) a male-sterile line comprising male-sterile parent plants which comprise a homozygous male-sterility genotype at a first genetic locus and which lack at least one functional regulatory gene required for anthocyanin production in the seeds, and (b) a maintainer line comprising male-fertile parent plants which comprise said homozygous male-sterility genotype at said first genetic locus, and which further comprise at a second genetic locus which segregates independently from said first genetic locus, a foreign DNA comprising: i) a restorer gene, the expression of which prevents the phenotypic expression of said homozygous male-sterility genotype, and ii) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in the cells of seeds of said plant which is capable of producing anthocyanin at least in the seeds of said plant so that anthocyanin production in the seeds is visible externally; wherein said foreign DNA is heterozygous at said second genetic locus, and wherein said male-sterile plants produce seeds producing anthocyanin, as well as seeds not producing anthocyanin, said anthocyanin being produced only in seeds comprising said at least one anthocyanin regulatory gene in said foreign DNA; (ii) harvesting seeds from said male-sterile parent plants; and (iii) selecting from the harvested seeds, either those seeds which do not produce anthocyanin, said selected seeds being capable of growing into a new generation of male-sterile plants, or selecting those seeds which produce anthocyanin, said selected seeds being capable of growing into a new generation of male-fertile plants. 2. The process of claim 1, in which said at least one anthocyanin regulatory gene is capable of producing anthocyanin at least in the aleurone of seeds. 3. The process of claim 1, in which said first genetic locus is a foreign genetic locus and comprises a male-sterility gene which, if present in a plant in the absence of said restorer genotype, would render the plant male-sterile, and wherein said male-sterility gene is homozygous at said first genetic locus. 4. The process of claim 3, wherein said male-sterility genotype at said first genetic locus comprises a foreign DNA which comprises a male-sterility gene comprising: a male-sterility DNA encoding an RNA, protein or polypeptide which, when produced or overproduced in a stamen cell, significantly disturbs the metabolism, functioning and/or development of said cell; and a sterility promoter capable of directing expression of said male-sterility DNA selectively in the stamen cells of the plant; the male-sterility DNA being in the same transcriptional unit as, and under the control of, the sterility promoter; and in which said restorer genotype at said second genetic locus comprises a foreign DNA which comprises a restorer gene which comprises: a fertility-restorer DNA encoding a restorer RNA, protein or polypeptide which, when expressed in the same cell as said male-sterility gene, prevents the phenotypic expression of said male-sterility gene; and a restorer promoter capable of directing expression of said fertility-restorer DNA at least in the same cells in which said male-sterility gene is expressed, so that the phenotypic expression of said male-sterility gene is prevented; said fertility-restorer DNA being in the same transcriptional unit as, and under the control of, said restorer promoter. 5. The process of claim 4, wherein said male-sterility DNA encodes a barnase and said fertility-restorer DNA encodes a barstar. 6. The process of claim 4, wherein said sterility promoter is a TA29 promoter or a CA55 promoter. 7. The process of claim 4, in which said sterility promoter is a PT72, PT42 or PE1 promoter. 8. The process of claim 4, in which said restorer promoter is identical to said sterility promoter. 9. The process of claim 8, in which said restorer promoter is a TA29 promoter or a CA55 promoter. 10. The process of claim 1, in which said first genetic locus is an endogenous male-sterility locus, comprising a recessive allele in homozygous condition, and in which said restorer gene is the dominant allele at said endogenous male-sterility locus. 11. The process of claim 1, in which said male-sterile parent plants contain a first anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in the cells of seeds that is functionally expressed in the seeds and said male-fertile parent plants contain a second anthocyanin regulatory gene which, when present with said first anthocyanin regulatory gene in the genome of a plant, is capable of conditioning the production of anthocyanin in the seeds which is visible externally. 12. The process of claim 1, wherein said at least one anthocyanin regulatory gene is selected from the group of a first gene that is expressed in seeds to produce a first active regulatory protein having the same activity as the protein encoded by a functional R or B gene, and a second gene that is expressed in seeds to produce a second active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 13. The process of claim 12, wherein said male-fertile parent plants comprise said first gene involved in the regulation of anthocyanin biosynthesis in the cells of seeds that is functionally expressed in the seeds in said foreign DNA, and wherein said male-sterile parent plants do not contain a gene that is functionally expressed in the seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional R or B gene. 14. The process of claim 13, in which said first gene comprises a DNA encoding a B-peru protein which is under the control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 358 promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 and a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 15. The process of claim 14, in which said first gene does not comprise an intron. 16. The process of claim 12, wherein said male-fertile parent plants comprise said second gene involved in the regulation of anthocyanin biosynthesis in the cells of seeds that is functionally expressed in the seeds in said foreign DNA, and wherein said male-sterile parent plants do not contain a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 17. The process of claim 16, in which said second gene comprises a DNA encoding the C1 protein which is under control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 35S promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 or a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 18. The process of claim 12, wherein said male-fertile parent plants comprise said first gene and said second gene in said foreign DNA, and wherein said male-sterile parent plants do not contain 1) a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional R or B gene, and 2) a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 19. The process of claim 18, in which said first gene comprises a DNA encoding the B-peru protein under the control of a promoter of the B-peru gene, a promoter comprising the nucleotides sequence of SEQ ID No. 6 between positions 1 and 188, a 35S promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 or a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG; and in which said second gene comprises a DNA encoding the C1 protein which is under the control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 35S promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 and a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 20. The process of claim 1, in which said anthocyanin regulatory gene is selected from the group of a C1 or C1-S gene having a nucleotide sequence corresponding to the sequence between positions 447 and 2418 of SEQ ID No. 1, a B-peru gene having a nucleotide sequence corresponding to the sequence between positions 1 and 3272 of SEQ ID No. 6, and the Eco-Sall fragment having a length of about 4000 bp of pCOL13 or a combination thereof which is functional for conditioning and regulating anthocyanin biosynthesis in seeds. 21. The process of claim 20, in which said anthocyanin regulatory gene does not comprise any introns. 22. A kit for maintaining a line of male-sterile or maintainer plants, said kit comprising: (i) a male-sterile parent plant of said line which comprises a homozygous male-sterility genotype at a first genetic locus and which lacks an anthocyanin regulatory gene required for anthocyanin production in seeds, and (ii) a maintainer parent plant of said line which comprises said homozygous male-sterility genotype at said first genetic locus, and, which further comprises, at a second genetic locus which segregates independently from said first genetic locus, a foreign DNA comprising: a) a restorer gene, whose expression prevents phenotypic expression of said homozygous male-sterility genotype, and b) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in the cells of seeds of said plant which is capable of producing anthocyanin at least in the seeds of said plant so that anthocyanin production in the seeds is visible externally; wherein said foreign DNA is heterozygous at said second genetic locus, and, wherein said male-sterile and male-fertile parent plant can be crossed to produce, on said male-sterile plants, seeds which produce anthocyanin, said anthocyanin being produced only in seeds comprising said at least one anthocyanin regulatory gene in said foreign DNA. 23. The kit of claim 22 in which said anthocyanin regulatory gene in said foreign DNA is expressed at least in the aleurone of seeds. 24. The kit of claim 22, in which said first genetic locus is a foreign genetic locus and comprises a male-sterility gene which, if present in the plant in the absence of said restorer genotype, would render the plant male-sterile, and wherein said male-sterility gene is homozygous at said first genetic locus. 25. The kit of claim 24 wherein said male-sterility genotype at said first enetic locus comprises a foreign DNA which comprises a male-sterility gene comprising: 1) a male-sterility DNA encoding an RNA, protein or polypeptide which, when produced or overproduced in a stamen cell, significantly disturbs the metabolism, functioning and/or development of said cell, and, 2) a sterility promoter capable of directing expression of said male-sterility DNA selectively in the stamen cells of the plant; the male-sterility DNA being in the same transcriptional unit as, and under the control of, the sterility promoter; and in which said restorer genotype at said second genetic locus comprises a foreign DNA which comprises a restorer gene which comprises: 1) a fertility-restorer DNA encoding a restorer RNA, protein or polypeptide which, when expressed in the same cell as said male-sterility gene, prevents the phenotypic expression of said male-sterility gene, and, 2) a restorer promoter capable of directing expression of said fertility-restorer DNA at least in the same cells in which said male-sterility gene is expressed, so that the phenotypic expression of said male-sterility gene is prevented; said fertility-restorer DNA being in the same transcriptional unit as, and under the control of, said restorer promoter. 26. The kit of claim 24, wherein said male-sterility DNA encodes a barnase and said fertility-restorer DNA encodes a barstar. 27. The kit of claim 24, wherein said sterility promoter is a TA29 promoter or a CA55 promoter. 28. The kit of claim 24, in which said sterility promoter is a PT72, PT42 or PE1 promoter. 29. The kit of claim 24, in which said restorer promoter is identical to said sterility promoter. 30. The kit of claim 29, in which said restorer promoter is a TA29 promoter or a CA55 promoter. 31. The kit of claim 23, in which said first genetic locus in an endogenous male-sterility locus, comprising a recessive allele in homozygous condition, and in which said restorer gene is the dominant allele at said endogenous male-sterility locus. 32. The kit of claim 23, in which said male-sterile parent plant contains a first anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in the cells of seeds that is functionally expressed in the seeds and said maintainer parent plant contains a second anthocyanin regulatory gene which when present with said first anthocyanin regulatory gene in the genome of a plant is capable of conditioning the production of anthocyanin in the seeds which is visible externally. 33. The kit of claim 23, wherein said at least one anthocyanin regulatory gene is selected from the group of a first gene that is expressed in seeds to produce a first active regulatory protein having the same activity as the protein encoded by a functional R or B gene, and a second gene that is expressed in seeds to produce a second active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 34. The kit of claim 33, wherein said maintainer parent plant comprises said first gene in said foreign DNA, and said male-sterile parent plant does not contain a gene that is functionally expressed in the seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional R or B gene. 35. The kit of claim 34, in which said first gene comprises a DNA encoding a B-peru protein which is under the control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 35S promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 and a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 36. The kit of claim 35, in which said first gene does not comprise an intron. 37. The kit of claim 33, wherein said maintainer parent plant comprises said second gene in said foreign DNA, and said male-sterile parent plants do not contain a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 38. The kit of claim 37, in which said second gene comprises a DNA encoding the C1 protein which is under control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 35S promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 or a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 39. The kit of claim 33, wherein said maintainer parent plant comprises said first gene and said second gene in said foreign DNA, and said male-sterile parent plants does not contain 1) a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional R or B gene, and 2) a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 40. The kit of claim 39, in which said first gene comprises a DNA encoding the B-peru protein under the control of a promoter of the B-peru gene, a promoter comprising the nucleotides sequence of SEQ ID No. 6 between positions 1 and 188, a 35S promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 or a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG; and in which said second gene comprises a DNA encoding the C1 protein which is under the control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 35S promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 and a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 41. The kit of claim 23, in which said anthocyanin regulatory gene is selected from the group of a C1 or C1-S gene having a nucleotide sequence corresponding to the sequence between positions 447 and 2418 of SEQ ID No. 1, a B-peru gene having a nucleotide sequence corresponding to the sequence between positions 1 and 3272 of SEQ ID No. 6, and the Eco-Sall fragment having a length of about 4000 bp of pCOL13 or a combination thereof which is functional for conditioning and regulating anthocyanin biosynthesis in seeds. 42. The kit of claim 41 in which said anthocyanin regulatory gene does not comprise any introns. 43. The process of claim 1, in which said male-sterile parent plants and said male fertile parent plants are plants of the same species selected from the group of corn, wheat and rice. 44. The kit of claim 23, in which said male-sterile parent plant and said maintainer parent plant are plants of the same species selected from the group of corn, wheat and rice. 45. A male-fertile parent plant for maintaining a male-sterile line of a plant comprising male-sterile parent plants which comprise a homozygous male-sterility genotype at a first genetic locus and which lack a functional anthocyanin regulatory gene required for anthocyanin production in seeds; wherein said male-fertile parent plant comprises said homozygous male-sterility genotype at said first genetic locus, and further comprises, at a second genetic locus which segregates independently from said first genetic locus, a foreign DNA comprising: a) a restorer gene, whose expression of which prevents the phenotypic expression of said homozygous male-sterility genotype, to render said plant male-fertile, and b) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in the cells of seeds of said plant which is capable of producing anthocyanin at least in the seeds of said plant, so that anthocyanin production in the seeds is visible externally; wherein said foreign DNA is heterozygous at said second genetic locus, and, wherein said male-fertile parent plant can be crossed to said male-sterile parent plants to produce, on said male-sterile plants, seeds which produce anthocyanin, said anthocyanin being produced only in seeds containing said at least one anthocyanin regulatory gene in said foreign DNA. 46. The plant of claim 45, in which said anthocyanin regulatory gene is capable of producing anthocyanin at least in the aleurone of seeds. 47. The plant of claim 45, in which said first genetic locus is a foreign genetic locus and comprises a male-sterility gene which, if present in the plant in the absence of said restorer genotype, would render the plant male-sterile, and wherein said male-sterility gene is homozygous at said first genetic locus. 48. The plant of claim 47, in which said first genetic locus is homozygous for a foreign DNA which comprises a male-sterility gene comprising: (1) a male-sterility DNA encoding an RNA, protein or polypeptide which, when produced or overproduced in a stamen cell, significantly disturbs the metabolism, functioning and/or development of said cell, and, (2) a sterility promoter capable of directing expression of said male-sterility DNA selectively in the stamen cells of the plant; said male-sterility DNA being in the same transcriptional unit as, and under the control of, said sterility promoter, and in which said restorer gene at said second genetic locus comprises at least: (1) a fertility-restorer DNA encoding a restorer RNA, protein or polypeptide which, when expressed in the same cell as said male-sterility gene, prevents the phenotypic expression of said male-sterility gene, and, (2) a restorer promoter capable of directing expressing of said fertility-restorer DNA at least in the same cells as those in which said male-sterility gene is prevented; said fertility-restorer DNA being in the same transcriptional unit as, and under the control of, said restorer promoter. 49. The plant of claim 48, wherein said male-sterility DNA encodes a barnase and said fertility-restorer DNA encodes a barstar. 50. The plant of claim 48, in which said sterility promoter is a TA29 promoter or a CA55 promoter. 51. The plant of claim 48 in which said sterility promoter is a PT72, PT42 or PE1 promoter. 52. The plant of claim 48, in which said restorer promoter is identical to said sterility promoter. 53. The plant of claim 52, in which said restorer promoter is a TA29 promoter or a CA55 promoter. 54. The plant of claim 18, in which said first genetic locus is an endogenous male-sterility locus comprising a recessive allele in homozygous condition, and in which said restorer gene is the dominant allele at said endogenous male-sterility locus. 55. The plant of claim 45, wherein said at least one anthocyanin regulatory gene is selected from the group of a first gene that is expressed in seeds to produce a first active regulatory protein having the same activity as the protein encoded by a functional R or B gene, and a second gene that is expressed in seeds to produce a second active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 56. The plant claim 55, which comprises said first gene in said foreign DNA, and which does not otherwise contain a gene that is functionally expressed in the seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional R or B gene. 57. The plant of claim 56 in which said first gene comprises a DNA encoding a B-peru protein which is under the control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 356 promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 or a promoter comprising the sequences of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 58. The plant of claim 57, in which said first gene does not comprise an intron. 59. The plant of claim 55, which comprises said second gene in said foreign DNA, and which plant does not otherwise contain a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 60. The plant of claim 59, in which said second gene comprises a DNA encoding the C1 protein which is under control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 358 promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 61. The plant of claim 55, which comprises said first gene and said second gene in said foreign DNA, and which plant does not otherwise contain 1) a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional R or B gene, and 2) a gene that is functionally expressed in seeds which encodes an active regulatory protein having the same activity as the protein encoded by a functional C1 gene. 62. The plant of claim 61, in which said first gene comprises a DNA encoding the B-peru protein under the control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 358 promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061, or a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 938 and 939 is changed to TTAGG; and in which said second gene comprises a DNA encoding the C1 protein which is under the control of a promoter of the B-peru gene, a promoter comprising the nucleotide sequence of SEQ ID No. 6 between positions 1 and 188, a 358 promoter, a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061, or a promoter comprising the sequence of SEQ ID No. 1 between positions 447 and 1061 in which TGCAG between positions 935 and 939 is changed to TTAGG. 63. The plant of claim 45, in which said anthocyanin regulatory gene is selected from the group of a C1 or C1-S gene having a nucleotide sequence corresponding to the sequence between positions 447 and 2418 of SEQ ID No. 1, a B-peru gene having a nucleotide sequence corresponding to the sequence between positions 1 and 3272 of SEQ ID No. 6, and the Eco-Sall fragment having a length of about 4000 bp of pCOL13 or a combination thereof which is functional for conditioning and regulating anthocyanin biosynthesis in seeds. 64. The plant of claim 63, in which said anthocyanin regulatory gene does not comprise an intron. 65. The plant of claim 45, which is selected from the group consisting of corn, wheat and rice.

Details for Patent 6,008,437

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26