Seeds

Abstract

This invention relates to a method for modifying cell proliferation in a plant which comprises the step of transforming a plant, or plant propagating material, with a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within the integuments and/or seed coat and at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby on expression of that nucleic acid sequence cell proliferation is modified. The invention also relates to a plant which includes a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within integuments and/or seed coat an at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulated cell proliferation, whereby on expression of that nucleic acid sequence cell proliferation is modified. The invention also relates to reproducible plant material including a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within integuments and/or seed coat and at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulated cell proliferation, whereby expression of that nucleic acid sequence cell proliferation is modified. According to another aspect of the invention, there is provided a method for modifying cell proliferation in a plant which comprises the step of modulating the response of the plant to an auxin in which cell proliferation is modified to produce larger or smaller seeds than wild-type.

1 . A method of modifying cell proliferation in a plant which comprises modulating the expression of a gene whose expression or transcription product is capable of directly or indirectly modulating cell proliferation in the plant or plant propagating material. 
   
   
       2 . A method of modifying cell proliferation in a plant which comprises the step of transforming a plant, or plant propagating material, with a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within the integuments and/or seed coat of at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation. 
   
   
       3 . A method according to  claim 1  or  2  in which the overall size of the integuments/seed coat is modified. 
   
   
       4 . A method according to  claim 1  or  2  in which the function of a gene or gene product that promotes cell division is enhanced or the function of a gene or gene product that represses cell division is inhibited. 
   
   
       5 . A method according to  claim 4  in which cell division in the integuments/seed coat is increased resulting in a larger seed of the plant compared to wild type. 
   
   
       6 - 9 . (canceled) 
   
   
       10 . A method according to  claim 5  in which the seed is at least 5% heavier than wild type 
   
   
       11 - 19 . (canceled) 
   
   
       20 . A method according to  claim 1  or  2  in which the diameter of the stem of the plant is at least 10% greater than wild type. 
   
   
       21 . (canceled) 
   
   
       22 . A method according to  claim 1  or  2  in which the number of cells in the integuments/seed coat is increased compared to wild type. 
   
   
       23 . (canceled) 
   
   
       24 . (canceled) 
   
   
       25 . A method according to  claim 1  or  2  in which the sepal length of the plant is sufficiently greater than wild type to inhibit flower opening. 
   
   
       26 . A method according to  claim 25  in which the sepal length is at least 20% greater than wild type. 
   
   
       27 . (canceled) 
   
   
       28 . A method according to  claim 1  or  2  in which the function of a gene or gene product that promotes cell division is inhibited or the function of a gene product that represses cell division is enhanced. 
   
   
       29 . A method according to  claim 28  in which cell division in the integuments/seed coat is decreased resulting in a smaller seed compared to wild type. 
   
   
       30 . A method according to  claim 29  in which the seed is at least 15% smaller than wild type. 
   
   
       31 . A method according to  claim 28  in which the number of cells in the integuments/seed cost is decreased compared to wild type. 
   
   
       32 . (canceled) 
   
   
       33 . (canceled) 
   
   
       34 . A method according to  claim 1  or  2  in which growth or development or function of any part of the plant other than the seed is not substantially affected. 
   
   
       35 . A method according to  claim 2  in which the regulatory sequence includes a promoter. 
   
   
       36 . A method according to  claim 35  in which the promoter is a constitutive promoter. 
   
   
       37 . A method according to  claim 36  in which the promoter directs gene expression in most or all cells of the plant. 
   
   
       38 . A method according to  claim 36  in which the promoter is the 35S promoter. 
   
   
       39 . A method according to  claim 35  in which the promoter is specific, directing expression exclusively or primarily in one organ, tissue, or cell type of the plant. 
   
   
       40 . A method according to  claim 39  in which the promoter directs expression exclusively or primarily in the integuments or seed coat. 
   
   
       41 . A method according to  claim 40  in which the promoter is expressed in the pre-fertilization integuments. 
   
   
       42 . A method according to  claim 41  in which the promoter is the promoter of the INO or BEL1 gene. 
   
   
       43 . A method according to  claim 42  in which the promoter is expressed in the seed coat after fertilization. 
   
   
       44 . A method according to  claim 43  in which the promoter is the promoter of the BAN, TT1, TT2, TT8, TT12, or TT16 gene. 
   
   
       45 . A method according to  claim 2  in which the nucleic acid sequence includes or is derived from a gene involved in hormone response, biosynthesis, translocation, or other aspects of hormone action. 
   
   
       46 . A method according to  claim 45  in which the gene is MNT, IPT1, or ARGOS or their orthologues. 
   
   
       47 . A method according to  claim 2  in which the nucleic acid sequence includes or is derived from a core cell cycle gene. 
   
   
       48 . A method according to  claim 47  in which the core cell cycle gene is CYCD3;1 or CYCB1;1 or their orthologues. 
   
   
       49 . A method according to  claim 2  in which the nucleic acid is a transcription factor involved in regulation of the extent or rate of cell proliferation. 
   
   
       50 . A method according to  claim 49  in which the transcription factor is ANT or its orthologue. 
   
   
       51 . A method according to  claim 3  in which the function of a gene that modulates cell proliferation is enhanced. 
   
   
       52 . A method according to  claim 51  in which transcription of the gene is activated. 
   
   
       53 . A method according to  claim 52  in which activation of transcription results in increased levels of mRNA and/or protein encoded by the gene. 
   
   
       54 . A method according to any  claim 53  in which levels of mRNA and/or protein encoded by the gene are increased by 20% or more. 
   
   
       55 . A method according to  claim 1  or  2  in which a plant promoter is operably linked to a coding region of the gene in the sense orientation. 
   
   
       56 . A method according to  claim 3  to  55  in which the function of the gene is modulated by operably linking a plant promoter to a nucleic acid fragment from the gene to form a recombinant nucleic acid molecule such that an antisense strand of RNA will be transcribed. 
   
   
       57 . A method according to  claim 56 , in which the function of a gene is modulated by introducing nucleic acid segments of the gene into an appropriate vector such that double-stranded RNA is transcribed where directed by an operably linked plant promoter. 
   
   
       58 . A method according to  claim 56  in which decreased levels of mRNA and/or protein encoded by endogenous copies of the gene are produced. 
   
   
       59 . A method according to  claim 58  in which levels of mRNA and/or protein encoded by endogenous copies of the gene are decreased by 20%, 50% or 75% or more. 
   
   
       60 . A method according to  claim 56  in which the levels of mRNA and/or protein encoded by homologues of the gene are reduced. 
   
   
       61 . A method according to  claim 60  in which levels of mRNA and or protein encoded by homologues of the gene are reduced by 20%, 50% or 75% or more. 
   
   
       62 . A method according to  claim 2  in which the function of the gene is modulated by operably linking a plant promoter to a ‘dominant negative’ allele of the gene, which interferes with the function of the gene product. 
   
   
       63 . A method according to  claim 2  in which the nucleic acid sequence comprises or is derived either from wild-type MNT or a mutant form of mnt or its orthologues. 
   
   
       64 . A method according to  claim 1  or  2  in which the plant is further modified to maintain desirable characteristics in other parts of the plant which may have been lost or modified after the modulation or transformation step. 
   
   
       65 . A method according to  claim 64  in which the desirable characteristic is fertility. 
   
   
       66 . A method according to  claim 65  in which wild type MNT function (or function of an MNT orthologue) is restored to petals and stamens of a transformed plant or an mnt mutant plant such that seeds have a modified phenotype but fertility is not impaired. 
   
   
       67 . A method according to  claim 66  in which the promoter of a gene that directs expression in petals and stamens but not carpels is operably linked to the wild-type MNT gene or an MNT orthologue. 
   
   
       68 . A method according to  claim 67  in which the promoter is the promoter of the AP3 gene. 
   
   
       69 . A method according to  claim 1  or  2  in which the plant is monocotyledonous. 
   
   
       70 . A method according to  claim 69  in which the plant is a crop plant. 
   
   
       71 . (canceled) 
   
   
       72 . A method according to  claim 1  or  2  in which the plant is dicotyledonous. 
   
   
       73 . (canceled) 
   
   
       74 . A method according to  claim 1  or  2  in which the plant is subsequently bred to be homozygous for the modulated gene. 
   
   
       75 . A method according to  claim 2  in which plant is bred to be heterozygous for the modulated gene. 
   
   
       76 . A plant which comprises, within the integuments and/or seed coats of the plant, a modulated gene whose expression or transcription product is capable of directly or indirectly modulating cell proliferation in the plant or plant propagating material. 
   
   
       77 . A plant which includes a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within the integuments and/or seed coat of at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation. 
   
   
       78 . A plant according to  claim 76  or  77  in which the overall size of the integuments/seed coat in the plant is modified. 
   
   
       79 . A plant according to  claim 76  or  77  in which the function of a gene or gene product that promotes cell division is enhanced or the function of a gene or gene product that represses cell division is inhibited. 
   
   
       80 . A plant according to  claim 79  in which cell division in the integuments/seed coat is increased resulting in a larger seed compared to wild type. 
   
   
       81 - 84 . (canceled) 
   
   
       85 . A plant according to  claim 80  to  84  in which the seed is at least 5% heavier than wild type. 
   
   
       86 - 94 . (canceled) 
   
   
       95 . A plant according to  claim 76  or  77  in which the diameter of the stem of the modified or transformed plant is at least 10% greater than wild type. 
   
   
       96 . (canceled) 
   
   
       97 . A plant according to  claim 76  or  77  in which the sepal length of the plant is sufficiently greater than wild type to inhibit flower opening. 
   
   
       98 . A plant according to  claim 97  in which the sepal length is at least 20% greater than wild type. 
   
   
       99 . (canceled) 
   
   
       100 . A plant according to  claim 76  or  77  in which the number of cells in the integuments/seed coat of the plant is increased compared to wild type. 
   
   
       101 . (canceled) 
   
   
       102 . (canceled) 
   
   
       103 . A plant according to  claim 76  or  77  in which the function of a gene or gene product that promotes cell division is inhibited or the function of a gene product that represses cell division is enhanced. 
   
   
       104 . A plant according to  claim 103  in which cell division in the integuments/seed coat is decreased resulting in a smaller seed compared to wild type. 
   
   
       105 . A plant according to  claim 104  in which the seed is at least 5% smaller than wild type. 
   
   
       106 - 108 . (canceled) 
   
   
       109 . A plant according to  claim 104  in which the seed is at least 5% lighter than wild type. 
   
   
       110 - 112 . (canceled) 
   
   
       113 . A plant according to  claim 76  or  77  in which the number of cells in the integuments/seed cost is decreased compared to wild type. 
   
   
       114 . A plant according to  claim 113  in which the number of cells in the integuments/seed coat is decreased by at least 30% compared to wild type. 
   
   
       115 . A plant according to  claim 76  or  77  in which growth or development of any part of the plant other than the seed is not substantially affected. 
   
   
       116 . A plant according to  claim 76  in which the regulatory sequence includes a promoter. 
   
   
       117 . A plant according to  claim 116  in which the promoter is a constitutive promoter. 
   
   
       118 . A plant according to  claim 117  in which the promoter directs gene expression in most or all cells of the plant. 
   
   
       119 . A plant according to  claim 117  in which the promoter is the 35S promoter. 
   
   
       120 . A plant according to  claim 116  in which the promoter is specific, directing expression exclusively or primarily in one organ, tissue, or cell type of the plant. 
   
   
       121 . A plant according to  claim 120  in which the promoter directs expression exclusively or primarily in the integuments or seed coat. 
   
   
       122 . A plant according to  claim 121  in which the promoter is expressed in the pre-fertilization integuments. 
   
   
       123 . A plant according to  claim 122  in which the promoter is the promoter of the INO or BEL1 gene. 
   
   
       124 . A plant according to  claim 120  in which the promoter is expressed in the seed coat after fertilization. 
   
   
       125 . A plant according to  claim 124  in which the promoter is the promoter of the BAN, TT1, TT2, TT8, TT12, or TT16 gene. 
   
   
       126 . A plant according to  claim 77  in which the nucleic acid sequence includes or is derived from a gene involved in hormone response, biosynthesis, translocation, or other aspects of hormone action. 
   
   
       127 . A plant according to  claim 126  in which the gene is MNT, IPT1, or ARGOS or their orthologues. 
   
   
       128 . A plant according to  claim 77  in which the nucleic acid sequence includes or is derived from a core cell cycle gene. 
   
   
       129 . A plant according to  claim 128  in which the core cell cycle gene is CYCD3;1 or CYCB1;1 or their orthologues. 
   
   
       130 . A plant according to  claim 77  in which the nucleic acid encodes a transcription factor involved in regulation of the extent or rate of cell proliferation. 
   
   
       131 . A plant according to  claim 130  in which the transcription factor is ANT or its orthologue. 
   
   
       132 . A plant according to  claim 76  or  77  in which the function of a gene that modulates cell proliferation is enhanced. 
   
   
       133 . A plant according to  claim 132  in which transcription of the gene is activated 
   
   
       134 . A plant according to  claim 133  in which activation of transcription results in increased levels of mRNA and/or protein encoded by the gene. 
   
   
       135 . A plant according to  claim 134  in which levels of mRNA and/or protein encoded by the gene are increased by at least 20%. 
   
   
       136 . A plant according to  claim 76  or  77  in which a plant promoter is operably linked to a coding region of the gene in the sense orientation. 
   
   
       137 . A plant according to  claim 76  or  77  in which the function of the gene is modulated by introducing a nucleic acid segments of the gene into an appropriate vector such that double-stranded RNA is transcribed where directed by an operably linked plant promoter. 
   
   
       138 . A plant according to  claim 137 , in which the function of the gene is modulated by operably linking a plant promoter to a nucleic acid segment from the gene, and introducing the resulting recombinant nucleic acid into an appropriate vector such that double-stranded RNA is transcribed. 
   
   
       139 . A plant according to  claim 137  in which decreased levels of mRNA and/or protein encoded by endogenous copies of the gene are produced. 
   
   
       140 . A plant according to  claim 139  in which levels of mRNA and/or protein encoded by endogenous copies of the gene are reduced by at least 50%. 
   
   
       141 . A plant according to  claim 137  in which the levels of mRNA and protein encoded by homologues of the gene are reduced. 
   
   
       142 . A plant according to  claim 141  in which the levels of mRNA and protein encoded by homologues of the gene are reduced by 50% or more. 
   
   
       143 . A plant according to  claim 76  or  77  in which the function of the gene is modulated by operably linking a plant promoter to a ‘dominant negative’ allele of the gene, which interferes with the function of the gene product. 
   
   
       144 . A plant according to  claim 76  or  77  in which the modulated gene or nucleic acid sequence comprises or is derived either from wild-type MNT or a mutant form of mnt or its orthologues. 
   
   
       145 . A plant according to  claim 76  or  77  in which the plant is further modified to maintain desirable characteristics in other parts of the plant which may have been lost or modified. 
   
   
       146 . A plant according to  claim 145  in which the desirable characteristic is fertility. 
   
   
       147 . A plant according to  claim 146  in which wild type MNT function (or the function of an MNT orthologue) is restored to petals and stamens of the plant or an mnt mutant plant such that seeds have a modified phenotype but so that fertility is not impaired. 
   
   
       148 . A plant according to  claim 147  in which the promoter of a gene that directs expression in petals and stamens but not carpels is operably linked to a wild type MNT gene or an MNT orthologue. 
   
   
       149 . A plant according to  claim 148  in which the promoter is the promoter of the AP3 gene. 
   
   
       150 . A plant according to  claim 76  or  77  in which the plant is monocotyledonous. 
   
   
       151 . A plant according to  claim 150  in which the plant is a crop plant. 
   
   
       152 . (canceled) 
   
   
       153 . A plant according to  claim 76  or  77  in which the plant is dicotyledonous. 
   
   
       154 . (canceled) 
   
   
       155 . A plant according to  claim 77  in which the plant has been bred to be homozygous for the modified gene. 
   
   
       156 . A plant according to  claim 77  which is heterozygous for the modified gene. 
   
   
       157 . A method for modifying cell proliferation in a plant which comprises the step of modulating the response of the plant to an auxin in which cell proliferation is modified to produce larger or smaller seeds than wild-type. 
   
   
       158 . A method according to  claim 157  in which the response to an auxin is modulated by altering the expression of an auxin response factor. 
   
   
       159 . A method according to  claim 158  in which the auxin response factor inhibits cell division. 
   
   
       160 . A method according to  claim 159  in which the auxin response factor inhibits cell division in the integuments/seed coat. 
   
   
       161 . A method according to  claim 160  in which the auxin response factor is ARF2. 
   
   
       162 . A method according to  claim 157  in which the function or expression of the auxin response factor is inhibited. 
   
   
       163 . A method according to  claim 162  in which the function or expression of the auxiliary response factor is specifically inhibited in the integument/seed coat of the plant. 
   
   
       164 . A method according to  claim 157  in which a gene encoding the auxin response factor is modified so as to affect expression of the factor. 
   
   
       165 . A method according to  claim 164  in which the gene is MNT or an orthologue thereof.