WO1997030581A1 - Plantes males steriles - Google Patents

Plantes males steriles Download PDF

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Publication number
WO1997030581A1
WO1997030581A1 PCT/AU1997/000102 AU9700102W WO9730581A1 WO 1997030581 A1 WO1997030581 A1 WO 1997030581A1 AU 9700102 W AU9700102 W AU 9700102W WO 9730581 A1 WO9730581 A1 WO 9730581A1
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Prior art keywords
plant
sequence
seq
gene
male
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PCT/AU1997/000102
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English (en)
Inventor
Julie Ann Glover
Stuart Craig
Mathilde Grelon
Elizabeth Salisbury Dennis
Abdul Mutakabbir Chaudhury
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Gene Shears Pty. Limited
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Priority to AU17600/97A priority Critical patent/AU1760097A/en
Publication of WO1997030581A1 publication Critical patent/WO1997030581A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates generally to male-sterility in plants and to methods of inducing male-sterility in plants.
  • the present invention is also directed to nucleic acid molecules which are useful in inducing male-sterility in plants and genetic constructs comprising same.
  • Male- sterile plants are particularly useful for producing hybrid plants by sexual or genetic crossing.
  • hybrid plants One important area of the agriculture and horticulture industries, in particular the seed production divisions thereof is the production of hybrid plants.
  • the production of hybrid plants from essentially homozygous parent piants permits the production of novel plant varieties, in particular via the introduction of a range of beneficial or desirable traits into a particular species, including disease-resistance, increased seed yield, frost-resistance, pest-resistance, and altered nutritional characteristics, amongst others.
  • a specific requirement in the production of hybrid plants is that the female parent plant does not self-fertilize. This is a particular problem in plant species which are predominantly or partially in-breeding populations.
  • the inventors investigated and characterised T- DNA mutants of Arabidopsis thaliana.
  • the inventors surprisingly discovered that disruption of a particular locus in A. thaliana leads to male-sterility. This result now permits the generation of male-sterile plants in a range of species and is a useful step in the generation of hybrid plants.
  • one aspect of the present invention provides a method of inducing or otherwise facilitating male-sterility in a plant, said method comprising targeting a genomic region of said plant to inhibit, reduce or otherwise disrupt expression of said genomic region, wherein said genomic region corresponds to the MS5 locus or JAG ⁇ S gene derived from Arabidopsis thaliana or an equivalent or homologue thereof.
  • genomic region shall be taken to refer to any sequence of nucleotides forming a part of the haploid genome, diploid genome, triploid genome, tetraploid genome, hexaploid genome or other genome complement of a plant species, or tissue or organ thereof, including any foreign genetic material introduced.
  • genomic region which may be manipulated genetically to induce male-sterility in a plant will be any sequence of nucleotides which, is a non-mutant, is required for the development of a viable male gametophyte (i.e. pollen grain) or male gametangium, or alternatively, any plant reproductive process which is required for said development or for fertilization of the female gametophyte other than a female-specific process, for example anther dehiscence.
  • targeting a genomic region is meant that the subject method of the invention comprises a step which has the end-result of reducing, inhibiting or otherwise disrupting the expression of a polypeptide encoded by a genomic region as hereinbefore defined.
  • the "targeting of a genomic region” does not require that the step taken to reduce, inhibit or otherwise disrupt expression of a polypeptide which is required for the development of viable pollen, anthers or plant reproductive process required for the development of same or for anther dehiscence, be a step in which the genomic region encoding said polypeptide is necessarily physically disrupted.
  • the present invention clearly contemplates the physical disruption of genomic region, for example by gene-targeting
  • the invention clearly extends to the disruption in trans of expression of a genomic region, for example by using antisense, ribozyme, co-suppression and immunoreactive molecular approaches.
  • the JAGIS gene is the Arabidopsis thaliana gene present at the MS5 locus on chromosome 4 of A. thaliana.
  • the nucleotide sequence of the JAG 18 gene is provided herein for the purposes of exemplification, as SEQ ID NO: 13.
  • the MS5 locus will be understood by those skilled in the art to refer to a genomic region as hereinbefore defined which includes a J ⁇ Gl ⁇ gene or a homologue, analogue or derivative thereof and, in the non-mutant form, is required for the development of fertile pollen, normal anther structures, anther dehiscence or other structures or processes required for male-fertility in plants, and/or when mutated, is associated with the expression of a male-sterile or semi- sterile phenotype.
  • equivalent as used herein shall be taken to refer to a genomic region derived from a plant species other than Arabidopsis thaliana which performs the same function as the MS5 locus or J G18 gene oi Arabidopsis thaliana or is at least 60% identical at the nucleotide sequence level to said MS5 locus orJ ⁇ Gl ⁇ gene.
  • homologue when used in the context of a genomic region as hereinbefore defined, shall be taken to refer to a sequence of nucleotides derived from any plant species including Arabidopsis thaliana which performs the same function as the MS5 locus or JAGIS gene exemplified herein, notwithstanding the occurrence in said sequence of nucleotide substitutions, additions or deletions, compared to the nucleotide sequences set forth in any one of SEQ ID NOS: 1, 3, 4 or 13.
  • Affi5 as used herein will be understood by those skilled in the art to mean the wild-type or non-mutant form of an MS5 locus as hereinbefore defined, wherein said locus is capable of encoding a functional polypeptide required for the development of male-fertility in plants.
  • mutant alleles of the corresponding wild-type MS5 locus which are associated with a male-sterile or semi-sterile phenotype.
  • mutant ms5-l and ms5-2 alleles is disclosed herein, in the Examples.
  • male-sterile or similar term shall be taken to refer to a plant or flower which is not capable of self-fertilisation, however possesses functional female reproductive organs such that, when pollen is introduced thereto from a second flower of the same species, fertilisation of the embryo sac will occur. Furthermore, a male-sterile plant or flower is not capable of fertilising a female reproductive organ on the same or another flower.
  • the terms "semi-fertile”, “semi-sterile” or similar term shall be taken to refer to a plant or flower which does not produce or release the same level of viable pollen as a fully male-fertile (wild-type) plant or flower of the same species or variety or alternatively, is not capable of fertilising a female reproductive organ on the same or another flower to the same extent as a fully male-fertile (wild-type) plant or flower of the same species or variety.
  • a semi- sterile plant or flower may be recognised by its reduced seed set compared to a wild-type plant or flower, whether or not said reduced seed set is manifested by fewer or small seed pods (siliques) on said semi-sterile plant.
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences);
  • mRNA or cDNA corresponding to the coding regions optionally comprising 5'- or 3 '-untranslated sequences of the gene;
  • a functional product is one which comprises a sequence of nucleotides or is complementary to a sequence of nucleotides which encodes a functional polypeptide, in particular the polypeptide encoded by the JAG ⁇ 8 gene exemplified by SEQ ID NO: 13 or a complementary strand thereto or a homologue, analogue or derivative thereof.
  • the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from a particular source, for example a particular organism or species, as specified herein, but has not necessarily been obtained directly from that source.
  • the present invention is directed to both monocotyledonous and dicotyledonous plants and are exemplified herein by Arabidopsis thaliana as this is the most convenient model to use to date. This is done, however, on the understanding that the present invention extends to all dicotyledonous plants.
  • the subject method is employed to induce or otherwise facilitate male-sterility in an agricultural or horticultural plant species, in particular a vegetable crop plant species, cereal crop plant species, tree species, or other agricultural plant species selected from the list comprising wheat, maize, soybean, cotton, sugar cane, oilseed rape, Canola ssp., Eucalyptus ssp., Linola ssp., chrysanthemum, rose, pine and poplar, amongst others.
  • an agricultural or horticultural plant species in particular a vegetable crop plant species, cereal crop plant species, tree species, or other agricultural plant species selected from the list comprising wheat, maize, soybean, cotton, sugar cane, oilseed rape, Canola ssp., Eucalyptus ssp., Linola ssp., chrysanthemum, rose, pine and poplar, amongst others.
  • targeting of the genomic region to induce male-sterility is accomplished by co-suppression.
  • Co-suppression is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell.
  • the present invention also extends to the use of co-suppression to inhibit, reduce or otherwise disrupt the expression of any genomic region which corresponds to the MS5 locus or JAG ⁇ 8 gene derived from Arabidopsis thaliana or an equivalent or homologue thereof.
  • targeting of the genomic region to induce male-sterility is accomplished using antisense technology.
  • an antisense molecule is an RNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed to produce a "sense" mRNA molecule capable of being translated into a polypeptide encoded by a JAG ⁇ % gene or a homologue or equivalent thereof.
  • the antisense molecule is therefore complementary to the sense mRNA or a part thereof.
  • the antisense RNA molecule possesses the capacity to form a double-stranded mRNA by base pairing with the sense mRNA, which may prevent translation of the sense mRNA and subsequent synthesis of a polypeptide gene product.
  • Ribozymes are synthetic RNA molecules which comprise a hybridising region complementary to two regions, each of at least 5 contiguous nucleotide bases in the target sense mRNA.
  • ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA.
  • Haseloff and Gerlach (1988) and contained in International Patent Application No. WO89/05852.
  • the present invention extends to ribozymes which target a sense mRNA encoding an FLF polypeptide, thereby hybridising to said sense mRNA and cleaving it, such that it is no longer capable of being translated to synthesise a functional polypeptide product.
  • the present invention provides a ribozyme or antisense molecule comprising at least 5 contiguous nucleotide bases which are able to form a hydrogen-bonded complex with a sense mRNA transcription product of the JAGIS gene or a homologue or equivalent thereof to reduce translation of said mRNA.
  • preferred antisense and/or ribozyme molecules hybridise to at least about 10 to 20 nucleotides of the target molecule
  • the present invention extends to molecules capable of hybridising to at least about 50-100 nucleotide bases in length, or a molecule capable of hybridising to a full-length or substantially full-length mRNA transcription product of the J4G18 gene or a homologue or equivalent thereof.
  • targeting of the genomic region to induce male-sterility is accomplished using gene-replacement (gene-targeting) approaches.
  • Gene targeting is the replacement of an endogenous gene sequence within a cell by a related DNA sequence to which it hybridises, thereby altering the form and/or function of the endogenous gene and the subsequent phenotype of the cell.
  • at least a part of the JAG ⁇ 8 gene or an equivalent or homologue thereof may be introduced into target cells containing an endogenous JAG 18 gene to replace said genetic sequence, thereby altering said endogenous gene.
  • the introduced nucleic acid molecule may comprise a missense or non-sense mutation relative to the corresponding sequence in the endogenous gene such that, when it replaces said corresponding sequence, the endogenous gene is no longer capable of expressing a functional gene produce.
  • the mutation may be introduced into a regulatory region of the gene, to prevent the binding of /raws-acting factors required for normal gene expression or alternatively or in addition, into the coding region of the gene such that an altered mRNA and/or polypeptide product is produced.
  • the resultant polypeptide product When mutations are designed such that an altered gene product is synthesised, the resultant polypeptide product possesses a different catalytic activity, substrate affinity or other polypeptide function compared to the endogenous J-4G18 gene product, such that when it is expressed in the target cell, the phenotype of said cell is altered.
  • the product of the gene targeting approach encodes a defective polypeptide
  • the phenotype of the plant will be male-sterile.
  • the phenotype may be semi- sterile if the altered polypeptide possesses different catalytic activity or substrate affinity compared to the non-mutated protein.
  • a method of inducing or otherwise facilitating male sterility in a plant comprising targeting a region in the genome of said plant which is adjacent to a region which encodes ⁇ -tubulin 9 or a homologue, analogue or derivative thereof to inhibit, reduce or otherwise disrupt expression of said adjacent region.
  • adjacent is meant a region contiguous to the JAGIS gene or in close proximity thereto such as approximately within about 100-200 kb of theJ Gl ⁇ gene.
  • ⁇ -tubulin 9 polypeptide is encoded by the TUB9 gene sequence, described by Snustad et al. (1992).
  • Reference herein to "TUB9” includes reference to a normally functional gene, a normally inactive gene and to adjacent DNA to such genes and includes a homologous or analogous DNA region in any plant.
  • the region adjacent to the 7 B9gene is the JAGIS gene.
  • JAG18 protein “JAG18 polypeptide” or similar term shall be taken to refer to the polypeptide encoded by the Arabidopsis thaliana JAGIS gene or a homologue or equivalent thereof.
  • the JAG18 polypeptide is conveniently defined by the amino acid sequence set forth in SEQ ID NO: 2 or encoded by one or more exon sequences comprising SEQ ID NO : 13 or a homologue, analogue or derivative polypeptide thereof.
  • homologues of a polypeptide refer to those polypeptides, enzymes or proteins which have a similar activity to a polypeptide encoded by the wild-type or mutant JAGIS gene or encoded by the closely-linked TUB9 gene, notwithstanding any amino acid substitutions, additions or deletions thereto.
  • a homologue may be isolated or derived from the same or another plant species.
  • amino acids of a homologous polypeptide may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.
  • Analogues encompass polypeptide products of the wild-type or mutant JAGl 8 gene or the 777-99 gene notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein.
  • derivative in relation to a polypeptide shall be taken to refer hereinafter to mutants, parts or fragments of a "parent molecule", from which it is derived.
  • the "parent molecule” may be known to possess a particular catalytic activity or binding activity, in which case the derivative thereof may or may not possess the same activity, or alternatively, may function with reduced activity compared to the parent molecule.
  • Derivatives include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention.
  • derivatives may comprise fragments or parts of an amino acid sequence, or alternatively, homopolymers or heteropolymers comprising two or more copies of the parent polypeptide or a fragment thereof.
  • substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a repressor polypeptide is replaced with another naturally-occurring amino acid of similar character, for example Gly ⁇ Ala, Val ⁇ Ile ⁇ Leu, Asp ⁇ Glu, Lys « ⁇ Arg, Asn*-*Gln or Phe « ⁇ Trp- « ⁇ Tyr
  • substitutions may also be "non-conservative", in which an amino acid residue which is present in a parent polypeptide is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.
  • an amino acid residue which is present in a parent polypeptide is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.
  • Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.
  • Amino acid deletions will usually be of the order of about 1-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino-or carboxyl-terminal fusions and of the order of 1 -4 amino acid residues.
  • a homologue, analogue or derivative of a polypeptide will comprise a sequence of amino acids that is at least 50% identical to at least 10 contiguous amino acids in primary amino acid sequence of the polypeptide to which it is homologous or analogous or from which it has been derived.
  • the percentage identity is at least 60-70%, even more preferably, at least 80- 90%, and still more preferably, at least 95%.
  • a further related embodiment of the present invention provides a method of inducing or otherwise facilitating male-sterility in a plant, said method comprising inducing an expression- reducing mutation in an endogenous plant genomic region, wherein said endogenous plant genomic region:
  • (iv) corresponds to a nucleotide sequence which is adjacent to a JAGIS gene and a TUB9 gene;
  • (v) corresponds to a homologue or equivalent of any one of (i) to (iv).
  • the step of inducing an expression-reducing mutation in the genomic region comprises the transformation of a plant cell with an isolated nucleic acid molecule which comprises a mutated genomic region capable of site-specific integration into the endogenous plant genomic region, using a gene-targeting approach.
  • the isolated nucleic acid molecule which comprises a mutated genomic region will comprise a non-sense point mutation compared to the endogenous plant genomic region, such that when it is integrated into said endogenous plant genomic region, a full-length JAGIS polypeptide is no longer produced.
  • the phenotypes of the Arabidopsis thaliana ms5-l and ms5-2 mutants which contain a spurious stop codons in the open reading frame of the JAG ⁇ S gene, exemplify the ability of such a mutation to induce a male-sterile phenotype.
  • the isolated nucleic acid molecule which comprises a mutated genomic region will comprise a mutation in a regulatory region of theJ_4G18 gene, wherein said mutation reduces transcription of the gene when inserted into the endogenous plant genomic region.
  • a further related embodiment of the present invention provides a method of inducing or otherwise facilitating male-sterility in a plant, said method comprising targeting the expression of an endogenous plant genomic region which comprise: (i) corresponds to an MS5 locus; or (ii) corresponds to aJ4G18 gene; or (iii) corresponds to a nucleotide sequence which is adjacent to an MS5 locus and a
  • (iv) corresponds to a nucleotide sequence which is adjacent to a JAG ⁇ 8 gene and a TUB9 gene;
  • the step of targeting the expression of an endogenous plant genomic region comprises the transformation of a plant cell with an isolated nucleic acid molecule which encodes an antisense, ribozyme or co-suppression molecule capable of preventing, reducing, delaying or otherwise disrupting the expression of said genomic region, wherein said antisense, ribozyme or co-suppression molecule further comprises a nucleotide sequence which corresponds or is complementary to a contiguous sequence of said genomic region.
  • each hybridising arm of a ribozyme molecule may comprise as few as 5-12 nucleotides complementary to the endogenous plant genomic region, whereas an antisense molecule may require at least 50 nucleotides or at least 100 nucleotides or at least 500 nucleotides complementary to the endogenous plant genomic region.
  • an antisense molecule may require at least 50 nucleotides or at least 100 nucleotides or at least 500 nucleotides complementary to the endogenous plant genomic region.
  • the genomic region being targeted comprise a sequence of nucleotides which is at least about 60% identical to any one of SEQ ID NOS: 1, 3, 4 or 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.
  • nucleotide sequence set forth in SEQ ID NO: 1 relates to the partial JAGIS cDNA sequence, corresponding to the region in the JAG] 8 gene spanning exon II to the 3 '-untranslated region.
  • SEQ LD NOS: 3-4 relate to the genomic sequences upstream and downstream of the T-DNA insertion site in exon V of the Arabidopsis thaliana ms5 mutant allele.
  • SEQ ID NO: 3 comprises JAGIS genomic sequences upstream of the insertion site and including T- DNA sequences.
  • SEQ LD NO: 4 comprises T-DNA sequences and the 3 '-end of the JAGIS gene downstream of the insertion site in exon V thereof, including sequences complementary to the 3 '-end of the adjacent TUB9 gene.
  • SEQ ID NO: 13 relates to the nucleotide sequence of the wild-type Arabidopsis thaliana JAGIS gene, which is localised within theMS5 locus, including the promoter region, exons I-V, intron sequences and 3 '-untranslated region.
  • a homologue, analogue or derivative of SEQ LD NO: l or SEQ LD NO: 3 according to any embodiments described herein, comprises a sequence of nucleotides which is at least 60% identical thereto or capable of hybridising to at least 10 contiguous nucleotides derived from SEQ ID NO: l or SEQ LD NO: 3 or its complementary strand under at least low stringency hybridisation conditions.
  • nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.
  • nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.
  • nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence identity to said sequence or a part thereof.
  • the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions.
  • Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5 ' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues.
  • Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed.
  • Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence.
  • Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.
  • the percentage identity to SEQ ED NO: 1, 3, 4 or 13 is at least about 70-80%, more preferably at least 80-90% and even more preferably at least about 90-99%.
  • a homologue, analogue or derivative of any one of SEQ ID NOS: 1, 3, 4 or 13 will comprise a sequence of nucleotides which is substantially identical thereto or to a complementary nucleotide sequence thereof.
  • a second aspectect of the present invention provides a method of inducing or otherwise facilitating male-sterility in a plant, said method comprising expressing in said plant a gene- targeting, antisense, ribozyme or co-suppression molecule which comprises a sequence of nucleotides capable of hybridising under at least low stringency conditions to at least 20 contiguous nucleotides in any one or more of SEQ ED NOS: 1, 3, 4 or 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.
  • the subject method comprises the additional first steps of transforming a plant cell with an isolated nucleic acid molecule or genetic construct which comprises the gene-targeting, antisense, ribozyme or co-suppression molecule and regenerating a whole plant therefrom.
  • the gene-targeting, antisense, ribozyme or co-suppression molecule comprises a sequence of nucleotides capable of hybridising under at least moderate stringency conditions, even more preferably at least high stringency conditions, to at least 20 contiguous nucleotides in any one of SEQ DD NOS: 1, 3, 4 a 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.
  • a high stringency wash is defined herein to be 0.1-0.2xSSC, 0.1% (w/v) SDS at 55-65°C for 20 minutes and a low level of stringency is considered herein to be 2xSSC, 0.1-0.5% (w/v) SDS at ⁇ 45 °C for 20 minutes.
  • Alternative conditions are applicable depending on concentration, purity and source of nucleic acid molecules.
  • the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and or wash.
  • SSC buffer concentration of SDS
  • concentration of SDS concentration of SDS
  • temperature of the hybridisation and or wash may vary depending upon the nature of the hybridisation membrane or the type of hybridisation probe used.
  • Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of the parameters affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.
  • Means for introducing recombinant DNA carrying a sense, antisense, gene-targeting, ribozyme or co-suppression molecule, into plant tissue include, but are not limited to, direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong etal, 1990) microparticle bombardment electroporation (Fromm et al., 1985), microinj ection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells (Christou etal, 1988; Sanford, 1988), vacuum-infiltration of plant tissue with nucleic acid, in planta transformation (Chang et al., 1994) or T-DNA-mediated transfer from Agrobacterium to the plant tissue.
  • Representative T-DNA vector systems are described in the following references: An et al.( ⁇ 9S5); Herrera-Estrella et al. (1983a,b);
  • a microparticle is propelled into a plant cell, in particular a plant cell not amenable to Agrobacterium-mediated transformation, to produce a transformed cell.
  • the cell is a plant cell
  • a whole plant may be regenerated from the transformed plant cell.
  • other non-animal cells derived from multicellular species may be regenerated into whole organisms by means known to those skilled in the art.
  • Any suitable ballistic cell transformation methodology and apparatus can be used in practising the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050).
  • the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
  • microparticles suitable for use in such systems include 1 to 5 ⁇ m gold spheres.
  • the DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
  • Plant species may be transformed with the genetic construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.
  • tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a ribozyme, antisense, gene-targeting or co- suppression molecule described herein.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • organogenesis means a process by which shoots and roots are developed sequentially from meristematic centers.
  • embryogenesis means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
  • Plants of the present invention may take a variety of forms.
  • the plants may be chimeras of transfo ⁇ ned cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species).
  • the transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T ) transformed plants may be selfed to give homozygous second generation (or T 2 ) transformed plants, and th ⁇ jT plants further propagated through classical breeding techniques.
  • the ribozyme, antisense, gene-targeting or co-suppression molecule is contained within a genetic construct- the genetic construct may further incorporate a dominant selectable marker, such as nptll, hygromycin-resistance gene, a phosphinothrium-resistance gene or ampicillin- resistance gene, amongst others, associated with the transforming DNA to assist in cell selection and breeding.
  • a dominant selectable marker such as nptll, hygromycin-resistance gene, a phosphinothrium-resistance gene or ampicillin- resistance gene, amongst others, associated with the transforming DNA to assist in cell selection and breeding.
  • Plants which may be employed in practising the present invention include all flowering plants such as but not limited to horticultural plant species, agricultural plant species, tree species and ornamental or horticultural plant species.
  • Preferred recipient plants for the genetic constructs and gene-targeting, antisense, ribozyme or co-suppression molecules of the present invention include, but are not limited to Arabidopsis thaliana, Thlaspi arvense, wheat, barley, rice, rye, maize, or sorghum, oil seed rape (Canola), Linola, cotton, sugar cane, Eucalyptus ssp, pine, poplar, rose and chrysanthemum, amongst others. Additional species are not excluded.
  • the expression of the introduced gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome.
  • Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants. Procedures for transferring the introduced genetic construct from the originally transformed plant into commercially useful cultivars are known to those skilled in the art.
  • a method of generating a plant which is substantially male-sterile comprising introducing into cells or callous of said plant a nucleic acid molecule and then regenerating a plant from said cells or callous wherein the nucleic acid molecule is capable of inserting into, disrupting expression of or acting as a ribozyme to a region of the genome of said plant, which region:
  • the nucleic acid molecule may be a gene-targeting molecule, causing an insertion, deletion or substitution mutation to the target region, or alternatively, an antisense molecule, ribozyme molecule or a sense molecule used in cosuppression.
  • the nucleic acid molecule may encode a "plantabody” or other targeting molecule capable of binding or interacting with the expression product of the JAGIS genomic region to prevent its function.
  • a still further aspect of the present invention extends to a transgenic plant carrying the foregoing sense, or antisense, or ribozyme, or co-suppression, or gene-targeting molecule and/or genetic constructs comprising the same.
  • the transgenic plant is one or more of the following: Arabidopsis thaliana, Thlaspi arvense, wheat, barley, rice, rye, maize, or sorghum, amongst others oil seed rape (Conoid), Linola, cotton, sugar cane, Eucalyptus ssp, pine, horticultural plants such as roses, chrysanthemum, etc. Additional species are not excluded.
  • the present invention further extends to the progeny of said transgenic plant.
  • transgenic plants and progeny thereof provided by the present invention display male- sterility or semi-sterility as hereinbefore defined, relative to untransformed plants which are otherwise isogenic.
  • the phenotype of the transgenic plant or its progeny may be non-conditional, in which case only slight variations in phenotype are observed under different growth conditions, or it may be conditional, in which case the level of male-sterility varies, depending upon environmental growth conditions to which said plants are subjected, for example photoperiod, temperature or light intensity, amongst others.
  • the level of male-sterility observed in the transgenic plants of the invention is increased by growing said plants in a short-day (SD) photoperiod or alternatively, reduced by growing said plants under continuous light or a long-day (LD) photoperiod.
  • SD short-day
  • LD long-day
  • a SD photoperiod comprises a light period of up to 8 hours duration in a 24 hour cycle
  • a LD photoperiod may comprise at least 12 hours of illumination in a 24 hour cycle.
  • the level of male-sterility observed may be altered by crossing said plant to a different variety, cultivar or genetic line of the same species.
  • conditional phenotypes are particularly useful where reversal of male-sterility (i.e. restoration of complete fertility) is required.
  • the level of male-sterility in the transgenic plants may vary, depending upon the nature of the genetic construct used to transform said plant, including the strength and specificity of any promoter sequence used to regulate expression of the transgene, the position of transgene insertion and copy number of the transgene.
  • this aspect of the invention defines, in a related embodiment, a male-sterile or semi-sterile plant or a plant which is substantially male-sterile, said plant carrying a mutation in a region:
  • (iii) substantially corresponds to a DNA sequence set forth in SEQ D NO:l, SEQ LD NO: 3 or SEQ ID NO: 4 or SEQ LD NO: 13 or which has at least about 60% identity thereto; (iv) is capable of hybridizing to the region in (i) or (ii) or (iii) under low stringency conditions.
  • a “mutation” in this context includes a chemically or genetically induced nucleotide substitution, addition and/or deletion, a disruption of expression caused by antisense or cosuppression or ribozyme or gene-targeting molecules or genetic constructs.
  • the foregoing antisense, or ribozyme, co-suppression or gene-targeting molecule is contained within a genetic construct, it will be understood that said molecule is placed operably under the control of a suitable promoter sequence capable of regulating the expression of the said nucleic acid molecule in a plant cell.
  • the said genetic construct optionally comprises, in addition to a promoter and an antisense, ribozyme, co-suppression, or gene-targeting nucleic acid molecule, a terminator sequence.
  • Terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3 '-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3 '-end of a primary transcript. Terminators active in plant cells are known and described in the literature.
  • terminators may be isolated from bacteria, fungi, viruses, animals and/or plants
  • terminators particularly suitable for use in the genetic constructs described herein include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays or the Rubisco small subunit (SSU) gene terminator sequences or subclover stunt virus (SCSV) gene sequence terminators, amongst others.
  • NOS nopaline synthase
  • CaMV Cauliflower mosaic virus
  • SSU Rubisco small subunit
  • SCSV subclover stunt virus
  • promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner.
  • a promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates.
  • the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said antisense, or ribozyme, or co-suppression nucleic acid molecule, in a plant cell.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the antisense or ribozyme or co-suppression molecule and/or to alter the spatial expression and/or temporal expression of said sense or antisense, or ribozyme, or co-suppression, or gene-targeting molecule.
  • regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense, or antisense, or ribozyme, or co-suppression, or gene-targeting molecule, thereby conferring copper inducibility on the expression of said molecule.
  • Placing a sense, ribozyme, antisense, co-suppression, or gene-targeting molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • promoters suitable for use in genetic constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant cells.
  • the promoter may regulate the expression of the said molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or plant pathogens, or metal ions, amongst others.
  • the promoter is capable of regulating expression of a sense, or ribozyme, or antisense, or co-suppression molecule or gene targeting, in a plant cell.
  • preferred promoters include the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, A. thaliana JAGl 8 promoter and the like.
  • the promoter is derived from the A. thaliana JAGIS gene, more preferably comprising a nucleotide sequence derived from SEQ LD NO: 13.
  • the promoter is capable of expression in a monocotyledonous or dicotyledonous plant cell, for example a cell in a horticultural, vegetable, cereal, tree or agricultural plant in particular a plant used in the cut-flower industry, a vegetable crop plant species, cereal crop plant species, tree species, or other agricultural plant species selected from the list comprising wheat, maize, soybean, cotton, sugar cane, oilseed rape, Canolassp., Eucalyptus ssp., Linolassp., chrysanthemum, rose, pine and poplar, amongst others.
  • one embodiment is directed to a genetic construct comprising a JAGIS promoter sequence or functional derivative, part fragment, homologue, or analogue thereof.
  • JAGIS promoter comprises a nucleotide sequence which is exemplified in SEQ ID NO: 13, in particular nucleotides 1-2000 thereof or other derivative thereof.
  • the genetic construct may further incorporate a dominant selectable marker, such as nptll, hygromycin-resistance gene, a phosphinothrium-resistance gene or ampicillin-resistance gene, amongst others, associated with the transforming DNA to assist in cell selection and breeding.
  • a dominant selectable marker such as nptll, hygromycin-resistance gene, a phosphinothrium-resistance gene or ampicillin-resistance gene, amongst others, associated with the transforming DNA to assist in cell selection and breeding.
  • Still another aspect of the present invention contemplates a method for producing a hybrid plant exhibiting a desired characteristic, said method comprising crossing a male-fertile plant having said desired characteristic with a male-sterile plant substantially as described herein.
  • said method comprises the further steps of obtaining progeny from the fertilized male-sterile plant and selecting therefrom plants which exhibit the desired characteristic.
  • the cross between the male-fertile plant (i.e. male-fertile parent) having a desired trait or characteristic (i.e. male-fertile parent) and the male-sterile plant (i.e. male-sterile parent) is referred to as "the original cross".
  • male-sterile parent used in the original cross was generated by expressing a ribozyme, antisense or co-suppression molecule therein, because such molecules generally exert their effect in trans, male-sterility will be dominant in the such plants and in their progeny which express the ribozyme, antisense or co-suppression molecule.
  • male-sterile plants will be readily detected in the heterozygous F, progeny of the original cross.
  • the use of such plants in the original cross may facilitate the selection of male-sterile hybrid plants in each generation of the back-crossing procedure.
  • the male-sterile parent was generated by insertion of an isolated nucleic acid molecule, such as a gene-targeting molecule or other nucleic acid molecule with or without T-DNA sequences, into th MS5 locus of JAGIS gene or a homologue or equivalent thereof
  • the male-sterile phenotype may be recessive or co-dominant, rather than being the dominant phenotype. In such cases, complete male-sterility may only be observed in plants which are homozygous for the inserted nucleic acid molecule.
  • the heterozygous F, progeny thereof may be male-fertile or only semi-sterile.
  • Re-establishment of male-sterility is also be required in each subsequent generation, to obtain a male-sterile hybrid comprising part or all of the genetic background of the male-fertile parent.
  • the desired trait of the male-fertile parent may be introduced into the male-sterile parent by recombinant genetic means.
  • a male-fertile plant which expresses a desired trait or characteristic may be transformed directly with a ribozyme, antisense, co-suppression or gene- targeting molecule or a genetic construct comprising same as described herein, to confer male- sterility thereto.
  • the present invention also provides hybrid seeds from the hybrid plant described herein.
  • Still another aspect of the present invention contemplates an isolated nucleic acid molecule comprising a sequence of nucleotides corresponding to or complementary to a genomic region, wherein said genomic region:
  • (iii) substantially corresponds to a DNA sequence set forth in any one of SEQ ID NOS: 1, 3, 4 or 14 or which has at least about 60% identity thereto; and/or (iv) is capable of hybridizing to the region in (i) or (ii) or (iii) under at least low stringency conditions.
  • the present invention extends to any plant JAG ⁇ 8 genes or JAG ⁇ 8-like genes and any functional genes, mutants, derivatives, parts, fragments, homologues or analogues thereof or non-functional molecules but which are at least useful as, for example, genetic probes, or primer sequences in the enzymatic or chemical synthesis of said gene, or in the generation of immunologically interactive recombinant molecules.
  • a related embodiment of the invention provides an isolated nucleic acid molecule which is at least 60% identical to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ LD NO: 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.
  • the percentage identity to SEQ LD NO: 1 or SEQ LD NO: 13 is at least about 75-80% and even still more preferably at least about 85-95%.
  • the isolated nucleic acid molecule of the invention is capable of inducing male-sterility on a plant when expressed therein as a gene-targeting, antisense, ribozyme or co-suppression molecule.
  • a further related embodiment of the invention provides an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in SEQ LD NO: 1 or SEQ LD NO: 13 or to a complementary strand, homologue, analogue or derivative thereof.
  • said nucleic acid molecule is capable of hybridising under at least moderate stringency conditions, even more preferably under at least high level stringency conditions as hereinbefore described.
  • the nucleic acid molecule of the invention further comprises a sequence of nucleotides which is at least 60% identical to the sequence set forth in SEQ LD NO: 1 or SEQ ID NO: 13 or a complementary sequence thereto, or a homologue, analogue or derivative thereof.
  • Preferred sources of the related JAG 18 gene or JAG 18-like gene include Arabidopsis thaliana, any tree, agricultural or horticultural plant species, in particular a plant used in the cut-flower industry, a vegetable crop plant species, cereal crop plant species, tree species, or other agricultural plant species selected from the list comprising wheat, maize, soybean, cotton, sugar cane, oilseed rape, Canola ssp., Eucalyptus ssp., Linola ssp., chrysanthemum, rose, pine and poplar, amongst others.
  • the present invention extends further to said plant FLF genes or FLF-like genes derived from cultured cells or tissues of plant origin.
  • genomic DNA, or mRNA, or cDNA is contacted with a hybridisation-effective amount ofaJAG ⁇ S genetic sequence, or a functional part thereof, and then said hybridisation is detected using a detection means.
  • the related genetic sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell.
  • the related genetic sequence originates from a plant species. More preferably, the related genetic sequence originates from an agricultural, vegetable, tree or horticultural plant species selected from the list comprising
  • the JAG ⁇ 8 genetic sequence comprises a sequence of nucleotides or at least 50 nucleotides, more preferably at least 100 nucleotides and even more preferably at least 20 500 nucleotides derived from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ED NO: 13 or its complement or a homologue, analogue or derivative thereof.
  • the JAG] 8 genetic sequence or probe is labelled with a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as 32 P or 35 S or a biotinylated 25 molecule).
  • a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as 32 P or 35 S or a biotinylated 25 molecule).
  • two opposing non-complementary nucleic acid "primer molecules” of at least 12 nucleotides in length derived from the nucleotide sequence of the JAG] 8 gene described herein is contacted with a nucleic acid "template” molecule and specific nucleic acid molecule copies 30 of the template molecule are amplified in a polymerase chain reaction.
  • the opposing primer molecules are selected such that they are capable of hybridising to complementary strands of the same template molecule, wherein DNA polymerase-dependant DNA synthesis occurring from a first opposing primer molecule will be in a direction toward the second opposing primer molecule.
  • both primers hybridise to said template molecule such that, in the presence of a DNA polymerase enzyme, a cofactor and appropriate substrate, DNA synthesis occurs in the 5' to 3' direction from each primer molecule towards the position on the DNA where the other primer molecule is hybridised, thereby amplifying the intervening DNA.
  • the nucleic acid primer molecule may further consist of a combination of any of the nucleotides adenine, cytidine, guanine, thymidine, or inosine, or functional analogues or derivatives thereof which are capable of being incorporated into a polynucleotide molecule.
  • the nucleic acid primer molecules may further be each contained in an aqueous pool comprising other nucleic acid primer molecules or alternatively, provided in a substantially pure form.
  • the nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell.
  • the related genetic sequence originates from an plant cell, tissue, or organ. More preferably, the related genetic sequence originates from a plant cell, tissue or organ.
  • the nucleic acid molecule may also be in a vector such as an expression vector and be capable of expression in a plant cell, bacterial cell, a mammalian cell or a yeast cell or an insert cell.
  • An expression vector may comprise a constitutive or inducible promoter.
  • the present invention further contemplates a recombinant product of the nucleic acid molecule.
  • the recombinant product comprises an amino acid sequence as set forth in SEQ ID NO:2 or having at least 60% identity thereto.
  • Another aspect of the present invention contemplates a method of restoring male-fertility to a male-sterile plant described herein, said method comprising expressing in said male-sterile plant a restorer gene which comprises an isolated nucleic acid molecule which encodes a functional JAG18 polypeptide or a functional homologue, analogue or derivative thereof.
  • the restorer gene functions to complement the male-sterile phenotype.
  • the restorer gene actually be expressed in planta to produce the functional JAG18 polypeptide.
  • the functional JAG18 polypeptide may comprise any amino acid substitution, addition or deletion, relative to SEQ ID NO: 2 or the polypeptide encoded by the exon sequences of SEQ ID NO: 13, subject to the proviso that said substitutions, additions or deletions do not eliminate functionality of the JAG18 polypeptide.
  • a restorer gene to include mutations which result in such substitutions, additions or deletions in the JAG 18 polypeptide may arise to prevent the activity of an antisense or ribozyme molecule already present in the male-sterile plant of the invention, from targeting expression of the restorer gene in addition to targeting expression of endogenous JAG] 8.
  • Figure 1 is a photographic representation showing the semi-dominance of the male-sterile phenotype in plants carrying the ms5-2 mutant allele.
  • A Wild-type - fertile
  • B heterozygotes - semi-fertile with only 1/5 siliques elongate
  • C homozygous ms5-2 mutants - completely male-sterile
  • Figure 2 is a graphical representation showing the number of siliques of a particular length in a segregating population of Arabidopsis thaliana plants, comprising fertile homozygous (MS5/MS5), semi-fertile heterozygous (MS5/ms5) and male-sterile homozygous (ms5/ms5) plants.
  • Silique length is indicated by the ranges of 3-5mm in length (grey-shaded boxes), 6- 9mm in length (black boxes) or greater than 9mm in length (open boxes).
  • Figure 3 is a photographic representation of a scanning electron micrograph showing floral structures derived from wild-type Arabidopsis thaliana and ms5-2 mutant plants at various developmental stages.
  • Panels A-C show floral structures of wild-type plants after dehiscence, in particular showing the depositing of pollen and germination.
  • Panels D-F show floral structures of ms5-2 homozygotes at similar stages.
  • wild-type flowers are shown to comprise stigmatic papillae and anthers capable of dehiscence
  • Panel D shows ms5-2 flowers to comprise shrivelled anthers and normal stigmatic papillae.
  • Panel B wild-type stigmatic papillae are either being deposited with pollen released from dehisced anthers, whereas Panel E shows that the pollen grains are collapsed and that no pollen deposition occurs in ms5-2 plants.
  • the pollen tubes snake their way down to the ovules (Panel C), whereas in ms5-2 flowers this does not occur (Panel F).
  • Figure 4 is a photographic representation of anther sections, showing tetrads of microspores in wild-type (WS ecotype) plants (Panel A) and abnormal tetrads in homozygous ms5-2 mutant plants (Panel B). Anther sections were stained with toluidine blue.
  • Figure 5 is a photographic representation of anther sections, showing the development of abnormal tetrads in homozygous ms5-2 mutant plants.
  • Panels B and C the microspores are seen to degenerate.
  • Panels E and F the microspores form large structures within the locules.
  • Panel G the locules are empty. Anther sections were stained with toluidine blue.
  • Figure 6 is a photographic representation of D API-stained developing microspores of wild- type plants (Panels A, C) and ms5-2 mutant plants (Panels B, D).
  • DAPI staining reveals chromosomes at the dyad stage (Panels A, B).
  • meiosis culminates in the production of four haploid nuclei encased in caliose (Panel C), which undergo cytokinesis to form tetrads of microspores.
  • Figure 7 is a graphical representation showing the percentage of double mutants scored from the F 2 progeny of a cross between a W100F marker line and ms5-l (in repulsion).
  • the W100F marker line was homozygous for the ap],py, hy2, gl], bp and cer2 mutant alleles.
  • the x-axis indicates the phenotype of double mutants observed in the F 2 progeny of the cross.
  • the y-axis indicates the frequency of each double mutant, as a percentage of the total observations.
  • Figure 8 is a diagrammatic representation showing the estimated chromosomal position of the MS5 locus.
  • Left genetic map showing the distance, in recombination units (cM), between MS5 and the markers m326 and pCITdl04.
  • Right genetic map showing the localisation of the MS5 locus on chromosome 4 of Arabidopsis thaliana, between the BPl and CER2 loci.
  • Distance between the MS5, BPl and CER2 loci is also indicated in recombination units (cM). Lines indicate the position of the left map within the right map.
  • Figure 9 is a photographic representation showing linkage between the ms5-2 mutation and RFLP marker m326.
  • the first 19 lanes contain £coRI-digested DNA obtained from pooled F 3 Arabidopsis thaliana plants derived from a cross between ms5-2 mutant plants in a Wassilewskija (Ws) background and wild-type Landsberg (La). Genotypes of the F 2 plants from which the F 3 pools were derived are indicated at the top of each lane: MM, homozygous for wild-type allele; Mm, heterozygous.
  • the last two lanes contain i-coRI-digested DNA obtained from the parental wild-type ecotypes, Wassilewskija (Ws) and Landsberg (La).
  • Plant DNA was hybridised with the RFLP marker m326.
  • the size of hybridising bands are indicated on the right-hand side of the figure, in kilobase pairs (kb).
  • the m326 probe hybridises to a 4.2kb fragment in Ws, and a 3.5kb fragment in La. This RFLP marker segregates with the ms5-2 mutation.
  • FIG 10 is a photographic representation of a Southern blot showing the presence of T-DNA linked to male-sterility in ms5-2 mutant plants.
  • Each lane contains l ⁇ g EcoRI-digested DNA obtained from pooled F 3 plants, representing fertile (F) and male-sterile (S) phenotypes. DNA was resolved on a 0.7% (w/v) agarose gel, transferred to a nylon membrane and hybridised to a 32 P-labelled T-DNA right border probe. The size of hybridising bands are indicated on the left-hand side of the figure, in kilobase pairs (kb).
  • FIG 11 is a photographic representation showing the strategy for crossing out of unlinked T-DNA insert in ms5-2. From left to right, an ms5-2 mutant plant (male-sterile, ms), containing both 16kb and 7.2kb hybridising .EcoRI T-DNA bands, is crossed to a wild-type plant and F 2 seeds derived therefrom are collected. DNA is isolated from ms F 2 plants and screened for the presence of the 16kb EcoRI fragment alone, by hybridising as described in the legend to Figure 11. Male-sterile stocks are then produced from seed which produced the desired DNA hybridisation profile. The sizes of hybridising bands are indicated on the right- hand side of each panel showing a Southern hybridisation, in kilobase pairs (kb).
  • Figure 12 is a diagrammatic representation of Left Border plasmid rescue. At the top of the
  • Plasmid pBR322-derived DNA contained within the T-DNA contains the ampicillin-resistance gene (Amp R ), bacterial origin of replication (Ori) and neomycin phosphotransferase gene (NPT 1 ) or tetracycline-resistance gene (Tet*) gene sequences. Further neomycin phosphotransferase gene sequences (NPT 2 ) and nopaline synthase gene regulatory sequences (NOS) are present in the T-DNA.
  • Amp R ampicillin-resistance gene
  • Ori bacterial origin of replication
  • NTT 1 neomycin phosphotransferase gene
  • Tet* tetracycline-resistance gene
  • plant DNA containing a T-DNA insert is digested with Sail, which cuts three times within the T-DNA and at a closely-linked site within the flanking plant DNA.
  • the digested DNA is re-ligated under conditions which promoter intra-molecular ligation, transformed into Escherichia coli and Amp R colonies are selected. Amp colonies will contain either a smaller internal plasmid comprising only T-DNA, or a larger external plasmid comprising T-DNA and plant DNA.
  • FIG. 13 is a diagrammatic representation showing the T-DNA integration site in the male- sterile mutant ms5-2.
  • the physical map of the T-DNA is indicated, showing the relative positions of the left-border (LB) sequences, EcoRI and H/ ' wdlLI restriction sites.
  • LB left-border
  • H/ ' wdlLI restriction sites In the centre of the Figure is a physical restriction map of the T-DNA integration region in the ms5-2 mutant, showing the relative location of EcoRI, Hindlil, Xhol and Pstl restriction sites. Numbers indicate the distance (bp) between the restriction sites.
  • the T-DNA has integrated in an inverted repeat pattern.
  • Directional arrows located above the physical map of the T-DNA and below the restriction map of the T-DNA integration region, indicate the position at which PCR primers hybridise.
  • the black box indicates a PCR probe comprising flanking plant DNA which is amplified using two PCR primers.
  • a map showing the organisation of the closely linked JAGIS and 7777i9 genes is indicated. The 5' and 3' ends of both genes are indicated. ⁇ xons are shown as filled boxes, stop codons as stars, and regions where the exact sequence is not known are lightly shaded.
  • Intron sequences are indicated by the bold black line. Numbers within the J .G18 sequence indicate the relative positions, within the genomic sequence, of intron/exon boundaries.
  • Figure 14 is a diagrammatic representation showing JAG] 8 gene organisation in wild-type, ms5-l and ms5-2 mutant plants.
  • the directional arrows indicate the relative orientations of theJ ⁇ Gl ⁇ and 777.99 genes.
  • the exons of theJ_4Gl8 and 777.39 genes are indicated by the light grey and dark grey boxes, respectively.
  • the positions of Ba Hl (B) and Clal ⁇ restriction sites is also indicated.
  • Translation stop positions in the ms5-l and ms5-2 mutant genes is indicated by the upward-facing arrows.
  • the bi-directional arrows indicate the genomic clones used in complementation analyses. The line at the top left indicates 1 kb of DNA sequence.
  • Figure 15 is a photographic representation of a Southern blot showing the presence of a small DNA fragment in plant containing the mutant ms5-2 allele.
  • DNA was isolated from individual fertile, semi-sterile and sterile plants, digested with EcoRI, resolved on an agarose gel, transferred to nylon membrane and hybridised to a 32 P-labeled DNA probe derived from the JAG]S cDNA clone.
  • the genotype (phenotype) of each plant is indicated at the top of the lanes: MM, homozygous Ms/Ms (wild-type); Mm, heterozygous Mslms5-2 (semi-sterile); mm, homozygous ms5-2lms5-2 (male-sterile).
  • hybridising bands are indicated on the right-hand side of the figure, in kilobase pairs (kb). Mm and mm plants have two small hybridising bands ( ⁇ 500bp in length), which are absent from MM plants, suggesting that the T-DNA interrupts the JA G 18 gene.
  • Figure 16 is a photographic representation of a gel showing amplified JAG] 8 DNA fragments obtained in RT-PCR experiments performed on flower buds from individual fertile (F), semi- sterile (SF) or male-sterile (S) plants. Amplification reactions were conducted in the presence (+) or absence (-) of reverse transcriptase. The primers used were either primers 3 and 4 (Top) or primers 2 and 3 (Below). Lane L contained size markers. The sizes of amplified DNA fragments are indicated on the left-hand side of the figure, in kilobase pairs (kb). Arrows to the right of each panel indicate the position of the amplification products obtained.
  • F fertile
  • SF semi- sterile
  • S male-sterile
  • FIG. 17 is a representation of the aligned amino acid sequences derived for the Arabidopsis thaliana JAGYS cDNA (JAG18 pep), rice expressed sequence tag (EST) SI 491 (Rice pep) and A. thaliana EST 202P9T7 (Ath pep) sequences. Numbering indicates amino acid residues from the start of the alignment. Shading represents amino acids which are conserved in at least two of the aligned sequences.
  • T-DNA-transformed Arabidopsis thaliana lines have been described previously (Feldmann and Marks, 1987; Forstoefel et al, 1992).
  • the ms5-2 mutant is the male-sterile T- DNA-transformed Arabidopsis thaliana Line 178 reported by Glover et al (1996).
  • ms5-l The EMS-generated male-sterile mutant ms5 has been described previously (Chaudhury et al, 1994).
  • ms5 mutant of Chaudhury et al (*1994) is hereinafter referred to as ms5-l.
  • Arabidopsis thaliana W100F marker line has been described previously (Koornneef et al, 1987) and was obtained from the ABRC.
  • A. thaliana strain csr-1 is an EMS-mutagenised line of the ecotype Columbia (Haughn and Somerville, 1986).
  • T 3 seed from Line 178 were grown in soil, self-fertilized and T 4 seed were collected from individual plants.
  • the T 4 families were grown at 22 ⁇ C under continuous fluorescent illumination in soil.
  • Germinating seeds were sprayed weekly with GA 3 (10 "5 M) until senescence. To determine 5 whether gibberelhn could restore the male-sterile plants to full-fertility, GA 3 was also applied onto the tips of four week old plants and plants were grown for a further 5 days prior to screening.
  • Tissue was mounted on a flat support with colloidal graphite conducting solution, introduced into the preparation chamber of a model E7400 cryo transfer unit (BioRad, Richmond, Cal, USA) and frozen under vacuum. After sputter-coating with gold, the tissue was examined at 15 15kV in a scanning electron microscope (model 6400;Jeol Tokyo, Japan) at around -150°C.
  • Tissue was embedded in ethanol: Spurrs resin [2:1 (v/v)] for 5 hours, followed by incubation overnight at 4°C in ethanol: Spurrs resin [1 :2 (v/v)], followed by incubation for 8 hours at room temperature or for 3 - 4 days at 4°C in 100% (v/v) Spurrs resin.
  • Sections (2 ⁇ m) were cut and stained with 0.5% toluidine blue.
  • Genomic DNA was isolated from plants essentially as described by Dean et al (1992), except that a purification step comprising ultra centrifugation of DNA on a CsCl cushion was added (Taylor and Powell, 1982).
  • DNA (2-5 ⁇ g) in a final volume of 100 ⁇ L, was digested with 60 units of EcoRI, in the presence of 1 mM spermidine, for up to 18 hours at 37 °C. Restriction digests were separated on an 0.7% (w/v) agarose gel and transferred onto Hybond N+ (Amersham) nylon filter and fixed thereto using 0.4N NaOH according to the manufacturer's protocol.
  • a T-DNA right border (RB) probe corresponding to HindUl fragment 23 derived from pTiC58 and containing the nopaline synthase (NOS) gene (EMBO J. 2, 21 3-2150, 1983), was used in all experiments requiring a right-border probe.
  • a T-DNA left border (LB) probe corresponding to the 3 kb EcoRI fragment derived from the left border of T-DNA vector, pGV3850: 1003, was used in experiments requiring a left-border probe.
  • Arabidopsis RFLP mapping set (ARMS) plasmids were hybridised to restriction endonuclease-digested plant DNA derived from a segregating population of plants containing the ms5-2 mutant allele.
  • the ARMS plasmids were obtained from the ABRC.
  • Plasmids were digested to release the ARMS insert fragments (Fabri and Schaffner, 1994), which were isolated from 0.7% (w/v) agarose/TBE gels using the PrepageneTM kit (Stratagene). Approximately 25 - 50 ng of ARMS insert DNA was labelled by random priming using the Standard MegaprimeTM protocol (Amersham) and 17pmol [ ⁇ - 32 P]dCTP.
  • Hybridization was performed in 0.5M NaHPO 4 (pH 7.2), ImM Na ⁇ DTA and 7% (w/v) SDS (Church and Gilbert, 1984) at 65°C for at least 18h.
  • Membranes were washed twice in 40mM NaHPO 4 (pH 7.2), 0.5% (w/v) SDS at 65°C for 15 min per wash, and then exposed to X-ray film. Autoradiographs were exposed for 1 -2 days.
  • membranes were required to be re-used, they were washed in boiling water containing 0.1% (w/v) SDS until no bound radioactivity could be detected.
  • Seeds were surface-sterilised with a solution of bleach:double-distilled H 2 O: 10% (w/v) SDS [33:66:1 (v/v/v)] for 15 min and rinsed several times in double-distilled H 2 O.
  • Surface-sterilised seeds were germinated by plating onto MS growth medium (Murashige and Skoog, 1962) containing 3% (w/v) sucrose, 0.4% (w/v) agar and 50 ⁇ g/ml kanamycin (kan).
  • plated seeds were kept in the dark at 4°C for at least 24 hours and then grown at 22 °C in a 16 hours light/8 hours dark photoperiod comprising an irradiance of 150 ⁇ mol quanta-m ' ⁇ s "1 PAR in the light cycle.
  • Kanamycin-resistant plants were then transferred to MS plates and grown a further 10 days and finally transferred to soil and grown to maturity.
  • l-2 ⁇ g of plant DNA was digested with Sail in the presence of lOmM spermidine for at least l ⁇ hours.
  • the restriction digest was purified by extraction with phenoLchloroform [1: 1 (v/v)], precipitated using ethanol and resuspended in 15 ⁇ l TE buffer.
  • the iS ⁇ r I-digested DNA was re-ligated in a 20 ⁇ L reaction, comprising the resuspended DNA, ImM ATP and 8 U of T 4 DNA ligase in ligation buffer. Ligation reactions were carried out at 14°C for at least l8 h.
  • Electrocompetent E. coli cells prepared as follows:
  • the electroporated cells were then pelleted for 1 min in a microfuge and resuspended in 100 ⁇ l of LB medium.
  • the transformation mixture or an aliquot thereof was plated onto LB plates containing 50 ⁇ g/ml ampicillin and incubated overnight at 37°C. In general, up to 100 ampicillin-resistant colonies were recovered per electroporation.
  • Double-stranded plasmid DNA was then prepared using standard procedures.
  • Nucleotide sequencing was performed by double-stranded sequence analysis on an Applied Biosystems Model 370ADNA sequencer using a fluorescent dye primer cycle sequencing kit, T ⁇ ql DNA polymerase and forward and reverse M13 primers (Applied Biosystems Inc). Computer analysis was performed with the GCG (Genetics Computer Group) software (Devereux, 1984). Data base searches were carried out using the BLAST programs (Altschul et al, 1990).
  • PCRs Polymerase Chain Reactions (PCRs) was performed using a Corbett FTSl Thermal Sequencer (Corbett Research, Sydney, Australia).
  • JMG7 5 ' CCCCTCGAGCATGTGATTGTAGTTTTG 3 ' [SEQ ID NO: 7]
  • Primer 1 5 ' AATGCGGAGATGAACCATT 3 ' [SEQ LD NO: 9]
  • Primer 2 5 'ATCTTGCTAACAGCTGGTAT 3 ' [SEQ LD NO: 10]
  • Primer 3 5'GGAATGGGATTTGGTGGTA 3' [SEQ ID NO: 11]
  • Primer 4 5'GACG_AAI_I£ATGGAGATATTG 3' [SEQ ID NO: 12]
  • genomic ⁇ library was constructed as follows Approximately 150 ⁇ g of wild-type plant DNA was partially digested with Sau3 Al, centrifuged on a glycerol gradient and fragments of size 9 - 20 kb were. The fragments were ligated into bacteriophage ⁇ EMBL4 arms, prepared by BamHl digestion to remove the stuffer fragment. Ligation mixtures were packaged and plated according to standard procedures. Approximately 30 000 plaques were screened with the PCR fragment.
  • the PCR fragment was also used directly as a probe to screen an amplified A. thaliana genomic library, constructed by ligating DNA from Arabidopsis thaliana strain csr-1 (Haughn and Somerville, 1986) into the vector pOCA18. Two positive clones (JAG10 and JAG16) were identified and isolated
  • Plant material was transformed essentially according to Chang et al (1994) Briefly, Agrobacterium tumefaciens strain AGL1, harbouring the cosmid clones JAG 10 or JAG 16, was grown in 100 ml of LB medium containing 5mg/ml tetracycline and 50 ⁇ g/ml rifampicin, for 18 - 36 hr at 28 °C with shaking. The cells were pelleted by a brief centrifugation and resuspended in 2 ml LB medium Water was withheld from soil-grown plants having primary bolts of 2-3 cm in height, for the period from two days preceding inoculation with the Agrobacterium tumefaciens culture to two days following inoculation.
  • Plants were inoculated with A. tumefaciens by excising the bolts off at the base with a scalpel blade and placing 50 - 100 ⁇ l culture on the wound site, using a micro pipette. Following continued growth of plants for two weeks, the inoculation procedure was repeated. Following the second inoculation, the plants were grown to maturity and seed collected.
  • Line 178 is allelic to the ms5-l mutation
  • Plants which were heterozygous for the Line 178 mutation were used as the pollen donor in crosses to the male-sterile mutants ms2, ms4, ms5-l, msl8 and antherless, in the homozygous state (Table 1). Additionally, plants which were heterozygous for the ms5-l mutation were used as the pollen donor in crosses with plants which were heterozygous for the Line 178 mutation (Table 1). In both cases, the F, progeny were scored for the male-sterile phenotype (Table 1).
  • the male-fertile plants were distinguishable from the semi-sterile plants and the male-sterile plants based on the length of the siliques for each phenotype.
  • silique length and seed set varied along the stem and from inflorescence to inflorescence.
  • the sterile plants had stunted siliques compared to both wild-type and semi-sterile plants.
  • male-sterile plants produced no seed in the absence of a male pollen donor.
  • the male-fertile, semi-sterile and male-sterile plants were present in the segregating population, in a 1 :2: 1 ratio (47:78:40 ⁇ .08, P>0.1), which is indicative of a semi-dominant mutation.
  • Seed were collected from selfed semi-sterile and male-sterile plants, germinated and the resulting plants were scored for the male-sterile phenotype as described in Example 2, to determine the parental genotypes.
  • Male-fertile plants were homozygous MS5/MS5 and semi- sterile plants were heterozygous MS5/ms5-2. Further crosses indicated that the completely male-sterile plants were homozygous ms5-2/ms5-2. These data indicate that the ms5-2 mutant allele is semi-dominant. The semi-dominance of ms5-2 is contrasted with the recessive nature of the ms5-l allele (Chaudhury et al, 1994).
  • the segregation pattern and mutant phenotype of the ms5-l allele is consistent with a nuclear recessive mutation in a range of different growth conditions.
  • the ms5-2 mutation was found to segregate differently in different environmental conditions.
  • SDs short days
  • the ms5-2 mutation appeared to segregate as a dominant allele, because heterozygous plants were very difficult to distinguish from homozygous male-sterile (ms5-2/ms5-2) plants.
  • heterozygous Fj plants were produced from a cross between a homozygous ms5-2lms5-2 mutant plant and a wild-type pollen donor, in a Wassilewskija (WS) genetic background.
  • the reduced fertility of MSIms5-2 heterozygotes in a short day photoperiod (SD; 8 hour light/16 hour dark cycle), compared to their fertility in a long day photoperiod (LD; 16 hour light/8 hour dark cycle) or continuous light photoperiod suggests that the ms5-2 mutation in Line 178 is dominant in SD but not in LD conditions.
  • SD short day photoperiod
  • LD 16 hour light/8 hour dark cycle
  • continuous light photoperiod suggests that the ms5-2 mutation in Line 178 is dominant in SD but not in LD conditions.
  • Fj plants were produced by crossing the homozygous ms5-2lms5-2 male-sterile mutant plant (Wassilewskija background) to a wild-type Landsberg erecta (Ler) pollen donor.
  • the Fj population comprising a random mixture of Ws and Ler backgrounds, were scored for the male-sterile phenotype, as described in Example 2, under different environmental growth conditions. Results are presented in Table 3.
  • the Ms/ms5-2 F, plants produced from the her X Ws cross were male-fertile when grown under a variety of environmental conditions for which the same genotype, in a Ws genetic background, is semi-sterile (Table 3).
  • the ms5-2 allele appears to be recessive in plants having a mixed Ws/Ler genetic background and grown under identical environmental conditions (Table 3).
  • exogenous GA 3 was applied to homozygous ms5-2lms5-2 plants, as described in Example 3, to determine whether the phytohormone was able to restore fertility to the male-sterile plants.
  • a series of flower bud sections was prepared from both Ws wild-type plants and homozygous ms5-2lms5-2 mutant plants at various developmental stages.
  • the data presented in Figures 4 and 5 indicate that, in the mutant plant, abnormal tetrads develop during meiosis.
  • the ms5-2 mutant allele produces a pre-meiotic or meiotic lesion that results in abnormal tetrad development in plants expressing the ms5-2 phenotype.
  • the microspores degenerate ( Figure 5, panels B and C), sometimes forming large structures ( Figure 5G). Occasional sections through the mutant plants showed an abnormal amount of tapetum ( Figure 4, panel B).
  • DAPI staining shows meiosis is disrupted in msS-2 mutant plants
  • EXAMPLE 20 Mapping the MS5 locus to A. thaliana chromosome 4
  • the genetic marker line, W100F ( Example 1) was used as a pollen donor for crosses with ms5-l male-sterile plants.
  • the resultant wild-type F plants were grown and F 2 seed collected.
  • the F 2 seeds were germinated in soil and, after 1 week, hy2 mutant plants were selected and were moved to different pots.
  • py mutants were removed and maintained by the daily addition of 1% (w/v) thymidine, applied directly to plants.
  • Mapmaker was used to convert these genetic distances into recombination frequencies and the MS5 locus was established to map between bp and cer-2 on chromosome 4 ( Figure 8).
  • the T-DNA genetic construct used to produce Line 178 contains, within the T-DNA left- and right-borders (LB and RB), the Escherichia coli neomycin phosphotransferase II (nptIT) gene which detoxifies kanamycin by phosphorylation (Bevan et al, 1983).
  • nptIT Escherichia coli neomycin phosphotransferase II
  • the presence of a T-DNA insert can be detected, in most cases, by scoring plants for a kanamycin resistant phenotype ("" • « *) , as described in Example 8. Accordingly, families of plants were produced from initial Line 178 transformant, comprising fourth generation segregants (i.e. T 4 families) and analysed for kanamycin resistance.
  • a wild-type plant was used as a pollen donor to fertilise an ms5-2 mutant plant derived from one of the two families which segregated for a single KANA marker.
  • the F] seed was sown and allowed to self-fertilise, F 2 seed was collected therefrom and the F 2 plants were then grown to maturity. Seed from 104 individual F 2 male-fertile plants was germinated to test, in the F 3 generation, for cosegregation of the male-sterile and kanamycin-resistant phenotypes.
  • the F 3 seed was also germinated and F 4 plants scored separately for kU ⁇ and male- sterility, to determine their F 3 parental genotypes.
  • DNA was isolated from F 3 plants, derived from 150 Ms Ms homozygotes and Mslms5-2 heterozygotes, and additionally from 60 ms5-2/ms5-2 homozygotes, derived from the Line 178 T 4 population, and hybridised to the T-DNA right- border (RB) sequence as described in Example 6. All of the ms5-2/ms5-2 homozygotes and Mslms5-2 heterozygotes studied exhibited the k * ⁇ * phenotype, as expected.
  • a second T-DNA insert evidenced by a 7.5kb EcoRI fragment hybridising to the RB sequence ( Figure 10), was also present in this population.
  • the second T-DNA insert segregated independently of the ms5-2 mutant allele, as it was absent in some male-sterile ms5-2/ms5-2 plants and present in some Ms/Ms homozygotes. Plants containing the second T-DNA insert alone were kan s , suggesting that this insert is not capable of conferring kanamycin-resistance on plants, possibly because it contained rearranged DNA.
  • the left border (LB) sequence of the T-DNA insert was hybridised to genomic DNA samples derived from individual male-sterile (ms5-2/ms5-2), heterozygous (Ms/ms5-2) and homozygous wild- type (Ms/Ms) plants, essentially as described in Example 6.
  • ms5-2/ms5-2 male-sterile
  • Ms/ms5-2 heterozygous wild- type
  • Ms/Ms homozygous wild- type
  • unlinked T-DNA was removed from the ms5-2 mutant using the procedure outlined in Figure 11. Briefly, a male-sterile mutant plant (ms5-2/ms5-2), derived from Line 178, was crossed with a wild-type homozygous plant (Ms/Ms) . The F, plants were grown and F 2 seed was collected. The F 2 seed was germinated without selection on kanamycin and DNA was isolated from 12 individual male-sterile plants. In all cases, a small inflorescence was retained from plants for crossing to a wild-type pollen donor, to obtain seed. DNA from the F 2 plants was digested then subjected to Southern hybridisation analysis as described in Example 6, to determine which pool contained only T- DNA linked to the ms5-2 mutation.
  • Ms/Ms wild-type homozygous plant
  • DNA amplified as described in the preceding Example was used to probe 300,000 clones from an Arabidopsis thaliana flower bud cDNA library (Weigel et al, 1992), essentially as described in Example 12.
  • Two positive cDNA clones were isolated and partially sequenced. Although both clones were identical in their nucleotide sequences at the 3' ends, one of the clones appeared to be chimeric or contain a rearrangement, as it contained a further poly(A) sequence at the 5' end of the transcript.
  • a database search was conducted to identify nucleotide or amino acid sequences related to S ⁇ Q ID Nos: 1-2. Data obtained from this search suggested that the 3 '-end of the JAG18 cDNA clone, in particular the last 222 nucleotides, are identical to the reverse complement of the 3' end of a genomic clone comprising the ⁇ -tubulin gene 777.39 (Snustad et al, 1992).
  • the 777-39 gene has also been localised to a region on chron ⁇ me 4 of Arabidopsis thaliana, between markers gl3838 and JGB9 in the region of MS5, ..._ng RFLP markers.
  • Eight other ⁇ -tubulin genes have also been cloned and sequenced (Marks et al, 1987, Oppenheimer et al, 1988, Snustad et al, 1992) but all differ in the 3' end from TUB9.
  • a ⁇ EMBL4 genomic DNA library of C24 DNA was constructed and genomic clones were isolated using the PCR fragment as a probe as described in Example 12. Two clones were isolated and estimated to contain 20 - 30 kb of plant DNA. These clones were mapped and the Nucleotide sequence analysis indicates that the JAGIS gene contains at least 5 exons (SEQ LD NO: 13).
  • the nucleotide sequence set forth in SEQ LD NO: 13 also comprises putative TATA box signals at positions 1864 to 1869, 1946 to 1954, and 1983 to 1989, suggesting that the genomic sequence comprises approximately 1.8-2.0 kbp of promoter sequence.
  • DNA flanking the T-DNA insertion site was cloned from the ms5-2 mutant plant and sequenced.
  • nucleotide sequence of the region of the ms5-2 mutant allele upstream of the T-DNA LB sequence is presented in SEQ ID NO:3.
  • Nucleotides 1-1112 of SEQ ID NO: 3 comprise the region spanning the 3 '-end of intronl to the 5 '-end of exonV of the ms5-2 mutant allele.
  • nucleotide sequence of the region of the ms5-2 mutant allele downstream of the T-DNA LB sequence ms5-2 mutant allele, including the T-DNA LB sequence, is presented in SEQ ED NO:4.
  • Nucleotides 91-1401 of SEQ ID NO:4 comprise plant DNA which includes the 3 '-end of exon V of the mutant ms5-2 allele.
  • T-DNA integration site is also 601 bp downstream of the stop codon of the 777i3 0 gene, the reverse-complement of which occurs at nucleotide positions 659 to 661 in SEQ ID NO:4.
  • the position of the T-DNA within the ms5-2 mutant allele is illustrated in Figure 13.
  • the region flanking the T-DNA integration site in the ms5-2 mutant was amplified by PCR, using the primers shown in Figure 13.
  • the forward primer annealed to genomic DNA immediately downstream of the Pstl site in the ms5-2 allele (i.e. SEQ LD Nos: 5 and 9), while the reverse primer annealed to genomic DNA immediately upstream of the Hindlll site in the 777.39 gene.
  • the amplified DNA was sub- cloned and sequenced.
  • Nucleotide sequence analysis of the amplified DNA showed no differences other than single base changes from the published sequence of the 777.39 gene (data not shown). It is possible that the single base changes observed represent ecotype polymorphisms, because the ms5-2 mutation is present in a Ws background, whereas the 777.39 gene was isolated from Columbia plants.
  • T-DNA integration in ms5-2 allele occurred in the 3' untranscribed region of 777.3 gene and in the last exon of JAGIS (Example 25).
  • JAG 18 genomic sequence from ms5-l allele showed that EMS mutagenesis which produced the ms5-l mutant resulted in the substitution of a cytosine at position 2914 of the gene sequence (SEQ ID NO: 13) for a thymidine, thereby changing a glutamine codon (CAA) into a stop codon (TAA) ( Figure 14). Therefore the ms5-l JAG 18 transcript encodes for a protein truncated of its C-terminal 305 amino acids.
  • the male- sterile phenotype is correlated with a mutation in the JAGIS gene.
  • primer 2 (SEQ ID NO: 10) was used in conjunction with primer 3 to amplify transcripts from the mutant ms5-2 RNA, because the primer 2 comprised nucleotide sequences in the junction region between the T-DNA and exon V and was not present in the wild-type transcript or gene sequences.
  • Primer 2 (SEQ ID NO: 10) is complementary to nucleotides 1 117 to 1136 of SEQ ID NO.3.
  • a schematic representation of the RT-PCR is presented in Figure 16.
  • Amplifications using primers 3 and 4 produced a fragment of 690 bp in length, in samples extracted from male-fertile plants which were either homozygous or heterozygous for the wild- type allele. No signal was amplified from cDNA coming from floral tissues of male-sterile ms5-2/ms5-2 homozygous plants ( Figure 16).
  • RT-PCR reactions performed using RNA extracted from vegetative tissues was always low and detectable only following hybridisation of the amplification products to a radio-labelled probe (data not shown).
  • MS 5 belongs to a multigene family highly conserved among species
  • the rice ⁇ ST (S1491) presented 46% identity overall to JAG18 at the amino acid level, however most of these similarities are found in the N-terminus of both proteins 56% identity to the first 200 amino acids of the JAG18 polypeptide).
  • the Arabidopsis ⁇ ST (202P9T7) showed more limited homology to JAG18 (only 39% identity between JAG18 and the first 100 amino acids encoded by the ⁇ ST clone).
  • Cosmid clones obtained by hybridisation screening of yeast artificial chromosome (YAC) libraries with the PCR-amplified probe (Example 22), were also used in complementation 10 experiments.
  • the genomic clone or cosmid DNA was transformed into MSIms5-l or Ms/ms5-2 heterozygous plants essentially as described in Example 13. Heterozygous plants were used as recipients for the DNA, as it was necessary to produce large 15 quantities of seed in order to observe sufficient numbers of transformants, using this method of transformation.
  • oligonucleotide 25 directed mutagenesis is employed to generate a fragment from the 5' end of the cDNA which contained the missing nucleotides.
  • the amplification reactions are carried out in a lO ⁇ l final volume containing 2 ⁇ M of each oligonucleotide primer, 200pg of cDNA as a template, 0.2 units of Taq ⁇ polymerase and
  • Amplification conditions are as follows: 95°C for 2 mins, followed by 5 cycles consisting of 15 sec denaturation 95 ⁇ C, annealing at 40°C for 30 sec, and polymerization at 72 ⁇ C for 1 min, followed by 25 cycles where the annealing temperature is raised to 50°C for 15 sec and finally, 30°C for 1 min.
  • the resulting PCR fragment is cloned into compatible restriction sites in the original cDNA plasmid and then sequenced to ensure that no mutations are introduced during the amplification procedure.
  • Antisense and sense binary constructs are made by sub-cloning the full length cDNA, in both orientations, into the expression vector pDH51 (Pietrzak et al . , 1986). This places the expression of the cDNA under the control of the CaMV 35S promoter. Recombinant plasmids, containing the cDNA in both orientations, are then sub- cloned between the right and left border sequences of the binary vector pBinl9 (Bevan, 1984).
  • binary constructs are transferred to Agrobacterium tumefaciens strain AGL1 (Lazo et al., 1991) by triparental mating, employing pRK2013 as the helper plasmid. Roots of wild-type C24 plants are transformed (Valvelkens et al., 1988) with both sense and antisense constructs. Alternatively, plant material is transformed according to Example 13. The npt ⁇ gene is used as a selectable marker to identify transgenic plants.
  • Constructs of the full-length 4G18 cDNA (Example 30) under the control of a 35S promoter and in either sense or antisense orientation, are introduced into Arabidopsis thaliana strain Ws, Ler and/or C24 plants. Tj seed is collected from primary transformants.
  • the expression level of the sense and antisense cDNA is analyzed in each of several families derived from the primary transformants. Those families expressing the antisense and sense mRNAs are retained for further analysis.
  • Transgenic plants expressing the full-length JAG7S cDNA in the sense orientation, under control of the 35S promoter, are obtained as described in Example 31.
  • the male-sterile phenotype is studied by scoring the T t and T 2 progeny derived from the initial transformants as described in Example 2.
  • the male-sterile phenotype is scored in families of transgenic plants which show detectable levels of antisense gene expression. Significant numbers of T, and T 2 progeny derived from the initial transformants exhibit the male-sterile of semi-sterile phenotype, compared to control plants which are otherwise isogenic.
  • Lys Lys Ser Gly Arg He Glu Glu Glu Ala Val Leu Leu Glu His Lys 100 105 110
  • ACAGCCTTAC AAAAGTAATA ATCAATCATC TTTTCTTTAG GCTCTTCTTC TTCTTCGTCC 720
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Arajbidopsis thaliana
  • CAGTAGAGCA AAAAGGGTTC AAQGAAAACA TGTTATTATG ACTATCGAGC AAGAGAAAGC 3180
  • CTCAGATGTT TCTTCTTCTC CAGCATCTGT GAGACCGAAC TCTGCAGGTC TATATACACA 3780

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Abstract

La présente invention concerne, d'une manière générale, la stérilité mâle dans des plantes ainsi que des procédés induisant une stérilité mâle dans des plantes. La présente invention concerne également des molécules d'acide nucléique utiles pour induire une stérilité mâle dans des plantes ainsi que des constructions génétiques les comprenant. Les plantes à stérilité mâle sont particulièrement utiles dans la production de plantes hybrides par des moyens sexués ou recombinés.
PCT/AU1997/000102 1996-02-22 1997-02-21 Plantes males steriles WO1997030581A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999023233A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Plantes presentant une sterilite male nucleaire, production de ces plantes et restauration de leur fertilite
US6183959B1 (en) 1997-07-03 2001-02-06 Ribozyme Pharmaceuticals, Inc. Method for target site selection and discovery
WO2015185514A1 (fr) * 2014-06-02 2015-12-10 Institut National De La Recherche Agronomique Mutation dominante dans le gène tdm menant à la production de diplogamètes dans les plantes
WO2019118342A1 (fr) * 2017-12-11 2019-06-20 Pioneer Hi-Bred International, Inc. Compositions et procédés de modification d'un génome de plante pour produire une plante mâle-stérile ms1 ou ms5
US11203752B2 (en) 2017-12-11 2021-12-21 Pioneer Hi-Bred International, Inc. Compositions and methods of modifying a plant genome to produce a MS9, MS22, MS26, or MS45 male-sterile plant

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMERICAN JOURNAL OF BOTANY, 82(8), (1995), LOUKIDES C.A. et al., "Two New Male-Sterile Mutants of Zea Mays (Poaceae) With Abnormal Tapetal Cell Morphology", pages 1017-1023. *
AVANCES IN CELLULAR AND MOLECULAR BIOLOGY OF PLANTS, 2, (1994), CHAUDHURY A.M. et al., "Genetics and Molecular Dissection of Male Fertily in Higher Plants", pages 403-422. *
SEX PLANT REPROD., 7, (1984), CHAUDHURY A.M. et al., "Genetic Control of Male Fertility in Arabidopsis Thaliana: Structural Analysis of Premeoitic Developmental Mutant", pages 17-28. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6183959B1 (en) 1997-07-03 2001-02-06 Ribozyme Pharmaceuticals, Inc. Method for target site selection and discovery
US6448009B1 (en) 1997-07-03 2002-09-10 Ribozyme Pharmaceuticals, Inc. Method for target site selection and discovery
WO1999023233A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Plantes presentant une sterilite male nucleaire, production de ces plantes et restauration de leur fertilite
US6603064B1 (en) 1997-10-30 2003-08-05 Syngenta Mogen B.V. Nuclear male sterile plants, method of producing same and methods to restore fertility
WO2015185514A1 (fr) * 2014-06-02 2015-12-10 Institut National De La Recherche Agronomique Mutation dominante dans le gène tdm menant à la production de diplogamètes dans les plantes
US10674686B2 (en) 2014-06-02 2020-06-09 Institut National De La Recherche Agronomique Dominant mutation in the TDM gene leading to diplogametes production in plants
WO2019118342A1 (fr) * 2017-12-11 2019-06-20 Pioneer Hi-Bred International, Inc. Compositions et procédés de modification d'un génome de plante pour produire une plante mâle-stérile ms1 ou ms5
US11203752B2 (en) 2017-12-11 2021-12-21 Pioneer Hi-Bred International, Inc. Compositions and methods of modifying a plant genome to produce a MS9, MS22, MS26, or MS45 male-sterile plant
US11926823B2 (en) 2017-12-11 2024-03-12 Pioneer Hi-Bred International, Inc. Compositions and methods of modifying a plant genome to produce a MS9, MS22, MS26, or MS45 male-sterile plant
US12054732B2 (en) 2017-12-11 2024-08-06 Pioneer Hi-Bred International, Inc. Compositions and methods of modifying a plant genome to produce a MS1 or MS5 male-sterile plant

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