WO1997030581A1 - Male-sterile plants - Google Patents

Abstract

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 recombinant means.

Description

MALE STERILE PLANTS

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.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The rapidly developing sophistication of recombinant DNA technology is greatly facilitating research and development in a range of industries and is important in many aspects of agriculture and human and veterinary medicine. The agriculture and horticulture industries are now beginning to benefit from the advances made in recombinant technology.

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. Due to the potential importance of hybrid plants to the agricultural and horticultural industries in general, much research has been undertaken to finding improved, more efficacious methods for their production. 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. A range of physical, chemical and genetic techniques have been used or proposed to prevent self-fertilization. For example, mechanical emasculation of flowers is a common, albeit labour-intensive and costly, method of preventing self-fertilisation Although some of these techniques have been partially successful, there is still a need to develop alternative, more broadly applicable methods of preventing self-fertilization.

In work leading up to the present invention, 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.

Accordingly, 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.

As used herein, the term "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.

In the context of the present invention, those skilled in the art will be aware that a 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.

By "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. Those skilled in the art will recognise that 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. Although 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.

For the purposes of nomenclature, 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 JΛGlδ 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.

The term "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 orJΛGlδ gene.

The term "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.

The designation "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.

The designations "ms5", "ms5-I" and "ms5-2" shall be taken to refer to mutant alleles of the corresponding wild-type MS5 locus which are associated with a male-sterile or semi-sterile phenotype. The nature of the mutant ms5-l and ms5-2 alleles is disclosed herein, in the Examples.

As used herein, the term "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.

As used herein, 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.

Reference herein to a "gene" is to be taken in its broadest context and includes:

(i) 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);

(ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) optionally comprising 5'- or 3 '-untranslated sequences of the gene; or

(iii) amplified DNA fragment or other recombinant nucleic acid molecule produced in vitro and comprising all or a part of the coding region and/or 5'- or 3 '-untranslated sequences of the gene.

The term "gene" is also used to describe synthetic or fusion molecules encoding all or part of a functional product. 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.

As used herein, 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. Preferably, 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.

In one embodiment of the invention, 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.

In a further embodiment, targeting of the genomic region to induce male-sterility is accomplished using antisense technology.

In the context of the present invention, 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. Although not limiting the mode of action of the antisense molecules of the present invention to any specific mechanism, 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.

In a further embodiment, targeting of the genomic region to induce male-sterility is accomplished using ribozymes. 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. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA. A complete description of the function of ribozymes is presented by 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.

According to this embodiment, 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. Although 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.

It is understood in the art that certain modifications, including nucleotide substitutions amongst others, may be made to the antisense and/or ribozyme molecules of the present invention, without destroying the efficacy of said molecules in inhibiting the expression of a JAG\ 8 gene. It is therefore within the scope of the present invention to include any nucleotide sequence variants, homologues, analogues, or fragments of the JAGIS gene encoding same, the only requirement being that said nucleotide sequence variant, when transcribed, produces an antisense and/or ribozyme molecule which is capable of hybridising to the said sense mRNA molecule.

In a further alternative embodiment of the invention, 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. According to this embodiment, 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. For example, 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. 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. Wherein the product of the gene targeting approach encodes a defective polypeptide, the phenotype of the plant will be male-sterile. Alternatively, the phenotype may be semi- sterile if the altered polypeptide possesses different catalytic activity or substrate affinity compared to the non-mutated protein.

In a related aspect of the present invention, there is provided a method of inducing or otherwise facilitating male sterility in a plant, said method comprising targeting a region in the genome of said plant which is adjacent to a region which encodes β-tubulin 9 or a homologue, analogue or derivative thereof to inhibit, reduce or otherwise disrupt expression of said adjacent region.

By "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 δ gene.

For the purposes of nomenclature the β-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.

Preferably, the region adjacent to the 7 B9gene is the JAGIS gene.

Hereinafter, the term "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.

In the present context, "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.

Furthermore, the 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.

The term "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.

Additionally, 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.

Procedures for derivatizing peptides are well-known in the art.

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.

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.

Preferably, 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.

More preferably, 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:

(i) corresponds to an MS 5 locus; or (ii) corresponds to a JAG 18 gene; or (iii) corresponds to a nucleotide sequence which is adjacent to anMS5 locus and a

TUB9 gene; or

(iv) corresponds to a nucleotide sequence which is adjacent to a JAGIS gene and a TUB9 gene; or

(v) corresponds to a homologue or equivalent of any one of (i) to (iv).

Preferably, 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. More preferably, 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.

Alternatively or additionally, 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

TUB9 gene; or

(iv) corresponds to a nucleotide sequence which is adjacent to a JAG\ 8 gene and a TUB9 gene; or

(v) corresponds to a homologue or equivalent of any one of (i) to (iv)

Preferably, 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.

Those skilled in the art will be aware of the precise length of nucleotide sequence which is required to be incoφorated into an antisense or ribozyme or co-suppression molecule for said molecule to effectively disrupt expression of the endogenous plant genomic region. For example, 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. Such optimisations of the present invention may be determined empirically, without undue experimentation.

According to the foregoing embodiments of the invention, it is particularly preferred that 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.

For the purposes of nomenclature, the 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. In particular, 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.

For the present purpose, "homologues" of a 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.

"Analogues" of a 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.

"Derivatives" of a 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. Generally, 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.

Preferably, 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%.

In a most particularly preferred embodiment, 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.

Preferably, 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.

More preferably, 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.

For the purposes of defining the level of stringency, reference can conveniently be made to

Maniatis et al (1982) at pages 387-389 which are incorporated herein by reference where the washing step at paragraph 11 is considered herein to be medium stringency. 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.

Generally, 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. Those skilled in the art will be aware that the conditions for 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); Herrera-Estrella et al. (1985).

For microparticle bombardment of cells, a microparticle is propelled into a plant cell, in particular a plant cell not amenable to Agrobacterium-mediated transformation, to produce a transformed cell. Wherein the cell is a plant cell, a whole plant may be regenerated from the transformed plant cell. Alternatively, 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). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to 5 μm 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.

Any plant 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).

The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers.

The term "embryogenesis", as used herein, 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 transfoπned 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 T₂) transformed plants, and thβjT plants further propagated through classical breeding techniques.

Wherein 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.

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.

Once introduced into the plant tissue, 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.

In a related embodiment, there is provided a method of generating a plant which is substantially male-sterile, said method 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:

(i) corresponds to JAGIS or an adjacent region; (ii) corresponds to a region adjacent TUB9; (iii) substantially corresponds to a DNA sequence set forth in SEQ ID NO: 1, SEQ

ID NO: 3 and/or SEQ ID NO: 4 or SEQ LD NO: 13 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 low stringency conditions.

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. Alternatively, 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.

Preferably, 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.

The 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.

In a particularly preferred embodiment, 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.

Those skilled in the art will be aware of the specific light-cycle requirements comprising a SD or LD photoperiod. Generally, a SD photoperiod comprises a light period of up to 8 hours duration in a 24 hour cycle, while a LD photoperiod may comprise at least 12 hours of illumination in a 24 hour cycle.

Alternatively, or in addition, 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.

Those skilled in the art will also be aware that conditional phenotypes are particularly useful where reversal of male-sterility (i.e. restoration of complete fertility) is required.

Those skilled in the art will also be aware that 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.

Accordingly, 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:

(i) corresponds to JAG\ 8 or an adjacent region; and/or (ii) corresponds to a region adjacent TUB9; and/or

(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.

Wherein 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.

The term "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. They may be isolated from bacteria, fungi, viruses, animals and/or plants Examples of 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.

Reference herein to a "promoter" is to be taken in its broadest context and 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. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term "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. For example, 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. Similarly, 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.

Examples of 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.

Preferably, 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. Examples of 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.

In a particularly preferred embodiment, the promoter is derived from the A. thaliana JAGIS gene, more preferably comprising a nucleotide sequence derived from SEQ LD NO: 13.

In a most preferred embodiment, however, 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.

According to this aspect, one embodiment is directed to a genetic construct comprising a JAGIS promoter sequence or functional derivative, part fragment, homologue, or analogue thereof.

Preferably, 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.

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.

In a preferred embodiment, said method comprises the further steps of obtaining progeny from the fertilized male-sterile plant and selecting therefrom plants which exhibit the desired characteristic.

Hereinafter, 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".

Further back-crossing of the F_t progeny of the original cross may be required to observe the desired trait which is present in the male-fertile parent, particularly where said trait is polygenic or due to the action of more than one locus or gene. Wherein the 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. In this case, male-sterile plants will be readily detected in the heterozygous F, progeny of the original cross. As a consequence, 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.

Altrnatively, wherein 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. Accordingly, wherein the male-sterile parent used in the original cross was generated by insertion of an isolated nucleic acid molecule, the heterozygous F, progeny thereof may be male-fertile or only semi-sterile. As a consequence, in order to obtain male-sterile hybrids, it may be necessary to re-establish male- sterile phenotype by selfing the heterozygous F, progeny plants to obtain male-sterile F₂ plants or alternatively, to emasculate the F, plants by other means, prior to back-crossing to the male- fertile parent. 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.

Alternatively, wherein the desired trait of the male-fertile parent is attributed to a single locus, back-crossing of the heterozygous Fj progeny plants produced by the original cross may not be required.

In an alternative embodiment, wherein the desired trait of the male-fertile parent is attributed to a single locus or a discrete number of loci, it may be introduced into the male-sterile parent by recombinant genetic means. In a further alternative embodiment, 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:

(i) corresponds to JAGIS or an adjacent region; (ii) corresponds to a region adjacent 777-39;

(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.

Those skilled in the relevant art will readily be capable of isolating one or more related JAGIS or J_4G18-like gene sequences from Arabidopsis thaliana or an alternative source without undue experimentation, when provided with a nucleotide sequence set forth herein or its complementary nucleotide sequence or a homologue, analogue or derivative thereof.

As a consequence, the present invention extends to any plant JAGλ 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.

Accordingly, 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.

In a preferred embodiment, 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%.

In a particularly preferred embodiment, 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.

Preferably, 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.

More preferably, 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.

Methods for identifying related JAG\ 8 genetic sequence, or plant JAG] 8-like genetic sequences 5 will be known to those skilled in the art.

In one approach genomic DNA, or mRNA, or cDNA is contacted with a hybridisation-effective amount ofaJAGλS genetic sequence, or a functional part thereof, and then said hybridisation is detected using a detection means.

10

The related genetic sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, 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

15 wheat, maize, soybean, cotton, sugar cane, oilseed rape, Canola ssp., Eucalyptus ssp., Linola ssp., chrysanthemum, rose, pine and poplar, amongst others.

Preferably, the JAG\ 8 genetic sequence (i.e probe) 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.

Preferably, 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 ³²P or³⁵ S or a biotinylated 25 molecule).

Alternatively, 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.

Accordingly, 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.

Those skilled in the art are aware of the technical requirements of the polymerase chain reaction and are capable of any modifications which may be made to the reaction conditions. The present invention encompasses all such variations.

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. Preferably, 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.

Preferably, 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.

In one embodiment, the restorer gene functions to complement the male-sterile phenotype. Those skilled in the art will be aware that such complementation requires that the restorer gene actually be expressed in planta to produce the functional JAG18 polypeptide. However, 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. The need for 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.

Other regulatory mechanisms may also be employed for restoration.

The present invention is further described by the following non-limiting figures and examples. In the Figures:

Figure 1 is a photographic representation showing the semi-dominance of the male-sterile phenotype in plants carrying the ms5-2 mutant allele. When seeds of ms5-2 were grown in soil under continuous light, three populations of plants were detected: Wild-type - fertile (A); heterozygotes - semi-fertile with only 1/5 siliques elongate (B) and homozygous ms5-2 mutants - completely male-sterile (C).

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. In Panel A, wild-type flowers are shown to comprise stigmatic papillae and anthers capable of dehiscence, whereas Panel D shows ms5-2 flowers to comprise shrivelled anthers and normal stigmatic papillae. In 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. In germinated wild-type flowers, the pollen tubes snake their way down to the ovules (Panel C), whereas in ms5-2 flowers this does not occur (Panel F). Scale bars: A=85μm; B=17μm; C=12μm; D=70μm; E=30μm; F=55μm.

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. In Panels B and C, the microspores are seen to degenerate. In Panels E and F, the microspores form large structures within the locules. In 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). In wild-type plants, meiosis culminates in the production of four haploid nuclei encased in caliose (Panel C), which undergo cytokinesis to form tetrads of microspores. Mutant anthers contain abnormal structures with five or more pools of chromosomes (Panel D). Scale bar= 3μm.

Figure 7 is a graphical representation showing the percentage of double mutants scored from the F₂ 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 F₂ 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 F₃ 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 F₂ plants from which the F₃ 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.

Figure 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 lμg EcoRI-digested DNA obtained from pooled F₃ 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 ³²P-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). In total, all male-sterile (ms5-2/ms5-2) plants and heterozygous (Ms/ms5-2) plants, but no wild-type (Ms/Ms) plants, from a total population of 60 male-sterile and 150 fertile and semi-sterile plants, contained the 16kb band. The 7.2kb hybridising fragment is unlinked to the msS-2 mutation.

Figure 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 F₂ seeds derived therefrom are collected. DNA is isolated from ms F₂ 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

Figure, a physical map of plant genomic DNA is indicated, showing the position of the T-DNA insert therein. 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 (NPT¹) or tetracycline-resistance gene (Tet*) gene sequences. Further neomycin phosphotransferase gene sequences (NPT²) and nopaline synthase gene regulatory sequences (NOS) are present in the T-DNA. To rescue the T-DNA left border, 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.

Figure 13 is a diagrammatic representation showing the T-DNA integration site in the male- sterile mutant ms5-2. Ai the top of the Figure, 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. 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. Immediately below the arrows, the black box indicates a PCR probe comprising flanking plant DNA which is amplified using two PCR primers. At the bottom of the Figure, 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. Εxons 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. At the top of the Figure, the directional arrows indicate the relative orientations of theJΛGlδ and 777.99 genes. Below, 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. Below the map of theJAG^]S and 777.99 genes, 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 ³²P-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). The size of 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. Below the photographic representations is a physical map of the J .G18 gene in the ms5-2 mutant, showing the nature and location of the T-DNA inserts, position, the location and direction of amplification primers (numbered arrows) and positions of JAGI S exons III, IV and V and introns there between. Lb, T-DNA left border; Rb, T-DNA right border. Figure 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.

EXAMPLE 1 Plant Lines

Several 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).

The EMS-generated male-sterile mutant ms5 has been described previously (Chaudhury et al, 1994). For the present purpose, the ms5 mutant of Chaudhury et al (*1994) is hereinafter referred to as ms5-l.

The 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).

EXAMPLE 2

Scoring the male-sterile phenotype of plants

T₃ seed from Line 178 were grown in soil, self-fertilized and T₄ seed were collected from individual plants. The T₄ families were grown at 22^βC under continuous fluorescent illumination in soil.

Male-sterile plants were easily detected in a population of wild-type plant because of their inability to self-fertilise plants and their short length of siliques.

Female-fertility of line 178 was confirmed by demonstrating the ability of the plant to set seed following pollination of flowers with pollen obtained from a wild-type fertile plant. EXAMPLE 3 GA₃ Treatment of male-sterile plants

Germinating seeds were sprayed weekly with GA₃ (10^"5 M) until senescence. To determine 5 whether gibberelhn could restore the male-sterile plants to full-fertility, GA₃ was also applied onto the tips of four week old plants and plants were grown for a further 5 days prior to screening.

EXAMPLE 4 10 Scanning Electron microscopy (SEM)

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.

EXAMPLE 5 Anther Sections

0 For bright field microscopy, whole flower buds were vacuum fixed in 3% (w/v) gluteraldehyde in 0.025 M NaPO₄ pH 7 buffer for 4 hours at room temperature. Tissues were then rinsed in buffer for 1 hour and then fixed in 2% (w/v) OsO₄ in buffer for 1.5 hours, rinsed in buffer for 1 hour and then dehydrated through a graded series of ethanol washes [ i.e. 25 % (v/v), 50 % (v/v), 70 % (v/v), 95 % (v/v) and 100% (v/v)], each being for 1 hour, with the exception that 5 the 70 % (v/v) ethanol wash was performed overnight at 4°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 (2μm) were cut and stained with 0.5% toluidine blue.

EXAMPLE 6 Southern hybridisation analyses

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 μg), in a final volume of 100 μL, 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. EXAMPLE 7 RFLP mapping

To identify an RFLP marker which is closely-linked to the ms5-2 locus in line 178, 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 Prepagene™ kit (Stratagene). Approximately 25 - 50 ng of ARMS insert DNA was labelled by random priming using the Standard Megaprime™ protocol (Amersham) and 17pmol [α-³²P]dCTP.

Hybridization was performed in 0.5M NaHPO₄(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 NaHPO₄(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.

Where 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.

EXAMPLE 8 Kanamycin selection of T-DNA tagged plants

Seeds were surface-sterilised with a solution of bleach:double-distilled H₂O: 10% (w/v) SDS [33:66:1 (v/v/v)] for 15 min and rinsed several times in double-distilled H₂O. 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 μg/ml kanamycin (kan). In order to overcome dormancy, 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 μmol quanta-m^'^s^"1 PAR in the light cycle.

After 7-10 days plants were scored for kanamycin resistance. Kanamycin-resistant plants were then transferred to MS plates and grown a further 10 days and finally transferred to soil and grown to maturity.

EXAMPLE 9 Plasmid Rescue of the T-DNA Left Border

To rescue the left border of T-DNA from ms5-2 mutant plants, l-2μg of plant DNA was digested with Sail in the presence of lOmM spermidine for at least lδhours. The restriction digest was purified by extraction with phenoLchloroform [1: 1 (v/v)], precipitated using ethanol and resuspended in 15 μl TE buffer.

The iSαr I-digested DNA was re-ligated in a 20μL reaction, comprising the resuspended DNA, ImM ATP and 8 U of T₄ 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:

A. Overnight starter cultures of E. coli (strain DH5α or PMC 104) were prepared in 1-5 ml of Luria-Bertani (LB) growth medium;

B. Four flasks, each containing 100ml LB growth medium, were each inoculated with 1ml of the overnight and incubated, at 37 °C with shaking, until cultures reached an absorbance at 600nm (Agnn) of 03 - 0.4;

C. Cells were harvested by centrifugation at 2500 x g , using a Beckman JA-14 rotor, for 10 min at 4°C;

D. Cell pellets were resuspended in 200 ml of ice-cold sterile distilled water, then re-centrifuged as described supra, E. Cells were washed twice more as described at step D;

F. Cells were resuspended in 50 ml of ice-cold 10% (v/v) glycerol;

G. Cells were transferred to Oakridge tubes and recentrifuged at 2000 x g for 10 min at 4°C; and H. Cells were finally resuspended in a total volume of 1.4 ml of ice-cold 10% (v/v) glycerol and stored as 200 μL aliquots -80^βC.

To transform Escherichia coli cells with the re-ligated DNA, 10 μL of the ligation reaction was mixed with 200 μl of the prepared electrocompetent E. coli and transferred to a pre-cooled sterile 0.2 cm gap electroporation cuvette (Bio-Rad). The mixture was electroporated using a Bio-Rad Gene Pulser set at 2.5EkV, 25EmF and 200E¹.. As quickly as possible 1 ml of LB was added to the cells, which were then transferred to a microfuge tube and placed at 37^βC for 1 hour.

The electroporated cells were then pelleted for 1 min in a microfuge and resuspended in 100 μl of LB medium. The transformation mixture or an aliquot thereof was plated onto LB plates containing 50μg/ml ampicillin and incubated overnight at 37°C. In general, up to 100 ampicillin-resistant colonies were recovered per electroporation.

EXAMPLE 10 Nucleotide sequence analysis of rescued left border clones

Rescued left border fragments were subcloned into pBlusecript Kan, which contains a functional kanamycin resistance gene. 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, Tαql 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).

EXAMPLE 11 Amplification reactions

Polymerase Chain Reactions (PCRs) was performed using a Corbett FTSl Thermal Sequencer (Corbett Research, Sydney, Australia).

Primers used in amplification reactions were as follows:

JAGF: 5 ' CGG Δπ_CAATGCGGAGATGAACCCATT 3 ' [SEQ LD NO: 5] JAGR: 5 ' CGCTTAAGATCTTGATAAC AGCTGGTAT 3 ' [SEQ LD NO:6]

JMG7: 5 ' CCCCTCGAGCATGTGATTGTAGTTTTG 3 ' [SEQ ID NO: 7]

LB T-DNA: 5 ' CCC_AAII_ _CTGTAATGACTCCGCGC 3 ' [SEQ LD NO:8]

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]

Some primers included the restriction sites EcoRI, BamHL or Xhol to facilitate subsequent cloning of amplified DNA (sites underlined supra). EXAMPLE 12 Genomic library construction and plaque hybridisations

Approximately 300,000 clones derived from an amplified Arabidopsis thaliana landsberg erecta (her) inflorescence cDNA library (Weigel et al, 1992) were screened by hybridisation to a PCR-amplified fragment prepared using primers JAGF and JAGR Two partial cDNAs of JAG18 were isolated and sequenced.

To isolate a corresponding genomic clone, a genomic λ library was constructed as follows Approximately 150 μg 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 λEMBL4 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

EXAMPLE 13 Plant transformation

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 50μg/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 μl 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.

EXAMPLE 14 Line 178 is allelic to the ms5-l mutation

Allelism tests were performed to test the possibility that the ms5-2 mutation in Line 178 was allelic to the previously described ms5-l mutation of Arabidopsis thaliana (Chaudhury et al, 1994).

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).

As shown in Table 1, all of the F, plants derived from crosses between Line 178 heterozygotes and the male-sterile mutants ms2, ms4, ms!8 and antherless were male-fertile. Additionally, the progeny of a cross between a pollen donor which is heterozygous for the msl8 mutation and line 178 only produced male-fertile progeny (Table 1). These data indicate that the mutation in line 178 is not allelic to any of the ms2, ms4, msl8 or antherless loci . In contrast, when the hne 178 heterozygote was crossed with homozygous ms5-l mutant plants, approximately 50% of the F_! progeny were male-sterile, suggesting that the mutation in line 178 is allelic to the ms5-l mutation. Similar results were obtained when the reverse cross was performed (i.e. heterozygous ms5-l plants were used to pollinate the line 178 heterozygote (Table 1).

This conclusion was supported by the 3: 1 ratio of male-fertile:male-sterile obtained when heterozygous ms5-l plants were crossed to the line 178 heterozygote (Table 1).

Table t: Results of allelism tests

Male parent Female Number of Male- Number of Male- χ¹ (ratio parent Fertile Progeny sterile Progeny fertile:sterile plants) line 178 ms2 36 0 heterozygote homozygote line 178 ms4 62 0 heterozygote homozygote line 178 ms5-l 10 8 0.222 (1:1) heterozygote homozygote line 178 msl8 52 0 heterozygote homozygote line 178 antherless 15 0 heterozygote homozygote msS'l line 178 43 15 0.023 (3:1) heterozygote heterozygote ms5-l line 178 54 60 0.316 (1:1) heterozygote homozygote msl8 line 178 31 0 heterozygote heterozygote EXAMPLE 15 The msS-2 mutant allele is semi-dominant

Genetic analyses of the ms5-l mutation confirm that it is a nuclear recessive sporophytic mutation (Chaudhury et al, 1994).

In contrast, when the F₂ progeny derived from F, plants which are heterozygous for the ms5-2 mutation (MS/ms5-2) were grown in soil under continuous fluorescent light at 22 °C, three types of plants could be scored: completely male-fertile, semi-sterile and male-sterile plants. These three phenotypes are shown in Figure 1.

As shown in Figure 2, 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. In the semi-sterile plants, 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.

Furthermore, male-sterile plants produced no seed in the absence of a male pollen donor.

Scanning electron micrographs of male-sterile ms5-2 plants demonstrate the absence of pollen being released from anthers and a mass of collapsed pollen is evident inside the anther (Figure 3).

The male-fertile, semi-sterile and male-sterile plants were present in the segregating population, in a 1 :2: 1 ratio (47:78:40 χ .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).

Further studies have confirmed that ms5-l segregates as a recessive allele under all the growth conditions which indicate the semi-dominance of ms5-2.

EXAMPLE 16 Conditionality of the msS-2 mutant phenotype

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.

In contrast, the ms5-2 mutation was found to segregate differently in different environmental conditions. When ms5-2 mutant seeds were grown under short days (SDs), an 8 hour light/ 16 hour dark photoperiod cycle, 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.

To confirm this observation and to determine the effect of other environmental conditions, 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 Fj progeny grown under a variety of conditions as shown in Table 2. The only condition which dramatically affected the phenotype of the heterozygous plants was day-length. 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. When plants were removed from a SD photoperiod to a LD photoperiod, the semi-fertile phenotype of heterozygotes was restored (data not shown).

In contrast, the ms5-l mutant allele segregated as a recessive mutation in all environmental conditions tested.

Table 2: Phenotype of ms5-2 heterozygotes under different growth conditions

Variable Temperature Light cycle Light level¹ Phenotype

Growth CQ

Condition

Temperature 16 continuous 200 Semi-sterile

19 continuous 200 Semi-sterile

24 continuous 200 Semi-sterile

Day Length 19 continuous 200 Semi-sterile

19 LD 200 Semi-sterile

19 SD 200 Sterile

Light Intensity 19 continuous 200 Semi-sterile

19 continuous 70 Semi-sterile

1. μmol quanta.m .s"¹ PAR; LD=long-day photoperiod; SD= short-day photoperiod

To determine the contribution of genetic background to the condition phenotype of the ms5-2 mutant, 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). Additionally, in marked contrast to the apparent dominance of the ms5-2 mutant allele in plants having a completely Ws genetic background and grown under a short day photoperiod at 19-20^βC, 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).

Table 3: Phenotype of heterozygote plants in different genetic backgrounds

μmol quanta.m^.s^*1 PAR; '^■ o*= pollen donor; ?=pollen receptor

These data indicate that ecotype-specific differences also play an important role in the conditional phenotype of ms5-2. In summary the dominance of the ms5-2 mutant allele is dependent upon both day-length and genetic background. The effect of ecotype differences was not tested for the ms5-l mutation.

EXAMPLE 17 Application of gibberellin to ms5-2lms5-2 plants does not restore fertility

Because of the involvement of hormones in the development of male-fertility in plants, exogenous GA₃ 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. The application of GA induced parthenocarpic pods similar to those observed previously (Chaudhury et al, 1994), however no pollen was produced (data not shown).

EXAMPLE 18 Pollen development in homozygous ms5-2/ms5-2 mutant plants

Cytological analysis of the effects of the ms5-l mutant allele on pollen development has been described elsewhere (Chaudhury et al. 1994). The results of Chaudhury et al (1994) suggest that male gametogenesis is altered early in the ms5-l mutant, leading the authors to classify it as a premeiotic mutant.

To characterise the ms5-2 mutant, cytological studies were performed on plants which were homozygous for the ms5-2 mutant allele (Figures 3-5).

Scanning electron micrographs (SEM) were produced, comparing the development of flowers in wild-type plants and ms5-2lms5-2 mutant plants (Figure 3). In wild-type plants, flowers fixed after dehiscence of the anthers contained released pollen germinating on the stigma surface (Figure 3, panels A- C). In contrast, no pollen is released at a similar stage in the homozygous ms5-2lms5-2 mutant plants (Figure 3, panels D - F). In the mutant, only a mass of debris, localised to the inside the anther, may be detected. Scanning of the anther surface of the ms5-2lms5-2 mutant, from the early stages of development, revealed obvious differences late in anther development, compared to the development of wild-type anthers (Figure 3, panels E-F). In particular, anthers collapsed late in development (Figure 3, panel F).

Similar results were obtained when heterozygous plants were examined that had been grown in SD medium in tissue culture instead of soil (data not shown).

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.

Sporogenic tissue, pollen mother cells (PMCs), tetrads, microspores and pollen grains were seen in a series of bud and anther sections from wild-type Ws plants (Figure 4, panel A). In marked contrast, anther sections of ms5-2lms5-2 mutant plants exhibited abnormal tetrads, without pollen grains therein (Figure 4, panel B).

As shown in Figure 5, 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. In ms5- 2lms5-2 mutant plants, 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).

EXAMPLE 19

DAPI staining shows meiosis is disrupted in msS-2 mutant plants

To better characterise the nature of the ms5-2 mutation, pollen mother cells of wild-type and mutant plants at different stage of floral development were stained using DAPI, essentially according to the method described by Maluszynska and Heslop-Harrison (1991). The product of the first meiotic division (dyads) were observed in both wild-type and mutant plants and there were no obvious differences, at least at this early stage, between the MS/MS and ms5-2lms5-2 genotypes (Figure 6, panels A and C).

Abnormal meiosis appears in the ms5-2 mutant, following the second meiotic division (Figure 6, compare panels C and D). In wild type plants the expected meiosis product for Arabidopsis thaliana, consisting of four pools of 5 chromosomes each, encased in a callose wall, is observed (Figure 6, panel C). In contrast, microspores containing more than 4 pools of chromosomes (5 to 8) were observed in the ms5-2 mutant, each pool apparently containing less than 5 chromosomes (Figure 6, panel D).

In summary, data presented in Figure 6 indicate that abnormal meiosis occurs in plants which express the ms5-2 mutant allele.

EXAMPLE 20 Mapping the MS5 locus to A. thaliana chromosome 4

To map the MS5 locus, 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 F₂ seed collected. The F₂ seeds were germinated in soil and, after 1 week, hy2 mutant plants were selected and were moved to different pots. One week later, py mutants were removed and maintained by the daily addition of 1% (w/v) thymidine, applied directly to plants.

Once plants reached maturity, they were scored for the male-sterile phenotype characteristic of ms5-l. Each male-sterile plant was also scored for each of the visible markers, ap 1 , hy2, py, gll, bp and cer2, in the W100F marker line. The transparent testa mutation tt-3, could not be scored, as male-sterile mutants do not set seed. Data on linkage between each marker and the male-sterile mutation were analysed using the Mapmaker program.

A large frequency of the F₂ generation plants expressed both the ms5-2 mutant allele and either apl, py, hy2 or gll. However, initial screening of the F₂ generation revealed only a very low percentage of male-sterile ms5-2 plants that were also scored as bp and cer2, even though these markers segregated in this F₂ population in the expected frequency. Among 140 ms5-l mutant plants, 6 plants were found to be homozygous for the bp mutation, and only 1 for the cer2 mutation (Figure 7). These data suggested that the MS5 locus is located on chromosome 4 of Arabidopsis thaliana.

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).

These data were confirmed using the RFLP mapping technique as described in Example 7. Of fourteen ARMS markers that were tested on DNA from the pooled F₃ plants described in Example 7, two markers from chromosome 4 (designated m326 and pCITdl04) detected RFLPs which segregated with the ms5-2 mutation. Data for the m326 marker are shown in Figure 9. By analysing the RFLP hybridisation data sets using Mapmaker, the ms5-2 mutant allele was positioned between these two markers, m326 and pCITdl04 (Figure 8).

EXAMPLE 21 Characterisation of the T-DNAs in the ms5-2 mutant

The T-DNA genetic construct used to produce Line 178 (the ms5-2 mutant) 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). As a consequence, 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. T₄ families) and analysed for kanamycin resistance.

Most of the segregating T₄ families derived from the ms5-2 mutant were sensitive to kanamycin (kan⁸). Two other families segregated for a single ^KANA marker , and one family segregated for two unlinked inserts.

To determine the linkage of the kanamycin resistance gene to the male-sterile phenotype of ms5-2 mutant plants, 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, F₂ seed was collected therefrom and the F₂ plants were then grown to maturity. Seed from 104 individual F₂ male-fertile plants was germinated to test, in the F₃ generation, for cosegregation of the male-sterile and kanamycin-resistant phenotypes. The F₃ seed was also germinated and F₄ plants scored separately for ^kUΛ and male- sterility, to determine their F₃ parental genotypes.

Of the 104 F₃ plants scored, 66 plants were heterozygous for the male sterility and heterozygous for the kanamycin-resistance marker gene and 38 plants were homozygous wild type plants and kan^s. No recombination between the ms5-2 mutant allele and the kanamycin-resistance marker gene were observed in this population of plants. These data suggest that the T-DNA comprising the kanamycin-resistance marker gene is closely-linked to the ms5-2 allele in Line 178.

In order to detect T-DNA inserts which were not responsible for conferring kanamycin- resistance on plants (i.e. silent T-DNA), DNA was isolated from F₃ 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 T₄ 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*^Λ* phenotype, as expected.

An example of the results of the Southern hybridisation analysis is shown in Figure 10. All of the ms5-2/ms5-2 homozygotes andMslms5-2 heterozygotes studied contained a 16kb EcoRI fragment which hybridised to the RB sequence. This hybridising fragment was not present in any of the male-fertile Ms/Ms homozygotes.

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.

EXAMPLE 22 Molecular cloning of the T-DNA left border (LB) in sS-2

1. The T-DNA integration pattern in the msS-2 mutant

To further characterise the integration pattern of the T-DNA in the ms5-2 mutant, 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. As there is an EcoRI site in the T- DNA LB sequence, located close to the LB-plant DNA junction, the Southern hybridisation was performed using H wdlll-digested plant DNA. There is only one Hwdlll site in the T-DNA sequence.

Three different-sized DNA fragments hybridised to the LB of the T-DNA in this population (data not shown). However, these same plants have only one Hindlll fragment which hybridised to the T-DNA RB sequence, suggesting that, in the ms5-2 mutant, two T-DNA inserts have integrated into plant genomic DNA at theMSS locus, in a tandem array.

Two of the three Hindlll fragments which hybridised to the LB sequence are linked and segregate with the ms5-2 mutant allele in male-sterile mutant and heterozygous plants. 2. Crossing out unlinked T-DNA from the ms5-2 mutant

To simplify the molecular cloning of the mutant ms5-2 allele, 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 F₂ seed was collected. The F₂ 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 F₂ 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.

Seed from plants containing the functional insert alone, as evidenced by the single 16kb hybridising band, was sown and used to produce stock seed for further experiments.

3. Molecular cloning of the left border (LB) from msS-2

To clone plant DNA adjacent to the T-DNA in the ms5-2 mutant, LB plasmid rescue experiments were performed as described in Example 9 and outlined in Figure 12. Re-ligated DNA was used to transform an mcr strain of Escherichia coli, to generate a bacterial colony containing a plasmid, designated JAG5, which comprises both T-DNA and plant DNA. By partial ly-sequencing the JAG5 clone, as described in Example 10, a 200 bp fragment of plant DNA directly adjacent to the T-DNA LB in ms5-2 was identified and subsequently amplified using the JAGF (SEQ ID NO:5) and JAGR (SEQ fD NO:6) primers, as described in Example 11.

The PCR-amplified clone was then used to probe a genomic Southern blot of plant DNA mutant, heterozygote and wild-type plants and shown to lie directly adjacent to the T-DNA in the mutant plant. EXAMPLE 23 Molecular cloning of wild-type MS5 cDNA sequences

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. The second cDNA clone, designated JAG18, was sequenced (SEQ LD NO: 1) and the amino acid sequence encoded thereby was determined (SEQ LD NO:2).

Sequence analysis of the second cDNA clone indicated that the longest open reading frame, excluding EcoRI linker/adaptor sequences, spanned from nucleotide position 16 to position 1167 of SΕQ LD NO: 1, thus encoding a polypeptide comprising 418 amino acids. However, the cDNA clone lacked a consensus ATG translation initiation codon and, as a consequence, was not a full-length sequence (SΕQ LD NO:2).

A database search was conducted to identify nucleotide or amino acid sequences related to SΕQ 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 β-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 β-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. These data suggest that the MS.) and TUB9 loci are closely-linked in the Arabidopsis thaliana genome, but that the genes may be transcribed in opposite directions such that the 3' end of 777.39 overlaps, or is at least adjacent to, the 3' end of the J.4G18 transcript (Figure 13).

EXAMPLE 24

Molecular cloning and nucleotide sequence analysis of the wild-type Arabidopsis thaliana MS5 (JAGIS) gene

A λEMBL4 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.

Corresponding genomic clones were also isolated from the ms5-2 mutant plant.

The organisation of the gene is presented in Figure 13. The region of overlap between the wild- type and mutant gene sequences is shown in Figure 14.

EXAMPLE 25 Nucleotide sequence analysis of the msS-2 mutant allele surrounding the T-DNA insertion site

DNA flanking the T-DNA insertion site was cloned from the ms5-2 mutant plant and sequenced.

The 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.

The 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.

These data indicate that the T-DNA was integrated within exon V of the mutant ms5-2 allele. The T-DNA integration site is also 601 bp downstream of the stop codon of the 777i3⁰ 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.

Comparison between the wild-type and mutant DNA sequences in the region surrounding the site of T-DNA integration showed that there was no deletion or jumbling of the plant DNA, however there was a duplication of the pentanucleotide sequence 5'-CAGCA-3', at positions 1108 to 1112 of SEQ LD NO:3 and at positions 91 to 95 of SEQ LD NO:4. The left border sequences were not completely conserved, and additional nucleotides (not present in either the plant or the T-DNA sequence at the target site) were also identified at both junctions (compare nucleotides 1 113 to 1216 of SEQ ID NO: 3 and the reverse complement of nucleotides 1 to 90 of SEQ ID NO:4).

To determine whether any rearrangements had occurred in the TUB9 gene or the ms5-2 allele sequence around the T-DNA integration site, the region flanking the T-DNA integration site in the ms5-2 mutant was amplified by PCR, using the primers shown in Figure 13. In particular, 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.

EXAMPLE 26 Nucleotide sequence analysis of the ms5-l mutant allele

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).

Furthermore, cloning and sequencing of 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.

In summary, in the two different characterised ms5 mutant alleles, ms5-l and ms5-2, the male- sterile phenotype is correlated with a mutation in the JAGIS gene.

EXAMPLE 27 Transcription of the JAGIS gene in the ms5-2 mutant

Northern experiments using RNA from buds and leaves performed with the EcoRI fragment specific to JAGIS failed to produce any signal even when a large amount (3μg) of poly(A)+ RNA isolated from flower buds was used in hybridisations, suggesting that this gene is probably expressed at very low levels and/or in specific floral tissues.

However, we were able to study J4G18 gene expression by RT-PCR experiments. Two sets of primers were used to amplify cDNA which was reverse-transcribed from total RNA extracted from leaves or flower buds of ms5-2 mutant plants grown in continuous light. Primers 3 and 4 (SΕQ LD Nos: 11-12) were designed to amplify part of the coding sequence of JAGIS, spanning nucleotides 355 to 1044 of SEQ ID NO: l. Alternatively, 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).

Similar experiments performed using primers 2 and 3 produced a smaller fragment, of only 490 bp in length, in floral tissue samples extracted from semi-sterile (i.e. Ms/ms5-2 heterozygous plants) or male-sterile (i.e. ms5-2/ms5-2 homozygous plants), indicating that the JAGIS transcript is truncated in the ms5-2 mutant plant. As expected, no amplification products were observed using these primers to amplify sequences derived from the floral tissues of wild- type plants (Figure 16).

These results indicate thatJAG!8 is expressed at a low level, mainly in reproductive tissues. Furthermore, the T-DNA integration in the last exon of JAGIS, in the ms5-2 mutant allele, does not prevent transcription of the JAG] 8 gene. However, the JAGIS transcript is truncated in the ms5-2 mutant plant, presumably resulting in the termination of translation at the first in frame stop codon, which occurs 18 bp after the integration site. As a consequence, it is expected that, in the ms5-2 mutant, the C-terminal 112 amino acids of the JAG18 protein are substituted with 5 amino acids encoded by the fusion sequence (Figure 14). Northern analysis on RNA isolated from buds or leaves using a 777739 specific probe did not detect any alterations in transcript level between ms5-2 mutant and wild-type plants (data not shown).

EXAMPLE 28

MS 5 belongs to a multigene family highly conserved among species

When the JAG18 cDNA clone was used as a probe on a genomic plant DNA digest, the expected band shift between the ms5-2 mutant and the wild-type DNA was detected as well as multiple hybridising bands with different intensities, suggesting this clone is part of a multigene family (Figure 15). However, we could obtain a JAG18 specific probe by using the 500 bp EcoRI subclone spanning the T-DNA insertion site (Figure 15).

Searches in nucleotides databases translated in all reading frames using TBLASTN program (ref) revealed significant homologies with 2 expressed sequence tags (ΕST) both from rice and Arabidopsis thaliana (Figure 17). These ΕSTs were obtained and fully sequenced.

The rice ΕST (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 ΕST (202P9T7) showed more limited homology to JAG18 (only 39% identity between JAG18 and the first 100 amino acids encoded by the ΕST clone).

The divergence observed between JAG18 and the rice and Arabidopsis ΕST sequences at their C-terminal ends suggests that they are members of a gene family rather than being orthologues. EXAMPLE 29 Complementation of the male-sterile phenotype in ms5-l and msS-2 plants

To confirm that JAGIS is indeed MS5, complementation experiments were carried out using 5 the wild-type genomic clone to complement the sterile phenotype of both the ms5-l and ms5-2 mutants.

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.

For the complementation 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.

Twenty ms5-l heterozygous plants were transformed and the seed collected.

20

EXAMPLE 30 Construction of Sense and Antisense clones

As the cloned cDNA (SEQ ID NO: 1) was lacking the ATG of the start codon, 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 lOμl final volume containing 2μM of each oligonucleotide primer, 200pg of cDNA as a template, 0.2 units of Taq\ polymerase and

30 125μM of each of the four deoxynucleotides. Amplification conditions are as follows: 95°C for 2 mins, followed by 5 cycles consisting of 15 sec denaturation 95^βC, annealing at 40°C for 30 sec, and polymerization at 72^βC 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 (co-supression or restorer) 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).

These 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.

EXAMPLE 31 Production of transformed sense and antisense 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.

EXAMPLE 32

Analysis of transgenic plants which over-express the JAGIS cDNA

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 T₂ progeny derived from the initial transformants as described in Example 2.

EXAMPLE 33

Analysis of transgenic plants which express JAGIS in the antisense orientation

Constructs of the full-length X G18 cDNA, in the antisense orientation, under the control of a 35S promoter, are introduced into plants as described in Example 31.

The male-sterile phenotype is scored in families of transgenic plants which show detectable levels of antisense gene expression. Significant numbers of T, and T₂ progeny derived from the initial transformants exhibit the male-sterile of semi-sterile phenotype, compared to control plants which are otherwise isogenic.

The severity of the male-sterile phenotype is correlated with the level of antisense RNA in these families. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

REFERENCES

1. Altschul SF, Gish W, Millar W, Myers EW, Lipman DJ (1990) J Mol Biol 215:403- 410.

2. An et al (1985) EMBO J. 4:277-284.

3. Armstrong, C.L., Peterson, W.L., Buchholz, W.G., Bowen, B.A. Sulc, S.L. (1990). Plant Cell Reports 9: 335-339.

4. Ausubel, F. M., Brent, R., Kingston, RE, Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987). In: Current Protocols in Molecular Biology. Wiley Interscience (ISBN 047150338).

5. Bevan MW, Flavell RB, Chilton MD (1983) Nature 394: 184-187.

6. Bevan, M. (1984). Nucl. Acids Res. 12: 8711-8721.

7. Chang SS, Park SK, Kim BC, Kang BJ, Kim DU, Nam HG (1994) The Plant Journal 5:551-558.

8. Chaudhury AM, Lavithis M, Taylor PE, Craig S, Singh, MB, Signer ER, Knox, RB, Dennis ES (1994) Sexual Plant Reproduction 7: 17-28.

9. Christou, P., McCabe, D.E., Swain, W.F. (1988) Plant Physiol 87: 671-674.

10. Church GM, Gilbert W (1984) Proc. Natl. Acad. Sci. (USA) 83: 1991-1995.

11. Crossway et al (1986) Mol. Gen. Genet. 202: 179-185.

12. Dean C, Sjodin C, Page T, Jones J, Lister C (1992) The Plant Journal 2:69-81.

13. Devereux J, Haeberli P, Smithies O. (1984) Nucleic Acids Research 12:387-395.

14. Fabri CO, Schaffner AR (1994) The Plant Journal 5: 149-156.

15. Feldmann KA (1991) The Plant Journal 1:71-82.

16. Feldmann KA and Marks (1987)

17. Forstoefel et al ( 1992)

18. Fromm et al. (1985) Proc. Natl. Acad. Sci. (USA) 82:5824-5828.

19. Gerlach, W. and Haseloff, J. (1988)

20. Glover JA et al (1996)

21. Haughn and Somerville (1986) Mol. Gen. Genet. 204:430.

22. Herrera-Estrella et al (1983a) Nature 303:209-213. 23. Herrera-Estrella et al (1983b) EMBO J. 2:987-995.

24. Herrera-Estrella et al (1985) In: Plant Genetic Engineering, Cambridge Umversity Press, NY, pp 63-93.

25. Koornneef M, Hanhart CJ, Van Loenen Martinet EP, Van der Veen JH (1987) Arabidopsis Information Service 23: 47-50.

26. Krens, F.A., Molendijk, L., Wullems, G.J. and Schilperoort, R.A. (1982). Nature 296: 72-74.

27. Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) Bio/technology 9: 963-967.

28. Maluszynska J, Heslop-Harrison J (1991) The Plant Journal 1:159-166.

29. Maniatis T, Fritsch EF, Sambrook J (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratories, Cold Spring Harbour, New York, U.S.A.

30. Marks et al (1987)

31. Murashige T, Skoog F (1962) Physiol Plant 15:473-497.

32. Oppenheimer DG t't α/ (1988)

33. Pazkowski et al (1984) EMBO J. 3:2717-2722.

34. Pietrzak, M., Shillito, R.D., Hohn, T. and Potrykus, I. (1986). Nucl. Acids Res 14: 5857-5868.

35. Sanford, J.C, Klein, T.M., Wolf, E.D., Allen, N. (1987). Particulate Science and Technology 5: 27-37.

36. Schmidt R, Cnops G, Bancroft I, Dean C (1992) Aust J Plant Physiol 19:341-351.

37. Snustad DP, Haas NA, Kopczak SD, Silflow CD (1992) The Plant Cell 4: 549-556.

38. Southern, E M. (1975). J. Mol. Biol. 98: 503-517.

39. Taylor B, Powell A (1982) FOCUS 4: 4-6.

40. Valvelkens, D., Van Montagu, M. and Van Lijesbettens, M (1988).Proc. Natl. Acad. Sci. (USA) 87: 5536-5540.

41. Weigel D, Alvarez J, Smyth DR, Yanofsky MF, Meyerowitz EM (1992) Cell 69: 843-859. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: GENE SHEARS PTY. LTD.

(ii) TITLE OF INVENTION: MALE-STERILE PLANTS

(iii) NUMBER OF SEQUENCES: 13

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DAVIES COLLISON CAVE

(B) STREET: 1 LITTLE COLLINS STREET

(C) CITY: MELBOURNE

(D) STATE: VICTORIA

(E) COUNTRY: AUSTRALIA

(F) ZIP: 3000

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT INTERNATIONAL

(B) FILING DATE: 21-FEB-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: HUGHES DR, E JOHN L

(C) REFERENCE/DOCKET NUMBER: EJH/EK

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +61 3 9254 2777

(B) TELEFAX: +61 3 9254 2770 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1271 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAATTCGGCA CGAGGTTTCA CATTGTTCAT AAAGTTCCTT CTGGTGATTC TCCTTACGTC 60

AGAGCCAAAC ACGCGCAGTT GATAGATAAA GATCCGAACA GAGCTATATC ATTGTTTTGG 120

ACTGCAATAA ACGCTGGAGA TCGAGTTGAT AGTGCTTTGA AGGATATGGC CGTTGTAATG 180

AAACAATTAG GTCGATCTGA TGAAGGGATT GAAGCTATTA AATCCTTTCG GTATCTCTGT 240

TCTTTCGAGT CTCAAGACTC GATTGATAAC TTGCTACTCG AGCTCTACAA GAAGTCAGGG 300

AGAATTGAAG AAGAAGCTGT TTTACTTGAG CACAAACTGC AAACACTCGA ACAAGGAATG 360

GGATTTGGTG GTAGAGTCAG TAGAGCAAAA AGGGTTCAAG GAAAACATGT TATTATGACT 420

ATCGAGCAAG AGAAAGCAAG GATACTAGGG AACTTGGGCT GGGTTCATTT ACAGTTACAT 480

AACTATGGAA TTGCAGAGCA GCATTACAGG AGAGCTTTGG GTTTGGAGCG AGACAAAAAC 540

AAACTCTGTA ACCTTGCAAT CTGCTTGATG CGTATGAGTC GAATTCCTGA AGCTAAATCT 600

CTGCTTGATG ATGTAAGAGA TTCTCCTGCA GAGAGTGAAT GCGGAGATGA ACCATTCGCA 660

AAGTCTTATG ACCGAGCCGT CGAAATGTTA GCAGAAATAG AATCGAAAAA GCCAGAAGCT 720

GATCTTTCGG AGAAGTTCTA CGCGGGATGT TCATTTGTGA ATAGGATGAA GGAAAATATA 780

GCTCCTGGAA CCGCAAACAA GAACTACTCA GATGTTTCTT CTTCTCCAGC ATCTGTGAGA 840

CCGAACTCTG CAGGTCTATA TACACAACCA CGCAGATGCA GATTGTTTGA AGAAGAGACG 900

AGAGGTGCTG CTCGAAAGCT ACTATTTGGA AAACCACAAC CTTTTGGCTC TGAACAGATG 960

AAGATCTTAG AGAGAGGAGA AGAAGAACCC ATGAAGCGAA AGAAACTGGA CCAGAACATG 10 0

ATTCAATATC TCCATGAATT CGTCAAAGAT ACAGCAGATG GTCCGAAGAG TGAATCAAAG 1080

AAAAGCTGGG CAGATATCGC AGAAGAGGAA GAAGCAGAAG AAGAAGAAGA AGAAAGATTG 1140

CAGGGAGAGC TTAAAACCGC AGAGATGTAG GATCTATTTG TACCGGTTAT CAAGTTTTTT 1200

GGATCATAGA AATTGTATAG GCTTTTGACA TTGAAATATT TTCATAGACA AAACAAAAAA 1260

AAAAAAAAAA A 1271 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Glu Phe Gly Thr Arg Phe His He Val His Lys Val Pro Ser Gly Asp 1 5 10 15

Ser Pro Tyr Val Arg Ala Lys His Ala Gin Leu He Asp Lys Asp Pro 20 25 30

Asn Arg Ala He Ser Leu Phe Trp Thr Ala He Asn Ala Gly Asp Arg 35 40 45

Val Asp Ser Ala Leu Lys Asp Met Ala Val Val Met Lys Gin Leu Gly 50 55 60

Arg Ser Asp Glu Gly He Glu Ala He Lys Ser Phe Arg Tyr Leu Cys 65 70 75 80

Ser Phe Glu Ser Gin Asp Ser He Asp Asn Leu Leu Leu Glu Leu Tyr 85 90 95

Lys Lys Ser Gly Arg He Glu Glu Glu Ala Val Leu Leu Glu His Lys 100 105 110

Leu Gin Thr Leu Glu Gin Gly Met Gly Phe Gly Gly Arg Val Ser Arg 115 120 125

Ala Lys Arg Val Gin Gly Lys His Val He Met Thr He Glu Gin Glu 130 135 140

Lys Ala Arg He Leu Gly Asn Leu Gly Trp Val His Leu Gin Leu His 145 150 155 160

Asn Tyr Gly He Ala Glu Gin His Tyr Arg Arg Ala Leu Gly Leu Glu 165 170 175

Arg Asp Lys Asn Lys Leu Cys Asn Leu Ala He Cys Leu Met Arg Met

180 185 190

Ser Arg He Pro Glu Ala Lys Ser Leu Leu Asp Asp Val Arg Asp Ser 195 200 205

Pro Ala Glu Ser Glu Cys Gly Asp Glu Pro Phe Ala Lys Ser Tyr Asp 210 215 220

Arg Ala Val Glu Met Leu Ala Glu He Glu Ser Lys Lys Pro Glu Ala 225 230 235 240

Asp Leu Ser Glu Lys Phe Tyr Ala Gly Cys Ser Phe Val Asn Arg Met 245 250 255 Lys Glu Asn He Ala Pro Gly Thr Ala Asn Lys Aβn Tyr Ser Asp Val 260 265 270

Ser Ser Ser Pro Ala Ser Val Arg Pro Asn Ser Ala Gly Leu Tyr Thr 275 280 285

Gin Pro Arg Arg Cys Arg Leu Phe Glu Glu Glu Thr Arg Gly Ala Ala 290 295 300

Arg Lys Leu Leu Phe Gly Lys Pro Gin Pro Phe Gly Ser Glu Gin Met 305 310 315 320

Lys He Leu Glu Arg Gly Glu Glu Glu Pro Met Lys Arg Lys Lys Leu 325 330 335

Asp Gin Asn Met He Gin Tyr Leu His Glu Phe Val Lys Asp Thr Ala 340 345 350

Asp Gly Pro Lys Ser Glu Ser Lys Lys Ser Trp Ala Asp He Ala Glu 355 360 365

Glu Glu Glu Ala Glu Glu Glu Glu Glu Glu Arg Leu Gin Gly Glu Leu 370 375 380

Lys Thr Ala Glu Met Xaa Asp Leu Phe Val Pro Val He Lys Phe Phe 385 390 395 400

Gly Ser Xaa Lys Leu Tyr Arg Leu Leu Thr Leu Lys Tyr Phe His Arg 405 410 415

Gin Asn Lys Lys Lys Lys Lys 420

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1216 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GAATTCTAGA TTCTTCCTCA TGTGATAAAA TCTCTTAAAG CTTTTCAAAT TTTGAGTGAT 60

CTTCTTGAAC CTGTCTATGA TTGAGGTTTT CTAACAAATG GATATTGTTT ATGTGGAAGA 120

ACAGTTGATA GATAAAGATC CGAACAGAGC TATATCATTG TTTTGGACTG CAATAAACGC 180

TGGAGATCGA GTTGATAGTG CTTTGAAGGA TATGGCCGTT GTAATGAAAC AATTAGGTCG 240

ATCTGAT^QAA GGGATTGAAG CTATTAAATC CTTTCGGTAT CTCTGTTCTT TCGAGTCTCA 300

AGACTCGATT GTAACTTGCT ACTCGAGCTC TACAAGGTAA TGCAGCCTCC TTCTTTGTTA 360

ATGCAGCAAG TGTATGGAGT TGTAAACTTG GAATGGGTTT GGTGTTTTGC AAGTCAGGAG 420 ACTTGAAGAA GAACTGTTTA CTTGAQCACA AACTGCAACA CTCGAACAAG GAATGGGATT 480

TGGTGGTAGA GTCAQTAGAG CAAAAAGGGT CAAGAAACAT GTTATTATGA CTATCGAGCA 540

AGAGAAAGCA AGGTGAGAAA GTTTCGGTGT ATCTCATGTT AGCTTGATGA GTGTTTTGTC 600

TCTTGAATTG ACGTTTCCAT TGTTGTTTGA ACATTTTAGG ATACTAGGGA ACTTGGGCTG 660

GGTTCATTTA CAGTTACATA ACTATGGAAT TGCAGAGCAG CATTACAGGT TTGGTTTTGT 720

TACCAAAATC CCAAATATTG ATTACTGTCT GGTCATGTGA GTTTTGCTTA TATTCAACTG 780

ATTATGAACA GGAGAGCTTT GGGTTTGGAG CGAGACAAAA CAAACTCTQT AACCTTGCAA 840

TCTGCTTGAT GCGTATGAGT CGAATTCCTG AAGCTAAATC TCTGCTTGAT GATGTAAGAG 900

ATTCTCCTGC AGAGAGTGAA TGCGGAGATG AACCATTCGC AAAGTCTTAT GACCGAGCCG 960

TCGAAATGTT AGCAGAAATA GAATCGAAAA AQCCAGAAGC TGATCTTTCG GAGAAGTTCT 1020

ACGCGGGATG TTCATTTGTG AATAGGATGA AGGAAAATAT AGCTCCTGGA ACCGCAAACA 1080

AGAACTACTC AGATGTTTCT TCTTCTCCAG CAGGTCATAC CAGCTGTTAG CAAGATCAGT 1140

TTATATTGTG GTGTAAACAA ATTGACGCTT AGACAACTTA ATAACACATT GCGGACGTTT 1200

TTAATGTACT GAATTC 1216

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1401 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

GAATTCAGTA CATTAAAAAC GTCCGCAATG TGTTATTAAG TTGTCTAAGC GTCAAATTGT 60

ATATCTCCAG CTGGTATGAT CTTGCTACGT CAGCATCTGT GAGACCGAAC TCTGCAGGTC 120

TATATACACA ACCACGCAGA TGCAGATTGT TTGAAAAAGA GACGAGAGGT GCTGCTCGAA 180

AGCTACTATT TGGAAAACCA CAACCTTTTG GCTCTGAACA GATGAAGATC TTAGAGAGAG 240

GAGAAGAAGA ACCCATGAAG CGAAAGAAAC TGGACCAGAA CATGATTCAA TATCTCCATG 300

AATTCGTCAA AGATACAGCA GATGGTCCGA AGAGTGAATC AAAGAAAAGC TGGGCAGATA 360

TCGCAGAAGA GGAAGAAGCA GAAGAAGAAG AAGAAGAAAG ATTGCAGGGA GAGCTTAAAA 420

CCGCAGAGAT GTAGGATCTA TTTGTACCGG TTATCAAGTT TTTTGGATCA TAGAAATTGT 480

ATAGGCTTTT GACATTGAAA TATTTTCATA GACAAAACAA AGAAGCATAG GCAAATGTGA 540 TTACACTCTG GAACAAATCT TAGQTTTQGG AAAGAAATTC AATAACAAAA CACACAAAGC 600

ACAAAAAGAG AGAATCCCAA TTCAGAAAAA GAGGTAAAAG QAAATGAGTG CGACGAACTT 660

ACAGCCTTAC AAAAGTAATA ATCAATCATC TTTTCTTTAG GCTCTTCTTC TTCTTCGTCC 720

TCCTCGTACT CCTCTTCACC GACTGTAGCA TCTTGATATT GCTGATACTC TGCAACAAGA 780

TCATTCATGT TACTCCTGCT TCAGTGAACT CCATCTCGTC CATCCTTCTC CTGTGTCCCA 840

TGAAGGAAGC CTTTCTCCTG AACATGGCAG TGAACTGTTC ACTCACACGC CTGAACATTT 900

CCTGGATTGA AGTTGAGTTT CCAATGAAAG TTGATGCCAT TTTCAAACCA GTGGGAGCTA 960

TTTCGCAGAC GCTTGATTTC ACGTTATTTG GAATCCATTC AACAAAGTAT GAGGAGTTTT 1020

TGTTCTGGAC GTTCATCATC TGTTCATCAA CTCTTTGGTA CTCATCTTTC CACGGAAGAC 1080

AGCAGAAGCT GTCAAGTAAC GACCATGACG AGGATCAGCT TCACACATAT GTTTTTGGCA 1140

TCCCACATCT TTGAGTTAGC TCAGGGACAC TCAAGGCACT GTATTGTTGA GATCCTCTTA 1200

TGTCAAAGGT CGAAACCAAC CATGAAGAAG TGGAGTCGTG GGAAAGGATT ATGTTCACTG 1260

CAATTTTTCT AAGGTCAGAG TTTAGTTGAC CAGGGAACCG AAGACAGCAT GTAACACCAC 1320

TCATTGTAGC TGAGATGAGA TGGCTAAGGT CACTAACTAA GATACACCAC CACATAAGAA 1380

AGAGAACAAT GTCAGGATTT T 1401

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CGGAATTCAA TGCGGAGATG AACCCATT 28

(2) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CGCTTAAGAT CTTGATAACA GCTGGTAT 28

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CCCCTCGAGC ATQTGATTGT AGTTTTG 27

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENQTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CCGAATTCCT GTAATGACTC CQCQC 25

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENQTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

AATGCGGAGA TGAACCATT 19 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ATCTTGCTAA CAGCTGGTAT 20

(2) INFORMATION FOR SEQ ID NO:ll:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GGAATGGGAT TTGGTGGTA 19

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GACGAATTCA TGGAQATATT G 21 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4433 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arajbidopsis thaliana

(vii) IMMEDIATE SOURCE:

(B) CLONE: JAG18

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 2343..2555

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2556..2739

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 2740..2952

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2953..3034

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3035..3183

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 3184..3271

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3272..3340

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 33 1..3423

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3424..4168

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

GGATCCATGA ATCCACATAT TGAATCAGAA GTTACTCAGA TGACTGTGGG TGAATATGCT 60

TCATTTAGGA TGACACCCCC TGATGCTGCT GAAGCTTTGA TTTTGGCTGT TGGTTCTGAT 120

ACTGTGAGAA TCCGCTCGCT CCTATCAGGT AATACTTATA ATGTTTACAT TTGTTGTAGG 180

ATAAATTTCT GAAAACGATT TTCTGGTTQA AAAAAAAAAA AAAAGGTCTT TATCATATGT 240 TAGACATTTG AACTGCCTTC ACCAGTGAAC TTAGGTCTAG TCTTACTAGT AACTACAAAG 300

CTCTGGTTCT CACTAATACT AAAAAGAGGC AAAGTTATGT AAGAACCTCA CGTAAAGTCT 360

TCTGGTGAAA CTTAGGATTQ ATTTAAAGTC TTGTGCATTT TATTTTTTCT ATTCTTTCTG 420

TTATGGAATA ATGTATTATT AGCATTTCAA CTTTACTGTG TCAGCTCTAG CTAGTTAGAT 480

GAATCTGTCG QTCTCTTATT TCTTTTTCAA ATCATAGCCA AAATTATCAA AGCTTACATG 540

GCTGTAAACA GAGGATGAAC AAAAATTAGA CCTATATCTA TGACTAAGTT AAGAGATATC 600

ATGAACATTT AAATQTQATA GTGATGGATT TCTCTGCTTT TTGTGCACAQ AACGTCCATG 660

TTTAAATTAC AATATACTTT TGTTGGGAGT GAAAGGGCCA TCAGAAGAAC GAATGGAGGC 720

GGCTTTTTTC AAACCTCCAC TTTCAAAACA ACGTGTTQAA TATGCACTGA AACATATAAG 780

AGAATCATCC GCTTCTACTT TGGTAAGCTC TATATATCTT AATGTCTTCC TGCTTACTAG 840

GATTATGAAT TCAAGACTTT GTTAAAACTC ATTTGTTTCA GTCTCTTTTT GTCCTTATTG 900

GTAAATCTTT GACAAGCATA AGATGGCACC TCTATTCTTG TTACAGGTTG ACTTTGGATG 960

CGGATCTGGA AGTTTATTAG ACTCTCTACT TGACTATCCA ACTTCACTTC AAACCATCAT 1020

CGGTGTTGAC ATTTCGCCAA AGGGTCTTGC CCGTGCTGCT AAGGTCTGCA AAATCACACA 1080

TCACTGTTTT TTATTCCCGC TCGTAGACAT AACTTGATGA GACTAATGCA TCAAAATAAT 1140

TGATAAATGT TTGATGAAGA ATGTATTTQC ACCACTCATG ATTCTCTGCA TTTTGTGTAA 1200

TTGAAAAGAA ATGGTAGATT TACTTATGCT TTTGTGTATC TTGTGTTTCA ACTTTAGATG 1260

CTACATGTAA AACTAAACAA GGAAGCTTGC AACGTCAAAT CTGCTACACT TTATGATGGT 1320

TCCATCCTTG AGTTTGACTC CAGGCTGCAT GACGTTGACA TCGGCACTTG CTTAGAGGTC 1380

CACCTCTCAT CTGCTCTCTA ATCCTTCAAA TTTTGTACAC GTTTATCAAA AGCAATCTGT 1440

CTGGTTTCGG CGTTAAATCT ATAATCTGTT CCTAATCTGT AGGTTATTGA ACACATGGAA 1500

GAAGATCAAG CCTGTGAATT TGGGGAAAAA GTTCTGAGCT TGTTCCACCC CAAGCTCCTG 1560

ATTGTCTCAA CACCAAACTA CGAGTTCAAC ACAATTCTCC AGCGGTCTAC ACCAGAAACC 1620

CAAGAAGAAA ACAACTCGGA GCCACAGCTT CCAAAGTTCA GGAACCATGA CCACAAATTC 1680

GAGTGGACAA GAGAACAGTT CAATCAATGG GCATCAAAGC TCGGCAAACG CCATAACTAC 1740

AGCGTCGAGT TTAGCQGTGT TGQTGGGTCT QGTGAAGTAG AACCCGGATT TGCTTCTCAG 1800

ATAGCTATTT TTAGACGGGA AGCTTCATCT GTTGAGAATG TTGCAGAAAG CTCAATGCAG 1860

CCTTATAAAG TCATCTGGGA GTGGAAGAAA GAAGATGTAQ AAAAGAAAAA GACTGATCTT 1920

TGACTAGAGT AGTATCTACC AGAGAATATA TATATTQCAA GCAATGTGAT GACGTGTTCA 1980

TCTATAAAAC TCAAAGCTCG TTTAGTTCTT TTGCCATGAG CTTTTGCAGA TTAACCATAA 2040

AATCGAACCT TTATGTTTAC AAGATCCTTT TGTCTCCAAG TCTACTGGTC CGTTGAAGTT 2100 TAAAGATTAG ATCAGTAGAT TACCCTTTTT GCCCCTACGT TAAAGTTTGT TTATTTAAAT 2160

ACAAGTCCTT GTQATTTTTA TCACTTTCTC ACTTCTTCAA CGACACTGAT TCGTTTTCAA 2220

AGCATTTGCG TTCTTQATCT TTTTCACGAG QGCGATTTCT GAAGAACACQ AGAATTTGAA 2280

TTCTGGGAAA AGCTTTCTCG AACTTTATCT GAGTAAATTG ACAGAGAGAA TCGAAAAAGA 2340

AAATGTGTCC CTGCGTAGAG CQTCGTGCTC CACCTGGAQT TTACTATACT CCGCCGCCGG 2400

CGAGAACAAG TGATGATGTG GCGGCGATGC CGATGACGGA GAGGAGGAGA CCACCQTATT 2460

CTTGTTCTTC GTCGTCGGAG AGACGTGATC CGTTTCACAT TGTTCATAAA GTTCCTTCTG 2520

GTGATTCTCC TTACGTCAGA GCCAAACACG CGCAGGTATA ATCAATTTTC CCGCGAAAAC 2580

AAGACAGQGG AATTTTTCTT TTGACCTAAT TTAGGAATTC TAGATTCTTC CTCATGTGAT 2640

AAAATCTCTT AAAAGCTTTT CAAATTTTGA GTGATCTTCT TGAACCTGTC TATGATTGAG 2700

GTTTTCTAAC AAATGGATAT TGTTTATGTG GAAGAACAGT TGATAGATAA AQATCCGAAC 2760

AGAGCTATAT CATTGTTTTQ GACTGCAATA AACGCTGGAG ATCGAGTTGA TAQTGCTTTG 2820

AAGGATATGG CCQTTGTAAT GAAACAATTA GGTCGATCTG ATGAAGGGAT TGAAGCTATT 2880

AAATCCTTTC GGTATCTCTG TTCTTTCGAG TCTCAAGACT CGATTGATAA CTTGCTACTC 2940

GAGCTCTACA AGGTAATGCA GCCTCCTTCT TTGTTAATGC AGCAAGTGTT ATGGAGTTGT 3000

TATAACTTGG AAATGGGTTT TGGTGTTTTT GCAGAAGTCA GGGAGAATTG AAGAAGAAGC 3060

TGTTTTACTT GAGCACAAAC TGCAAACACT CGAACAAQGA ATGGGATTTG GTGGTAQAGT 3120

CAGTAGAGCA AAAAGGGTTC AAQGAAAACA TGTTATTATG ACTATCGAGC AAGAGAAAGC 3180

AAQGTGAGAA AGTTTCGGTG TATTCTCATG TTAGCTTGAT GAGTGTTTTQ TCTCTTGAAT 3240

TGACGTTTCC ATTGTTGTTT GAACATTTTA GGATACTAGG GAACTTGGGC TGGGTTCATT 3300

TACAGTTACA TAACTATGGA ATTGCAGAGC AGCATTACAG GTTTGGTTTT GTTACCAAAA 3360

TCCCAAATAT TGATTACTGT CTGGTCATGT GAGTTTTGCT TATATTCAAC TGATTATQAA 3420

CAGGAGAGCT TTGGGTTTGG AGCGAGACAA AAACAAACTC TGTAACCTTG CAATCTGCTT 3480

GATGCGTATG AGTCGAATTC CTGAAGCTAA ATCTCTQCTT GATGATGTAA GAGATTCTCC 3540

TQCAGAGAGT GAATGCGGAG ATGAACCATT CGCAAAGTCT TATGACCGAG CCGTCGAAAT 3600

GTTAGCAGAA ATAGAATCGA AAAAGCCAGA AGCTGATCTT TCGGAGAAGT TCTACGCGGG 3660

ATGTTCATTT GTGAATAGGA TGAAGGAAAA TATAGCTCCT GGAACCGCAA ACAAGAACTA 3720

CTCAGATGTT TCTTCTTCTC CAGCATCTGT GAGACCGAAC TCTGCAGGTC TATATACACA 3780

ACCACGCAGA TGCAGATTGT TTGAAGAAGA GACGAGAGGT GCTGCTCGAA AGCTACTATT 3840

TGGAAAACCA CAACCTTTTG GCTCTGAACA GATGAAGATC TTAQAGAGAQ GAGAAQAAGA 3900

ACCCATGAAG CGAAAGAAAC TGGACCAGAA CATGATTCAA TATCTCCATG AATTCGTCAA 3960 AGATACAGCA GATGQTCCGA AGAGTGAATC AAAGAAAAGC TGGGCAGATA TCGCAGAAGA 4020

GGAAGAAGCA GAAGAAGAAG AAGAAGAAAQ ATTGCAGQQA GAQCTTAAAA CCGCAGAGAT 4080

GTAGGATCTA TTTGTACCGG TTATCAAGTT TTTTGGATCA TAGAAATTGT ATAGGCTTTT 4140

GACATTGAAA TATTTTCATA GACAAAACAA AGAAGCATAG GCAAATGTGA TTACACTCTG 4200

GAACAAATCT TAGGTTTGGG AAAGAAATTC AATAACAAAA CACACAAAGC ACAAAAAGAG 4260

AGAATCCCAA TTCAGAAAAA GAGGTAAAAG GAAATGAGTG CGACGAACTT ACAGCCTTAC 4320

AAAAGTAATA ATCAATCATC TTTTCTTTAG GCTTCTTCTT CTTCTTCGTC CTCCTCGTAC 4380

TCCTCTTCAC CGACTGTAGC ATCTTGATAT TGCTGATACT CTGCAACAAG ATC 4433

Claims

CLAIMS:

1. 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] 8 gene derived from Arabidopsis thaliana or an equivalent or homologue thereof.

2. The method according to claim 1, wherein the step of targeting a genomic region comprises the expression of a co-suppression molecule comprising nucleotide sequences derived from said genomic region in the plant.

3. The method according to claim 1, wherein the step of targeting a genomic region comprises expressing in the plant a ribozyme molecule which comprises a nucleotide sequence complementary to mRNA encoded by said genomic region.

4. The method according to claim 1, wherein the step of targeting a genomic region comprises expressing in the plant an antisense molecule which comprises a nucleotide sequence complementary to mRNA encoded by said genomic region.

5. The method according to claim 1, wherein the step of targeting a genomic region comprises the insertion a nucleic acid molecule into said genomic region to disrupt expression of the JAG18 polypeptide.

6. The method according to claim 5, wherein the nucleic acid molecule is a gene-targeting molecule which is inserted into the genomic region in a site-specific manner.

7. The method according to claim 5 or 6, wherein the nucleic acid molecule or gene- targeting molecule comprises a sequence of nucleotides which, when inserted into the genomic region, disrupts the open reading frame of theJ_4Gl8 gene within said genomic region.

8. The method according to claim 7, wherein the nucleic acid molecule or gene-targeting molecule is inserted into the exonV sequence of the JAG]S gene.

9. The method according to claim 5 or 6, wherein the nucleic acid molecule or gene- targeting molecule comprises a sequence of nucleotides which, when inserted into the genomic region, disrupts a 5' or 3' regulatory sequence in the J-4 l8 gene.

10. The method according to any one of claims 1 to 9, wherein the genomic region targeted comprises a sequence of nucleotides which is at least about 60% identical to the nucleotide sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 13 or a complementary sequence thereto.

11. The method according to any one of claims 1 to 9, wherein the genomic region targeted comprises a sequence of nucleotides which is at least about 70-80% identical to the nucleotide sequences set forth in SEQ ED NO: 1 or SEQ ID NO: 13 or a complementary sequence thereto.

12. The method according to any one of claims 1 to 9, wherein the genomic region targeted comprises a sequence of nucleotides which is at least about 90-99% identical to the nucleotide sequences set forth in SEQ ID NO: 1 or SEQ DD NO: 13 or a complementary sequence thereto.

13. A method of inducing or otherwise facilitating male sterility in a plant, said method comprising targeting a genomic region of said plant which is adjacent to a TUB9 gene or a homologue, analogue or derivative thereof, to inhibit, reduce or otherwise disrupt expression of said adjacent region.

14. The method according to claim 13, wherein the step of targeting a genomic region comprises the expression of a co-suppression molecule comprising nucleotide sequences derived from SEQ ID NO: 1 or SEQ ED NO: 13 or a homologue, analogue or derivative thereof.

15. The method according to claim 13, wherein the step of targeting a genomic region comprises expressing in the plant a ribozyme molecule which comprises a nucleotide sequence complementary to mRNA encoded by SEQ ID NO: l or SEQ ID NO: 13 or a homologue, analogue or derivative thereof.

16. The method according to claim 13, wherein the step of targeting a genomic region comprises expressing in the plant an antisense molecule which comprises a nucleotide sequence complementary to mRNA encoded by SEQ ID NO:l or SEQ ED NO: 13 or a homologue, analogue or derivative thereof.

17. The method according to claim 13, wherein the step of targeting a genomic region comprises the insertion of a nucleic acid molecule into said genomic region to disrupt expression of the JAG18 polypeptide encoded by SEQ ID NO: l or SEQ ID NO: 13 or a homologue, analogue or derivative thereof.

18. The method according to claim 17, wherein the nucleic acid molecule is a gene-targeting molecule which is inserted into the genomic region in a site-specific manner.

19. The method according to claim 17 or 18, wherein the nucleic acid molecule or gene- targeting molecule disrupts the open reading frame of the JAG 18 gene.

20. The method according to claim 19, wherein the nucleic acid molecule or gene-targeting molecule is inserted into the exonV sequence of the JAG]S gene.

21. The method according to claim 17 or 18, wherein the nucleic acid molecule or gene- targeting molecule comprises a sequence of nucleotides which, when inserted into the genomic region, disrupts a 5' or 3' regulatory sequence in the J-4G18 gene.

22. The method according to any one of claims 5-12 or 17-21, wherein the nucleic acid molecule or gene-targeting molecule which is inserted into the genomic region comprises nucleotide sequences derived from T-DNA.

23. The method according to claim 22, wherein the T-DNA-derived nucleotide sequences comprise left border (LB) or right border (RB) T-DNA sequences.

24. The method according to claim 23, wherein the LB sequence at least comprises nucleotides 1113-1216 of SEQ ID NO:3 or nucleotides 1-95 of SEQ ID NO:4 or a complmentary sequence thereto or a homologue, analogue or derivative thereof.

25. A method of inducing or otherwise facilitating male-sterility in a plant, said method comprising expressing in said plant a gene-targeting molecule which comprises a sequence of nucleotides capable of hybridising under at least low stringency conditions to any one of SEQ ID NOS: 1, 3, 4 or 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.

26. A method of inducing or otherwise facilitating male-sterility in a plant, said method comprising expressing in said plant an antisense 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 of SEQ ID NOS: 1 , 3, 4 or 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.

27. A method of inducing or otherwise facilitating male-sterility in a plant, said method comprising expressing in said plant a ribozyme 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 of SEQ ED NOS: 1, 3, 4 or 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.

28. A method of inducing or otherwise facilitating male-sterility in a plant, said method comprising expressing in said plant a co-suppression molecule which comprises a sequence of nucleotides capable of hybridising under at least low stringency conditions to any one of SEQ ID NOS: 1, 3, 4 or 13 or a complementary sequence thereto or a homologue, analogue or derivative thereof.

29. The method according to any one of claims 24 to 28, comprising 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.

30. The method according to any one of claims 24 to 29, wherein the gene-targeting, antisense, ribozyme or co-suppression molecule further comprises a nucleotide sequence which is substantially the same as any one of the sequences set forth in SEQ ID Nos:l, 3, 4 or 13 or which is at least about 60% identical to said sequence or a complement thereof.

31. A male-sterile or semi-sterile plant produced by the method according to any one of claims 1 to 30.

32. The male-sterile or semi-sterile plant according to claim 31 , wherein the phenotype of said plant is conditional upon the photoperiod under which said plant is grown.

33. The male-sterile or semi-sterile plant according to claim 32, wherein said plant is male- sterile when grown under a SD photoperiod.

34. The male-sterile or semi-sterile plant according to claim 33, wherein the male-sterile phenotype is made less severe or male-fertility is restored by growing plants under a LD photoperiod or continuous light.

35. The male-sterile or semi-sterile plant according to claim 31, wherein the phenotype of said plant is conditional upon its genetic background.

36. The male-sterile or semi-sterile plant according to any one of claims 31 to 35, selected from the list comprising Arabidopsis thaliana, Thlaspi arvense, wheat, barley, rice, rye, maize, or sorghum, oil seed rape (Conoid), Linola, cotton, sugar cane, Eucalyptus ssp, pine, roses and chrysanthemum, amongst others.

37 A method of producing a hybrid plant which expresses a desired trait, said method comprising crossing the male-sterile or semi-sterile plant according to any one of claims 31 to 36 with a pollen donor plant possessing or expressing said desired trait and obtaining progeny there from

38 The method according to claim 37 comprising the further step of selecting from the progeny those plants which express the desired trait.

39 An isolated nucleic acid molecule which is at least 60% identical to SEQ LD NO 1 or SEQ ID NO.13 or a complementary sequence thereto or a homologue, analogue or derivative thereof

40. The isolated nucleic acid molecule according to claim 39, wherein the percentage identity is at least about 75-80%

41 The isolated nucleic acid molecule according to claim 39, wherein the percentage identity is at least about 85-95%

42 The isolated nucleic acid molecule according to any one of claims 39 to 41, further capable of inducing male-sterility in a plant when expressed therein as a gene-targeting, antisense, ribozyme or co-suppression molecule

43 An isolated nucleic acid molecule which hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ LD NO 1 or SEQ ID NO 13 or to a complementary strand, homologue, analogue or derivative thereof

44 An isolated nucleic acid molecule which hybridises under at least moderate stringency conditions to the nucleic acid molecule set forth in SEQ LD NO 1 or SEQ ID NO. 13 or to a complementary strand, homologue, analogue or derivative thereof high

45. An isolated nucleic acid molecule which hybridises under high stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 1 or SEQ ID NO: 13 or to a complementary strand, homologue, analogue or derivative thereof.

46. The isolated nucleic acid molecule according to any one of claims 43 to 45, wherein said molecule further comprises a sequence of nucleotides which is at least 60% identical to the sequence set forth in SEQ LD NO: 1 or SEQ LD NO: 13 or a complementary sequence thereto, or a homologue, analogue or derivative thereof.

47. An isolated nucleic acid molecule comprising a sequence of nucleotides corresponding to or complementary to a genomic region, wherein said genomic region:

(i) corresponds to JAGl 8 or an adjacent region;

(ii) corresponds to a region adjacent TUB9,

(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.

48. An isolated nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence which is at least 60% identical to SEQ LD NO:2.

49. The isolated nucleotide sequence according to claim 48, wherein the percentage identity to SEQ LD NO:2 is at least 80-90%.

50. The isolated nucleotide sequence according to claim 48, wherein the percentage identity to SEQ LD NO:2 is at least 95%.

51. An isolated nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence which identical to SEQ LD NO:2 or a homologue, analogue or derivative thereof.

52. A genetic construct comprising the isolated nucleic acid molecule according to any one of claims 39 to 51.

53. The genetic construct according to claim 52, wherein the isolated nucleic molecule is placed operably in connection with a promoter sequence capable of regulating gene expressin in a plant cell.

54. The genetic construct according to claim 53, wherein the promoter is selected from the list comprising the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, A. thaliana JAGIS promoter, amongst others.

55. The genetic construct according to claim 54, wherein the A. thaliana JAG]S promoter comprises a sequence of nucleotides derived from nucleotides 1-2000 of SEQ LD NO: 13 or a complementary sequence thereto.

56. The genetic construct according to any one of claims to 52 to 55, further comprising a transcription terminator sequence.

57. The genetic construct according to claim 56, wherein the transcription terminator sequence is selected from the list comprising 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, the Rubisco small subunit (SSU) gene terminator sequences and subclover stunt virus (SCSV) gene sequence terminator, amongst others.

58. The genetic construct according to any one of claims 52 to 57, further comprising a dominant selectable marker gene.

59. A plant transformed with the isolated nucleic acid molecule according to any one of claims 39 to 51.

60. A plant transformed with a genetic construct according to any one of claims 52 to 58.

61. The plant according to claims 59 or 60, further defined as a male-sterile or semi-sterile plant.

62. The plant according to claim 60 or 61, selected from the list comprising Arabidopsis thaliana, Thlaspi arvense, wheat, barley, rice, rye, maize, or sorghum, amongst others oil seed rape (Conoid), Linola, cotton, sugar cane, Eucalyptus ssp, pine, roses, and chrysanthemum, amongst others.

63. A method of restoring male-fertility to the male-sterile plant according to any one of claims 31 to 36 or claim 61, 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.

64. The method according to claim 62, wherein the JAG18 polypeptide comprises an amino acid sequence which is at least about 60% identical to SEQ LD NO:2.