WO2006125678A2

WO2006125678A2 - Reversible male-sterile plants

Info

Publication number: WO2006125678A2
Application number: PCT/EP2006/006150
Authority: WO
Inventors: Michiel Van Lookeren Campagne; Robert Dirks; Roger Peeters; Chun-Ming Liu; Shipeng Li; Erwin Heberle-Bros; Alisher Touraev
Original assignee: Nunhems Netherlands Bv; Plant Research International; Universität Wien
Priority date: 2005-05-23
Filing date: 2006-05-23
Publication date: 2006-11-30
Also published as: WO2006125678A3

Abstract

The invention relates to the production of male-sterile plants, preferably conditionally reversible male-sterile plants. In particular, the invention is directed to these male-sterile plants and their use in the production of hybrid plants. The invention also encompasses methods for reproducing said male-sterile plants, and methods for producing male-fertile hybrid plants.

Description

Reversible male-sterile plants

[0001] The invention relates to the production of male-sterile plants, preferably conditionally reversible male-sterile plants. In particular, the invention is directed to these male-sterile plants and their use in the production of hybrid plants. The invention also encompasses methods for reproducing said male-sterile plants, and methods for producing male-fertile hybrid plants.

[0002] Plant male sterility is an attractive trait for plant breeders, since it is an essential component of hybrid seed production in many crops (Budar and Pelletier, 2001). Hybrid varieties are produced from controlled crosses between generally two inbred parental lines, the pollen of one parental line (the male parent) being used to pollinate the other parental line (the female parent). In order to efficiently perform this cross, pollination of the female patent must be controlled so as to prevent self-pollination.

[0003] Several method to control pollination of the female parent have been developed, like manual emasculation of the male organs, treatment of plants with chemical compounds toxic to pollen development, or the use of mutations preventing normal pollen development (Williams, 1995, Trends in Biotech., 13, 344-349). However, these methods present several disadvantages. The widely used cytoplasmic male sterility demands fertility restorer lines for maintenance and is not available for all crops (Cross and Ladyman, 1991). Also, chemical hybridizing agents have been shown to be inefficient and include application of partially toxic chemicals in order to induce pollen abortion or restore fertility (Perez-Prat and van Lookeren Campagne, 2002).

[0004] Plant reproduction is a complex process implying coordinate action of metabolic pathways and requiring a precisely timed series of developmental steps (McCormick, 1993; Bedinger et al., 1994). Male gametophyte formation occurs in close interaction with surrounding sporophytic tissues, in particular with the tapetum. The tapetum, representing the inner layer of the anther locule, has many important functions (Pacini and Franchi, 1993) and plays an essential role in pollen formation, particularly by nourishing developing pollen (Pacini et al., 1985; Goldberg et al., 1993; McCormick, 1993). Its significance becomes evident in male sterile plants, as male sterility is frequently caused by tapetal dysfunctions (Denis et al., 1993; Sanders et al., 1999; Kapoor et al., 2002). Discovery of tapetum-specific genes has enabled researchers to engineer male sterility into plants through selective destruction of tapetal cells (Mariani et al., 1990; van der Meer et al., 1992; Worrall et al., 1992; Yui et al., 2003). [0005] Several molecular biological approaches have led to alternative ways for introduction of male sterility in crop plants. Mariani et al. (1990) introduced a RNAse that leads to tapetum ablation and results in transgenic plants that are male sterile.

[0006] Another approach consisted in the use of auxotrophic mutants. Auxotrophic mutants of plants are characterized by the inability to synthesise one or more enzymes involved in metabolic pathways. Such mutants have been recovered in plant cells via a variety of techniques (for a review, see Pythoud and King, 1990).

[0007] One method has consisted in the creation of auxotrophic plants by targeted inhibition of one enzyme involved in the production of essential metabolic compounds (US Patent No. 6,262,339). Male-sterile plants have been obtained by targeting these enzymes in the male organ. One advantage of this method is that male sterility is reversible by supplying the plant with the missing metabolic compound. US Patent No. 6,262,339 described the targeted inhibition of the enzyme acetolactate synthase (ALS) in the tapetum by transformation of the plant with a genetic construct comprising an antisens nucleic acid of the targeted ALS enzyme. The main drawback of this technology is that fertility can only be restored by providing the missing metabolic compound(s) to the plant, i.e. valine, isoleucine, and leucine for ALS, which is not a very practical means possibly being used by breeders and hybrid producers.

[0008] Glutamine synthetase (hereinafter "GS", E.C. 6.3.1.2) catalyses the ATP-dependent conversion of glutamate to glutamine utilizing ammonia as substrate (Lea et al., 1989). Glutamine is a precursor of many different compounds, including proteins and several amino acids (Mousdale and Coggins, 1991) and possesses a central position in plant amino acid metabolism (Lea et al., 1989). The importance of glutamine for pollen development has been shown by the disruption of gametophytic development in tobacco microspore cultures (Kyo and Harada, 1986; Benito Moreno et al., 1988; Harada et al., 1988), the induction of male sterility by inhibition of glutamine synthase (Kimura et al., 1994) and the presence of its isoforms in tobacco male reproductive tissues (Becker et al., 1992; Dubois, 1996).

[0009] Plant GS is an octameric enzyme and has a very high affinity for ammonia (Stewart et al., 1980; Lea and Ridley, 1989; Lea, 1991). In higher plants, two isoenzymic forms, GSl and GS2, are known, which are located in the cytoplasm (GSl) and chloroplast (GS2), respectively (McNaIIy et al., 1983; Hirel et ah, 1993). In most plant species, GS2 is encoded by a single nuclear gene per haploid genome (Lightfoot et al., 1988), while GSl is represented by a multigene family (Peterman and Goodman, 1991; Li et al., 1993; Lam et al., 1995), members of which are differentially regulated (Dubois et al., 1996; Temple et al., 1998; Carvalho et al., 2000). Both GSl and GS2 are expressed in tobacco anthers with predominance of GSl (Becker et al., 1992; Dubois et al., 1996). Chloroplastic GS2 is primarily active in leaves (Becker et al., 1992), where it re-assimilates ammonia during the process of photorespiration (Leegood et al., 1995). Plants deficient in GS2 accumulate high concentrations of ammonia in leaves (Blackwell et al., 1987, 1988; Wallsgrove et al., 1987). Inhibition of GS depletes plants of crucial amino acids, including glutamine and glutamate and arrests photosynthesis (Sauer et al., 1987; Wild and Wendler, 1991; Downs et al., 1994). Effects of GS suppression have gained much attention, since it is the target enzyme of the commercial non-selective herbicides bialaphos and glufosinate (Wild and Manderscheid, 1984).

Description of the figures

Figure 1: Alignment of GSl and GS2 proteins from different plant species. The amino acids selected for mutation (D56 and R291) are highlighted in boxes. Some GS2 specific AA are indicated by *.

Description

[0010] The present invention is directed to a male sterile plant, wherein said plant comprises a chimeric gene comprising a male organ specific promoter operably linked to a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ.

[0011] According to the present invention, a plant which is male-sterile is a plant which does not produce any functional pollen. A male-sterile plant according to the invention is a plant whose male sexual organs are blocked in their normal development so that they do not produce any, or sufficient amounts of, functional pollen. A "functional pollen" is a pollen grain which is able to fertilize a plant female embryo sac of the same species. The male-sterile plants of the invention are conditionally reversible. A conditionally reversible male-sterile plant is a plant whose male sterility can be reserved by the conditional application of a mean enabling the reversal of the male sterility to male fertility and functional pollen production.

[0012] According to the invention, a "chimeric gene" is an artificial gene comprising the basic elements of a gene, i.e. a promoter, a coding sequence and a terminator, said elements being derived from at least two different genes as found in nature, and/or from the same gene (i.e. encoding a protein having the same function) of at least two different organisms.

[0013] The expression "operably linked" means that the different elements of the individual chimeric genes are linked to one another in such a manner to have a coordinated functioning and to enable the expression of the coding sequence. As an example, a promoter is operably linked to a coding sequence when it is able to drive expression of said coding sequence. The creation of chimeric genes according to the invention and the operable assembling of the different elements composing them can be done using classical biotechnological methods well-known to the skilled person, in particular those described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press).

[0014] A "dominant negative mutant" is a mutated protein whose mutation and expression into a cell causes the inactivation of another protein naturally interacting with its native non-mutated counterpart in cells. When overexpressed in a cell with the native protein, it adversely affects the functioning of the normal, native protein within the same cell, usually through protein-protein interactions. Preferably, a dominant negative mutant according to the invention is a mutated and inactivated monomer of a multimeric protein, whose overexpression in the cell leads to the complete inactivation of the whole multimeric protein.

[0015] According to the invention, the expression of a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in the biosynthesis of glutamine under the control of a male organ specific promoter selectively inhibits the production of glutamine in said male organ. Therefore, expression of the dominant negative mutant induces the inhibition of glutamine in the plant cells. Accordingly, the native protein whose activity is affected by the dominant negative mutant is a protein involved in glutamine biosynthesis. A protein involved in glutamine biosynthesis is to be understood as a protein whose enzymatic activity influences the biosynthesis of glutamine in the plant cells, in particular an enzyme involved in one of the biochemical steps leading to the production of glutamine by the plant cells. A preferred native protein to be inactivated by the dominant negative mutant according to the invention is the enzyme glutamine synthetase (hereinafter "GS"), also referenced in the art as EC 6.3.1.2.

[0016] Most preferably, the targeted GS to be inhibited by the dominant negative mutant according to the invention, i.e. the native protein involved in glutamine biosynthesis, is GSl. Among higher plants, two GS isoforms have been identified, GSl and GS2. GSl is known to be located in the cytosol of the cells, whereas GS2 is known to be localized in plastids, although both are encoded by nuclear genes. In order to be naturally transported to the plastid cellular compartment, GS2 is naturally encoded with a chloroplast transit peptide. GSl and GS2 show a high degree of sequence similarity at the protein level. However, GS2 proteins in different plant species showed several features that are different from GSl, in particular, 1) all GS2 have an about 50- to 60-AA chloroplast-targeting signal at their N-termini; 2) all GS2 have a short 8 to 18-AA extension at their C-termini; 3) hi the middle of these proteins, several GS2-specific sites could be recognized, such as two S instead of A and V in the middle of the proteins; and two L instead of F and I at their C-termini. All these features allow a skilled person distinguishing GSl from GS2. Alignments of GSl and GS2 amino acid sequences are provided in Figure 1.

[0017] According to a particular embodiment of the invention, the nucleic acid sequence encoding a dominant negative mutant of a native protein encodes a mutated GSl. A preferred mutated GSl is a GSl mutated at the conserved amino acid position corresponding to the asparagjne of position 56, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

[0018] In a preferred embodiment, the nucleic acid sequence encoding a mutated GSl encodes a GSl comprising an alanine at amino acid position 56, said position number being relative to the tobacco GS 1 sequence defined by SEQ TD NO: 2.

[0019] In a most preferred embodiment, the nucleic acid sequence encoding a mutated GSl encodes a GSl having an alanine at amino acid position 56, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2, said mutated GSl being selected from the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12, in which sequences the asparagine of position 56 is replaced by an alanine. Most preferably, said nucleic acid sequence encoding a mutated GSl is selected from the nucleic acid sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11, wherein the codon encoding the asparagine at position 56 is replaced by an alanine-encoding codon selected from GCA, GCC, GCG, or GCT.

[0020] Another preferred mutated GSl is a GSl mutated at the conserved amino acid position corresponding to the arginine of position 291, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

[0021] In a preferred embodiment, the nucleic acid sequence encoding a mutated GSl encodes a GSl comprising a leucine at amino acid position 291, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

[0022] In a most preferred embodiment, the nucleic acid sequence encoding a mutated GSl encodes a GSl having a leucine at amino acid position 291, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2, said mutated GSl being selected from the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12, in which sequences the arginine of position 291 is replaced by a leucine. Most preferably, said nucleic acid sequence encoding a mutated GSl is selected from the nucleic acid sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11, wherein the codon encoding the arginine at position 291 is replaced by a leucine-encoding codon selected from CTA, CTC, CTG, CTT, TTA, or TTG.

[0023] A most preferred mutated GSl comprises two mutations, and is a GSl mutated at the conserved amino acid positions corresponding to the asparagine of position 56 and the arginine of position 291, said position numbers being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

[0024] Preferably, the nucleic acid sequence encoding a mutated GSl comprising two mutations encodes a GSl comprising an alanine at amino acid position 56 and a leucine at amino acid position 291, said position numbers being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

[0025] In a most preferred embodiment, the nucleic acid sequence encoding a mutated GSl comprising two mutations encodes a GSl having an alanine at amino acid position 56 and a leucine at amino acid position 291, said position numbers being relative to the tobacco GSl sequence defined by SEQ ID NO: 2, said mutated GSl being selected from the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12, in which sequences the asparagine of position 56 is replaced by an alanine, and the arginine of position 291 is replaced by a leucine. Most preferably, said nucleic acid sequence encoding a mutated GSl is selected from the nucleic acid sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11, wherein the codon encoding the asparagine at position 56 is replaced by an alanine-encoding codon selected from GCA, GCC, GCG, or GCT, and the codon encoding the arginine at position 291 is replaced by a leucine- encoding codon selected from CTA, CTC, CTG, CTT, TTA, or TTG.

[0026] According to another particular embodiment of the invention, the nucleic acid sequence encoding a dominant negative mutant of a native protein encodes a mutated GS2. A preferred mutated GS2 is a GS2 having its chloroplast transit peptide deleted. Accordingly, the preferred mutated GS2 is truncated so as to be limited to its mature part. A most preferred mutated GS2 is a GS2 deleted of its 59 N-terminal amino acids, said numbering being relative to the tobacco GS2 sequence defined by SEQ ID NO: 14.

[0027] In a most preferred embodiment, the nucleic acid sequence encoding a mutated GS2 encodes a GS2 deleted of its 59 N-terminal amino acids, said numbering being relative to the tobacco GS2 sequence defined by SEQ ID NO: 4, said mutated GS2 being selected from the sequences set forth in SEQ ID NO: 14, 16, 18, 20, or 22, in which sequences the 59 N-terminal amino acids are deleted. Most preferably, said nucleic acid sequence encoding a mutated GS2 is selected from the nucleic acid sequences set forth in SEQ ID NO: 13, 15, 17, 19, or 21, wherein the codons encoding the 59 N-terminal amino acids are deleted. [0028] Another most preferred mutated GS2 is a GS2 deleted of both its 59 N-terminal amino acids and its 45 C-terminal amino acids, said numbering being relative to the tobacco GS2 sequence defined by SEQ ID NO: 14.

[0029] In a most preferred embodiment, the nucleic acid sequence encoding a mutated GS2 encodes a GS2 deleted of both its 59 N-terminal amino acids and its 45 C-terminal amino acids, said numbering being relative to the tobacco GS2 sequence defined by SEQ ID NO: 4, said mutated GS2 being selected from the sequences set forth in SEQ ID NO: 14, 16, 18, 20, or 22, in which sequences the 59 N-terminal amino acids and the 45 C-terminal amino acids are deleted. Most preferably, said nucleic acid sequence encoding a mutated GS2 is selected from the nucleic acid sequences set forth in SEQ ID NO: 13, 15, 17, 19, or 21, wherein the codons encoding the 59 N-terminal amino acids and the 45 C-terminal amino acids are deleted.

[0030] The nucleic acid sequence encoding a dominant negative mutant of a native protein involved in the biosynthesis of glutamine is under the control of a male organ specific promoter, so that expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ. According to the invention, a "male organ specific promoter" is a promoter of a gene naturally and specifically expressed in cells of the male organs of a plant, a "male organ" being understood as a male-specific sexual organ like the anthers, the anther filament, the tapetum, the microspore mother cell, and/or any cell of the male gametophyte.

[0031] A preferred male organ specific promoter according to the invention is a tapetum- specific promoter. One preferred tapetum-specific promoter is the promoter of the TA29 gene as described in Seurinck et al. ( 1990) and Koltunov, et al. ( 1991 ).

[0032] A preferred male organ specific promoter according to the invention is a microspore- specific promoter. One preferred microspore-specific promoter is the promoter of the NTMl 9 gene as described in Oldenhof et al. (1996, Plant MoI. Biol. 31(2), 213-225).

[0033] Accordingly, the tapetum-specific or microspore-specific expression of the nucleic acid sequence encoding a dominant negative mutant of a native protein involved in the biosynthesis of glutamine leads to the selective inhibition of the production of glutamine in the tapetum or microspore cells. The selective inhibition of glutamine production in the tapetum or microspore cells then leads to the inhibition of pollen development, and to male-sterility of the developed plants.

[0034] The plants according to the invention are transformed plants. To obtain the transformed plants according to the invention, those skilled in the art can use one of the many known methods of transforming plants.

[0035] Preferably, the plants according to the invention are transformed with a cloning, expression and/or transformation vector comprising a chimeric gene according to the invention.

[0036] The vectors that may be useful for implementing the invention are, for example, plasmids, cosmids, bacteriophages or viruses. Preferably, the vectors for transforming the plant cells or the plants according to the invention are plasmids. In general, the main qualities of a vector should be an ability to maintain itself and to self-replicate in plant cells, in particular by virtue of the presence of an origin of replication. With the aim of obtaining stable transformation of a host organism, the vector may also integrate into the genome. The choice of such a vector and also the techniques for inserting the gene according to the invention into said vector are widely described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press) and are part of the general knowledge of those skilled in the art. Advantageously, the vector used in the present invention also contains, in addition to the gene according to the invention, another gene encoding a selection marker. The selection marker makes it possible to select the host organisms that are effectively transformed, i.e. those that have incorporated the vector. Among the selection markers that can be used, mention may be made of markers containing genes for resistance to antibiotics, such as, for example, that of the hygromycin phosphotransferase gene (Gritz et al., 1983, Gene 25: 179- 188), but also markers containing herbicidal tolerance genes, such as the bar gene (White et al., 1990, Nucleic Acid Res. 18(4): 1062) for tolerance to bialaphos, the EPSPS gene (US 5,188,642) for tolerance to glyphosate or alternatively the HPPD gene (WO 96/38567) for tolerance to isoxazoles. Mention may also be made of genes encoding readily identifiable enzymes such as the GUS enzyme, or genes encoding pigments or enzymes that regulate the production of pigments in the transformed cells. Such selection marker genes are in particular described in patent applications WO 91/02071, WO 95/ 06128, WO 96/38567 and WO 97/04103.

[0037] Among the transformation methods that can be used to obtain transformed plants according to the invention, one of these consists in placing the cells or tissues of the plants to be transformed in the presence of polyethylene glycol (PEG) and of the vectors described above (Chang and Cohen, 1979, MoI. Gen. Genet. 168(1), 111-115; Mercenier and Chassy, 1988, Biochimie 70(4), 503-517). Electroporation is another method, which consists in subjecting the cells or tissues to be transformed and the vectors to an electric field (Andreason and Evans, 1988, Biotechniques 6(7), 650-660; Shigekawa and Dower, 1989, Aust. J. Biotechnol. 3(1), 56- 62). Another method consists in injecting the vectors directly into the cells or the tissues by microinjection (Gordon and Ruddle, 1985, Gene 33(2), 121-136). Advantageously, the "biolistic" method may be used. It consists in bombarding cells or tissues with particles onto which the vectors are adsorbed (Bruce et al., 1989, Proc. Natl. Acad. Sci. USA 86(24), 9692- 9696; Klein et al., 1992, Biotechnology 10(3), 286-291; US Patent No. 4,945,050). Preferably, the transformation of plant cells or tissues can be carried out using bacteria of the Agrobacterium genus, preferably by infection of the cells or tissues of said plants with A. tumefaciens (Knopf, 1979, Subcell. Biochem. 6, 143-173; Shaw et al., 1983, Gene 23(3):315- 330) or A. rhizogenes (Bevan and Chilton, 1982, Annu. Rev. Genet. 16:357-384; Tepfer and Casse-Delbart, 1987, Microbiol. Sci. 4(1), 24-28). Preferably, the transformation of plant cells or tissues with Agrobacterium tumefaciens is carried out according to the protocol described by Ishida et al. (1996, Nat. Biotechnol. 14(6), 745-750). Those skilled in the art will choose the appropriate method according to the nature of the plant to be transformed.

[0038] The invention also encompasses parts of the above-described plants, and the progeny of these plants. The expression "part of these plants" is intended to mean any organ of these plants, whether aerial or subterranean, including its meiotic products (i.e. pollen and embryo sac). The aerial organs are essentially the stems, the leaves and the flowers. The subterranean organs are mainly the roots, but they may also be tubers. The term "progeny" is intended to mean mainly the seeds containing the embryos derived from the sexual reproduction of these plants with a male fertile plant. The male fertile plant can be another plant, or a male sterile plant according to the invention which has been reversed to male fertility. By extension, the term "progeny" applies to the seeds formed at each new generation derived from crosses between two plants, at least one of them being a male sterile plant according to the invention.

[0039] The present invention is also directed to a method for the generation of plants that are male sterile, said method comprising: a) transforming plant cells with a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis under the control of a male organ specific promoter, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ; b) regenerating plants from said plant cells; and c) selecting said male sterile plants from said regenerated plants.

[0040] The invention also encompasses male sterile plants which are obtainable by the above method.

[0041] The invention is also directed to a method for reversing the male sterile plants according to the invention to male fertility, said method comprising the step of supplying said male sterile plants with a composition comprising glutamine or a derivative thereof.

[0042] The invention is also directed to a method for reproducing the male sterile plants according to the invention, said method comprising the steps of supplying said male sterile plants with a composition comprising glutamine or a derivative thereof, then enabling the recovered pollen to fertilize the female sexual organs of said male sterile plant, harvesting the seeds obtained from the fecundation, and then growing a new generation of male sterile plants from said seeds.

[0043] The invention is also directed to another method for reproducing the male sterile plants according to the invention, said method comprising the steps of culturing in vitro some microspores obtained from the male organ of said male sterile plant into a culture medium comprising glutamine or a derivative thereof so as to enable pollen development, then enabling the recovered pollen to fecundate the female sexual organs of said male sterile plant, harvesting the seeds obtained from the fecundation, and then growing a new generation of male sterile plants from said seeds.

[0044] According to another embodiment, the invention is directed to a method for reproducing the male sterile plants according to the invention, said method comprising the steps of culturing in vitro some microspores obtained from the male organ of said male sterile plant into a culture medium comprising an embryogenesis agent so as to enable embryos formation from the cultured microspores, then growing said plant embryos into haploid plants. If necessary, such plants can be rendered diploid by treatment of the embryos with a diploidizing agent, for example colchicine. The said diploid plants are therefore homozygous male-sterile diploid plants, also called male-sterile doubled-haploid plants.

[0045] According to another embodiment, the invention is directed to a method for reproducing the male sterile plants according to the invention, said method comprising the steps of culturing in vitro some undeveloped microspores obtained from the male organ of said male sterile plant into a culture medium enabling embryos formation from the cultured microspores, then growing said plant embryos into haploid plants. A preferred male-sterile plant to carry out the present method is a male sterile plant according to the invention whose male-organ specific promoter is a microspore-specific promoter. In that respect, undeveloped microspores are microspores whose normal development into mature pollen has been blocked by the targeted expression of the dominant negative mutant in the microspores. This targeted expression of the dominant negative mutant in the microspores and the associated glutamine starvation prevents said microspores to develop into mature pollen, thus leading to male-sterility, and favors their development into haploid embryos if cultured in a medium enabling said embryogenic development. If necessary, such plants can be rendered diploid by treatment of the embryos with a diploidizing agent, for example colchicine. The said diploid plants are therefore homozygous male-sterile diploid plants, also called male-sterile doubled-haploid plants.

[0046] The invention is also directed to a method for producing a male-fertile hybrid plant, said method comprising the steps of crossing the female sexual organs of a male-sterile plant according to the invention with the male sexual organs of a male- fertile plant, said male-sterile plant comprising a chimeric gene itself comprising a male organ specific promoter operably linked to a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ, and said male-fertile plant comprising a chimeric gene itself comprising a male organ specific promoter operably linked to a nucleic acid sequence encoding the native protein corresponding to said dominant negative mutant of the male-sterile plant, wherein said male-organ specific promoter of the male-fertile plant is a promoter enabling overexpression of the native protein at a level higher than the one at which the male-organ specific promoter of the male-sterile plant enables overexpression of the dominant negative mutant. [0047] According to the above method, the hybrid plant produced is male fertile because of the presence of a higher quantity of active protein, i.e. the native protein, compared to the inactive protein, i.e. the dominant negative mutant, in the male organ, thereby allowing production of glutamine in said male organ and functional pollen formation.

[0048] According to a particular embodiment, the male-organ specific promoter of the male- sterile plant is a tapetum-specific promoter, more particularly the TA29 promoter.

[0049] According to a particular embodiment of the above method, the male-organ specific promoter of the male-fertile plant can be any male-organ specific promoter as described above.

A bacteriophage T7 RNA polymerase expression system can also be used as described in

Nguyen at al. (2004, Plant Biotech. J. 2(4), p. 301). According to such a bacteriophage T7 RNA polymerase expression system, the male-organ specific promoter of the male-fertile plant would be operably linked to a T7 RNA polymerase, and said male-fertile plant would also contain another chimeric gene comprising a T7 expression signal, i.e. recognized by the T7 RNA polymerase, operably linked to the nucleic acid sequence encoding the native protein corresponding to said dominant negative mutant of the male-sterile plant.

[0050] The invention is also directed to a method for producing a male-fertile hybrid plant, said method comprising the steps of crossing the female sexual organs of a microspore-inhibited, male-sterile plant with the male sexual organs of a male-fertile plant, said male-sterile plant comprising a chimeric gene itself comprising a microspore-specific promoter operably linked to a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ, and said male- fertile plant being an untransformed plant.

[0051] According to a particular embodiment of the above-method, the microspore-specific promoter is the NTM 19 promoter. [0052] The following examples aim at illustrating the invention, without limiting its scope. All the methods or operations described below in these examples are given by way of example and correspond to a choice made from the various available methods for achieving the same result. This choice has no bearing on the quality of the result and, consequently, any suitable method may be used by those skilled in the art to achieve the same result. In particular, and unless otherwise specified in the examples, all the recombinant DNA techniques used are carried out according to the standard protocols described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Second edition, Nolan C. ed., Cold Spring Harbor Laboratory Press, NY), in Sambrook and Russel (2001, Molecular cloning: A laboratory manual, Third edition, Cold Spring Harbor Laboratory Press, NY), in Ausubel et al. (1994, Current Protocols in Molecular Biology, Current protocols, USA, Volumes 1 and 2), and in Brown (1998, Molecular Biology LabFax, Second edition, Academic Press, UK). Standard materials and methods for plant molecular biology are described in Croy R.D.D. (1993, Plant Molecular Biology LabFax, BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK)). Standard materials and methods for PCR (Polymerase Chain Reaction) are also described in Dieffenbach and Dveksler (1995, PCR Primer: A laboratory manual, Cold Spring Harbor Laboratory Press, NY) and in McPherson et al. (2000, PCR - Basics: From background to bench, First edition, Springer Verlag, Germany).

Example 1: Generation of plasmids with dominant negative mutations in GS genes

[0053] The dominant negative mutant (DNM) versions of cytosolic (GSl) and chloroplastic (GS2) glutamine synthetases were generated in order to inactivate GS in anthers of transgenic tobacco plants. Tobacco chloroplastic glutamine synthetase (GS2) was modified by deleting the N-terminal sequence targeting the enzyme to chloroplasts (TA29.GS2).

[0054] The dominant negative mutations of cytosolic glutamine synthetase gene (GSl) were created by point mutations, based on the previous work in plant and E.coli (Clemente and Marquez, 1999; Eisenberg et al., 2000). To obtain maximum poisoning effects in the glutamine synthetase activity and the lowest interruption with the sub-unit interaction, two amino acids have been changed. In a first construct the arginine residue at position 291 was replaced by a leucine residue (TA29GS1L291) and in a second construct, two mutations were introduced, whereby the arginine residue at position 291 was replaced by a leucine residue and the asparagine residue at position 56 was replaced by an alanine residue (TA29GS1A56L291). All constructs were placed under the control of the tapetum-specific TA29 promoter which has been shown to be active from the tetrad stage until first pollen mitosis (Koltunow et al., 1990), cloned into the binary vector pBIN and transferred into Agrobacterium tumefaciens strain LBA 4404 via electroporation.

Example 2: Tobacco transformation and selection of transgenic plant lines

[0055] Nicotiana tabacum cv. Petite Havana SRl was used for transformation experiments. Agrobacterium-mediateά leaf disc transformation was essentially done as described by Horsch (1985). Transgenic shoots were selected on MS medium containing 50 mg/1 kanamycin and 500 mg/1 cefotaxime sodium (Duchefa, NL). Rooted plantlets were transferred to soil for further growth under the same conditions as described for wild type plans in the greenhouse until flowering.

[0056] Transgenic lines were indistinguishable from wild type plants regarding the vegetative development until flowering. 100 % male sterility was observed in six lines harbouring either TA29GS1L291 or TA29GS1A56L291 and in five lines containing TA29.GS2, characterised by the complete lack of seed set. Stable integration of constructs was confirmed by Southern blot analyses and segregation of the kanamycin-resistance marker gene in seedlings derived from crosses between wild type and transformed male sterile plants.

[0057] Expression levels of GS in extracts from anthers of wild type and male sterile tobacco plants were assessed by Western blot using an antibody recognising both chloroplastic GS2 and cytosolic GSl. Two distinct bands corresponding to GSl (molecular mass 38 - 40 kDa) and GS2 (molecular mass 44 - 45 kDa) (Becker et al., 1992; Dubois et al., 1996) were detected.

[0058] The TA29 promoter has been confirmed to be active specifically in the tapetum (Koltunov, et al., 1991; Mariani et al., 1990; 1992). Transgenic lines with DNM GSl and GS2 showed unaltered phenotypes except male sterility. Anthers, however, appeared smaller compared to the wild type, and colour of anther walls started changing to olive around first pollen mitosis.

Exemple 3: Analysis of pollen viability

[0059] Pollen viability was analysed using translucent light and fluorescent microscopy. The nuclear status of pollen was determined by 4',6-damidino-2-phenylindole (DAPI) staining (Vergne et al., 1987), while pollen viability was scored by fluorescein diacetate (FDA) (Heslop- Harrison and Heslop -Harrison, 1970) staining.

[0060] Cytologjcal preparations from transgenic anthers contained much debris, caused by the release of degraded tapetal layers, not observed in those of wild-type plants. Anthers from control plants contained uniform starch-filled, viable pollen grains upon maturity with well defined vegetative and generative nuclei. In contrast, pollen grains isolated from transgenic GS male sterile lines appeared highly heterogeneous in size and shape in all stages with frequently observed nuclear irregularities and fragmentation upon maturity.

[0061] Although early microsporogenesis was completed regularly with the formation of homogenous population of microspores, pollen plasmolysed around the first pollen mitosis and a few viable pollen grains developed to maturity in all transgenic lines analysed. Upon dehiscence, pollen had aborted almost completely (91 - 99.5 %), and in vitro germination assays revealed no pollen grains able to germinate.

Example 4: Restoration of fertility by in vitro pollen maturation and subsequent in situ pollination with in vitro matured pollen

[0062] Microspores, isolated from tobacco male sterile transgenic lines, were matured in medium Tl rich medium supplemented with necessary nitrogen sources, including glutamine, as described by Touraev and Heberle-Bors (1999).

[0063] Upon isolation, up to 40 % of microspores appeared viable and morphologically indistinguishable from wild type microspores. When cultured, 60 % of viable microspores from transgenic lines, i.e. around 20 % of the total number of cells, divided asymmetrically, giving rise to vegetative and generative nuclei, accumulated starch and completed maturation after six days of incubation. The phenotype of in vitro matured pollen grains resembled that of wild type pollen, and they appeared starch filled, transparent and triangular in shape upon maturity. Although the frequency of in vitro maturation in male sterile lines was lower compared to control plants, in vitro matured pollen grains from sterile lines could be germinated in vitro and subsequently used for pollination of receptive stigmas of the wild type and male sterile line, leading to seed set. [0064] After six days of incubation, a well developed mature pollen population was collected by centrifugation 2 min at 200xg, resuspended in germination medium GV (Touraev and Heberle- Bors, 1999) and three μl of suspension containing approximately 5-10000 pollen grains were for in situ pollination of previously emasculated flowers. Seeds were collected after three-four weeks from matured seed pods and subjected for further segregation analysis. These seeds were kanamycin resistant when germinated in the presence of antibiotic confirming that sterile pollen in male sterile plants can be rescued to fertility by in vitro maturation and used for pollination of sterile plants to restore their fertility.

Example 5: Restoration of fertility by in vivo spraying of the plant with the missing glutamine

[0065] Another approach to restore pollen fertility was to spray male sterile plants with the missing metabolites. Transgenic male sterile tobacco plants which, due to nearly complete pollen death at maturity, did not yield seeds after self-pollination were provided twice a day with 0.05 mM glutamine. Glutamine was dissolved 0.01 - 0.05 mM in water and, after filter sterilization, sprayed every day two times (early morning and late afternoon) on the bottom of the leaf and inflorescence of young healthy glass house grown plants at meiosis stage and continued for seven to ten days. Functionality of up to 10 % of the total pollen population was restored after seven to ten days, as assessed by in vitro germination. These in vivo matured pollen grains morphologically resembled mature wild type pollen, showing that developing microspores could acquire the missing nutrient. After achieving seed set, seeds were harvested and subjected to segregation analysis on kanamycin containing medium. Successful seeds set, i.e. restoration of fertility has been achieved in two lines harbouring mutated GSl and three lines transformed with mutated GS2 gene. These data confirmed that pollen abortion was indeed caused by nitrogen starvation, and that external addition of glutamine by spraying could restore fertility in 100 % male sterile lines.

Example 6: Maintaining of male sterile plants by in vitro microspore embryogenesis [0066] Male gametophyte development in transgenic male sterile plants had been disturbed around first pollen mitosis, and the majority of microspores were not affected and viable. These microspores were isolated and subjected to stress pre-treatment in medium B at 33°C for the formation of embryogenic cells according to Touraev and Heberle-Bors (1999). The embryogenic microspores were cultured in embryogenesis medium AT3 and haploid embryos and plants were obtained from all male sterile lines used for experiments. These plants were diploidized by the treatment with colchicine, and homozygous doubled haploid plants have been obtained, which were completely sterile, confirming the transmission of the transgene into the progeny. These results showed the possibility of rescuing and maintaining male sterile plants via microspore embryogenesis.

Example 7: Inhibition of microspore development

[0067] New constructs are designed, in which the coding sequences encoding the dominant negative mutants of GSl and GS2 described in Example 1 are put under the control of the microspore-specific promoter NTM 19, instead of the tapetum-specific promoter TA29. One example of such new constructs is pNTM19-GSlA56L291. Transformation is achieved according to the methods described in Example 2. Ten out of nineteen pNTM19-GSlA56L291 lines exhibit 50% pollen viability as expected for single insertions of the transgene. The anthers of the wild-type and pNTM19-GSlA56L291 lines look similar until the dehiscence stage. However, the transgenic anthers clearly release less pollen. Cross-pollination of the male-sterile To plants with wild-type pollen lead to normal seed set, showing that female fertility is not impaired. Primary pNTM19-GSlA56L291 plants set as many seeds as wild-type plants after self- and cross-pollination.

[0068] Homozygosity is particularly crucial to achieve 100% male sterility in pNTM19- GS1A56L291 lines. In a simulation of F₁ hybrid seed production homozygous pNTM19- GS1A56L291 plants were used as female parents, and pollinated with wild-type pollen (male donor line). All T₂ hybrid seedlings were kanamycin-resistant, produced 50% viable, non- transgenic pollen, and self-pollination resulted in full seed set.

[0069] Gametophytic male sterility using the constructs driven by the NTM19 promoter can be applied to seed and non-seed crops, as 50% of the pollen in the resulting hybrids is wild-type, fertile, and sufficient for full seed set.

Claims

1. A male sterile plant, wherein said plant comprises a chimeric gene comprising a male organ specific promoter operably linked to a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ.

2. A male-sterile plant according to claim 1 , wherein the dominant negative mutant of a native protein is a dominant negative mutant of the enzyme glutamine synthetase.

3. A male-sterile plant according to claim 2, wherein the dominant negative mutant of a native protein is a dominant negative mutant of the GSl glutamine synthetase.

4. A male-sterile plant according to claim 3, wherein the dominant negative mutant of the

GSl glutamine synthetase is a GSl glutamine synthetase mutated at the conserved amino acid position corresponding to the asparagine of position 56, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

5. A male-sterile plant according to claim 4, wherein the mutated GSl glutamine synthetase encodes a GSl glutamine synthetase comprising an alanine at amino acid position 56, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

6. A male-sterile plant according to claim 5, wherein the mutated GSl glutamine synthetase is a GSl glutamine synthetase having a sequence selected from the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12, in which sequences the asparagine of position 56 is replaced by an alanine.

7. A male-sterile plant according to claim 6, wherein the mutated GSl glutamine synthetase is encoded by a nucleic acid sequence selected from the nucleic acid sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11, wherein the codon encoding the asparagine at position 56 is replaced by an alanine-encoding codon selected from GCA, GCC, GCG, or GCT.

8. A male-sterile plant according to claim 3, wherein the dominant negative mutant of the GSl glutamine synthetase is a GSl glutamine synthetase mutated at the conserved amino acid position corresponding to the arginine of position 291, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

9. A male-sterile plant according to claim 8, wherein the mutated GSl glutamine synthetase encodes a GSl glutamine synthetase comprising a leucine at amino acid position 291, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

10. A male-sterile plant according to claim 9, wherein the mutated GSl glutamine synthetase is a GSl glutamine synthetase having a sequence selected from the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12, in which sequences the arginine of position 291 is replaced by a leucine.

11. A male-sterile plant according to claim 10, wherein the mutated GSl glutamine synthetase is encoded by a nucleic acid sequence selected from the nucleic acid sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11, wherein the codon encoding the arginine at position 291 is replaced by a leucine-encoding codon selected from CTA, CTC, CTG, CTT, TTA, or TTG.

12. A male-sterile plant according to claim 3, wherein the dominant negative mutant of the GSl glutamine synthetase is a GSl glutamine synthetase mutated at the conserved amino acid positions corresponding to the asparagine of position 56 and the arginine of position 291, said position numbers being relative to the tobacco GSl sequence defined by SEQ

ID NO: 2.

13. A male-sterile plant according to claim 12, wherein the mutated GSl glutamine synthetase encodes a GSl glutamine synthetase comprising an alanine at amino acid position 56 and a leucine at amino acid position 291, said position number being relative to the tobacco GSl sequence defined by SEQ ID NO: 2.

14. A male-sterile plant according to claim 13, wherein the mutated GSl glutamine synthetase is a GSl glutamine synthetase having a sequence selected from the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12, in which sequences the asparagine of position 56 is replaced by an alanine, and the arginine of position 291 is replaced by a leucine.

15. A male-sterile plant according to claim 14, wherein the mutated GSl glutamine synthetase is encoded by a nucleic acid sequence selected from the nucleic acid sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11, wherein the codon encoding the asparagine at position 56 is replaced by an alanine-encoding codon selected from GCA, GCC, GCG, or GCT, and the codon encoding the arginine at position 291 is replaced by a leucine- encoding codon selected from CTA, CTC, CTG, CTT, TTA, or TTG.

16. A male-sterile plant according to claim 2, wherein the dominant negative mutant of a native protein is a dominant negative mutant of the GS2 glutamine synthetase.

17. A male-sterile plant according to claim 16, wherein the GS2 glutamine synthetase is a

GS2 glutamine synthetase having its chloroplast transit peptide deleted.

18. A male-sterile plant according to claim 17, wherein the GS2 glutamine synthetase is a GS2 glutamine synthetase deleted of its 59 N-terminal amino acids, said numbering being relative to the tobacco GS2 sequence defined by SEQ ID NO: 14.

19. A male-sterile plant according to claim 18, wherein the mutated GS2 glutamine synthetase is a GS2 glutamine synthetase having a sequence selected from the sequences set forth in SEQ ID NO: 14, 16, 18, 20, or 22, in which sequences the 59 N-terminal amino acids are deleted.

20. A male-sterile plant according to claim 19, wherein the mutated GS2 glutamine synthetase is encoded by a nucleic acid sequence selected from the nucleic acid sequences set forth in SEQ ID NO: 13, 15, 17, 19, or 21, wherein the codons encoding the 59 N- terminal amino acids are deleted.

21. A male-sterile plant according to claim 16, wherein the GS2 glutamine synthetase is a GS2 glutamine synthetase deleted of both its 59 N-terminal amino acids and its 45 C- terminal amino acids, said numbering being relative to the tobacco GS2 sequence defined by SEQ ID NO: 14.

22. A male-sterile plant according to claim 21, wherein the mutated GS2 glutamine synthetase is a GS2 glutamine synthetase having a sequence selected from the sequences set forth in SEQ ID NO: 14, 16, 18, 20, or 22, in which sequences the 59 N-terminal amino acids and the 45 C-terminal amino acids are deleted.

23. A male-sterile plant according to claim 22, wherein the mutated GSl glutamine synthetase is encoded by a nucleic acid sequence selected from the nucleic acid sequences set forth in SEQ ID NO: 13, 15, 17, 19, or 21, wherein the codons encoding the 59 N- terminal amino acids and the 45 C-terminal amino acids are deleted.

24. A male-sterile plant according to anyone of claims 1 to 23, wherein the male organ specific promoter is a tapetum-specific promoter.

25. A male-sterile plant according to claim 24, wherein the tapetum-specific promoter is the promoter of the TA29 gene.

26. A male-sterile plant according to anyone of claims 1 to 23, wherein the male organ specific promoter is a microspore-specific promoter.

27. A male-sterile plant according to claim 26, wherein the microspore-specific promoter is the promoter of the NTMl 9 gene.

28. Method for the generation of plants that are male sterile, said method comprising: a) transforming plant cells with a chimeric gene comprising a male organ specific promoter operably linked to a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ; b) regenerating plants from said plant cells; and c) selecting said male sterile plants from said regenerated plants.

29. A method for reversing male sterile plants according to anyone of claims 1 to 27 to male fertility, said method comprising the step of supplying said male sterile plants with a composition comprising glutamine or a derivative thereof.

30. A method for reproducing male sterile plants according to anyone of claims 1 to 27, said method comprising the steps of supplying said male sterile plants with a composition comprising glutamine or a derivative thereof so as to enable pollen development, enabling the recovered pollen to fertilize the female sexual organs of said male sterile plant so as to produce seeds, harvesting the seeds obtained from the fertilization, and then growing a new generation of male sterile plants from said seeds.

31. A method for reproducing male sterile plants according to anyone of claims 1 to 27, said method comprising the steps of culturing in vitro some microspores obtained from the male organ of said male sterile plants into a culture medium comprising glutamine or a derivative thereof so as to enable pollen development, enabling the recovered pollen to fertilize the female sexual organs of said male sterile plant so as to produce seeds, harvesting the seeds obtained from the fertilization, and then growing a new generation of male sterile plants from said seeds.

32. A method for reproducing male sterile plants according to anyone of claims 1 to 27, said method comprising the steps of culturing in vitro some microspores obtained from the male organ of said male sterile plant into a culture medium comprising an embryogenesis agent so as to enable embryos formation from the cultured microspores, and then growing said plant embryos into haploid plants.

33. A method for reproducing male sterile plants according to anyone of claims 26 or 27, said method comprising the steps of culturing in vitro some undeveloped microspores obtained from the male organ of said male sterile plant into a culture medium enabling embryos development, and then growing said plant embryos into haploid plants.

34. A method according anyone of claims 32 or 33, wherein said plants are made diploid by treatment of the embryos with a diploidizing agent.

35. A method for producing a male-fertile hybrid plant, said method comprising the steps of crossing the female sexual organs of a male-sterile plant according to anyone of claims 1 to 25 with the male sexual organs of a male-fertile plant, said male-sterile plant comprising a chimeric gene itself comprising a tapetum-specific promoter operably linked to a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ, and said male-fertile plant comprising a chimeric gene itself comprising a tapetum-specific promoter operably linked to a nucleic acid sequence encoding the native protein corresponding to said dominant negative mutant of the male-sterile plant, wherein said tapetum-specific promoter of the male-fertile plant is a promoter enabling overexpression of the native protein at a level higher than the one at which the tapetum- specific promoter of the male-sterile plant enables overexpression of the dominant negative mutant.

36. A method for producing a male-fertile hybrid plant, said method comprising the steps of crossing the female sexual organs of a male-sterile plant according to claims 26 or 27 with the male sexual organs of a male-fertile plant, said male-sterile plant comprising a chimeric gene itself comprising a microspore- specific promoter operably linked to a nucleic acid sequence encoding a dominant negative mutant of a native protein involved in glutamine biosynthesis, whereby expression of said nucleic acid sequence selectively inhibits the production of glutamine in said male organ, and said male-fertile plant being an untransformed plant.