WO2002052924A2

WO2002052924A2 - Method to maintain a genic male-sterile female parental lines for the production of hybrid wheat

Info

Publication number: WO2002052924A2
Application number: PCT/US2002/000549
Authority: WO
Inventors: Moshe Feldman; Eitan Millet
Original assignee: Yeda Research And Development Co. Ltd.; Fleit, Lois
Priority date: 2001-01-04
Filing date: 2002-01-02
Publication date: 2002-07-11
Also published as: WO2002052924A3; EP1347678A2; AU2002248325A1; EP1347678A4

Abstract

The present invention provides a method for stably maintaining a genic male-sterile female parental line of bread and durum wheat for the production of hybrid wheat. It also provides a male-sterile female line homozygous for a recessive male sterility allele, and a maintainer line which is readily and stably propagated. The maintainer line is isogenic to the female line but has an alien chromosomal arm, added to the wheat complement, carrying a dominant male-fertility allele that restores fertility to the maintainer line, a heterologous microspore-suicide gene that kills microspores or pollen grains carrying it thereby preventing the transmission of this chromosome arm to the female line, and a heterologous selectable marker that facilitates the selection of maintainer plants among the progeny of the selfed maintainer line and thus, maintenance of the maintainer itself.

Description

METHOD TO MAINTAIN A GENIC MALE-STERILE FEMALE PARENTAL LINES FOR THE PRODUCTION OF HYBRID WHEAT

BACKGROUND OF THE INVENTION

Field Of The Invention

The present invention relates to the production of hybrid lines of common and durum wheat, and more specifically, relates to a new method for maintaining a male-sterile female parental line for use in the production of hybrid wheat seeds, which female line is homozygous for a recessive male-sterility mutant allele, and relates to a new maintainer line for maintaining the female parental line, which is isogenic to the female line, but has an alien chromosomal arm, as a monotelosomic addition, carrying a dominant male-fertility allele homoeoallelic to the recessive male-sterility allele, a heterologous microspore-suicide gene capable of killing the microspores or pollen grains carrying it, and a heterologous selectable marker. These three genes on the alien chromosomal arm are permanently linked due to lack of pairing and recombination between the alien chromosomal arm and the wheat chromosomes. The presence of the microspore-suicide gene on the alien chromosomal arm ensures that all the maintainer viable male gametes (about 80% of the gametes) lack this chromosomal arm and consequently, the dominant male-fertility allele and the selectable marker allele. Hence, pollination of male-sterile female plants with such a maintainer will yield only male- sterile female plants. On the other hand, since female gametes carrying the alien chromosomal arm are viable, self pollination will yield a mixture of seeds, all of which are homozygous for said male-sterility allele, about 80% of which lack the alien chromosome arm, said seeds, when grown, developing into male-sterile female plants, and about 20%, of which contain the alien chromosome arm, said seeds, when grown, developing into male-fertile maintainer plants. When the selectable marker is a gene conferring resistance to a specific herbicide, it is possible by way of spraying with this herbicide, to kill all plants lacking the alien chromosomal arm thus allowing the growth of only maintainer plants, all of which contain the alien chromosomal arm and therefore are resistant to said herbicide. On the other hand, when the selectable marker is seed color such as blue grains, it is possible, by way of seed sorting, to separate the maintainer seeds (the blue seeds) from the female seeds (the red or white seeds). The invention further provides the maintainer line. The resulting hybrid plants do not contain the heterologous genes and are all heterozygous for the dominant male-fertility allele and for the recessive male-sterility allele and are, therefore, male-fertile.

Prior Art

Hybrid wheat lines yield higher than pure, true breeding lines, and exhibit improved quality and greater tolerance to environmental and biotic stresses (Wilson and Driscoll, 1983; Pickett, 1993; Bruns and Peterson, 1998; Jordaan et al., 1999). Common (bread) wheat, Triticum aestivum L. ssp. aestivum MacKey, and durum (macaroni) wheat, T. turgidum L. ssp. durum (Desf.) Husn., are predominantly self-pollinating species and every flower contains both female and male organs. To produce hybrid seeds, it is therefore necessary to male-sterilize the female parent. Since hand emasculation is impractical in wheat, male-sterility may be brought about by application of chemical hybridizing agents (CHAs) or by genetic means. Utilization of a CHA to male-sterilize wheat plants is expensive, inefficient and pollutant.

The following conditions are required for the production of hybrid wheat seeds by genetic means: 1 ) complete and stable male-sterility of the female parent; 2) complete and stable fertility restoration by the male parent; and 3) easy propagation of the male-sterile female parent by the male-fertile maintainer line. Although these conditions are well known to wheat geneticists there has, however, not been a breakthrough in hybrid wheat production during the 50 years since the first male-sterile wheat was described (Kihara, 1951).

There are two main types of genetic male-sterility that can be exploited for hybrid seed production: cytoplasmic male-sterility (CMS) in nuclear substitution or alloplasmic lines, caused by the incompatible interaction of an alien cytoplasm with the common wheat nuclear genome, and genie male-sterility (GMS) in euplasmic lines, caused by a recessive mutation or a deletion of a nuclear gene(s) that is essential to male-fertility in common wheat.

Whereas in many commercial crops it is the genie male-sterility that prevails, this type has not yet been fully exploited in common or durum wheat. Most attempts in common wheat have been directed to producing hybrid seeds on the basis of cytoplasmic male-sterility. However, the use of an alien cytoplasm as a sterilizing factor in common wheat has a major drawback since various important traits including grain yield are negatively affected by the interaction between the wheat nuclear genome and the alien cytoplasm. In addition, it has been difficult to find stable fertility restoration genes for the alloplasmic male-sterile lines, which are highly effective in a wide range of genotypes. Moreover, the system requires breeding of the male parent too (e.g. introduction of genes that can restore male-fertility to the alien cytoplasm), thus rendering hybrid seed production more expensive and limiting the number of male parents that can be tested for combining ability (contribution to a significant heterosis).

Genie male sterility, on the other hand, is expressed in a normal common or durum wheat cytoplasm and does not involve deleterious effects on plant performance. Further, using a female parent homozygous for a recessive male-sterility allele, any wheat cultivar which is by its nature homozygous for the dominant allele conferring male-fertility, can be used as a male parent that will restore complete fertility to the Fi hybrids. There is no need to breed for male lines and no limitation exists for the number of males which can be crossed with the male-sterile females and evaluated for their combining ability. Several chromosome arms have been described in common wheat which carry genes affecting male-fertility, e.g. chromosome arms of group 4: the long arm of chromosome 4A (4AL), the short arm of chromosome 4B (4BS) and the short arm of chromosome 4D (4DS), carrying the normal male-fertility Ms-A1, Ms-B1 and Ms-D1 genes, respectively, and the long arms of the group 5 chromosomes: 5A, 5B and 5D (5AL, 5BL and 5DL, respectively), carrying the Ms-A2, Ms-B2 and Ms-D2 genes, respectively. However, until now, only in the ^"Ms-B1 locus, on the distal region of chromosome arm 4BS three recessive alleles that cause male sterility were found or induced. These alleles, namely, ms-B1-a, ms-B1-b and ms-B1-c (often also called msla, mslb and ms1c, respectively), were reported not to cause any effect, beyond male-sterility, on plant performance (reviewed by Wilson & Driscoll, 1983).

Maintenance of the male-sterile female lines remains the major obstacle for a successful hybrid production system based on GMS. One way is to equip the maintainer line with an alien male-fertility allele homoeoallelic to the recessive mutant male-sterility allele, which is not transmitted into the female line. An example of this approach of maintaining the male-sterile female is the XYZ system of Driscoll (1972). Three decades ago he suggested to add into the male-sterile female Z line (homozygous for the recessive mutant allele ms-B1-c) an extra single (in Y line) or a pair (in X line) of an alien chromosome carrying the dominant Ms homoeoallele which, in turn, confers fertility to X and Y lines. The alien chromosome does not pair with its wheat homoeologous chromosomes and in the Y line (maintainer) is transmitted through the pollen in a very low frequency and thus the pollinated male-sterile female line produces seeds, most of which will germinate into male-sterile plants. Since the maintainer (Y) is not a true-breeding line, it is produced by pollinating the male-sterile female (Z) by the disomic alien addition line (X). This system was characterized by two major drawbacks: some transmission of the alien chromosome occurred through the pollen of the maintainer line which introduced male fertility to the new generation of the male-sterile female line; and addition decay occurred in the X line impairing its purity. These are possibly the reasons why this system has never come into practical (commercial) use.

More recently, Driscoll (1985) proposed a modification of the above XYZ system of producing hybrid wheat. In this system, a selfed Y-line replaces the Y-line to maintain and propagate the male-sterile Z-line. This modification eliminates the need for the X-line that was originally needed to generate a large quantity of Y-line plants. Moreover, the newly proposed Y-line carries an alien isochromosome so that the compensating male-fertility homoeoallele is in two doses. While the modified XYZ system requires fewer crosses between the various parental plants in order to maintain and propagate the male-sterile female plants, than the original XYZ system, the drawbacks characterizing the original XYZ system as noted above, do however, also exist in the modified XYZ system and limit its use in commercial production of hybrid seeds.

In view of the above, it therefore seems that traditional methods of hybrid production are not efficient enough and new approaches are needed. One such new approach, based on an improvement of the above XYZ system of Driscoll (1972), has been described in the International PCT Patent Application Nos. PCT/AU91/00319 (WO 92/01366) and PCT/AU93/00017 (WO93/13649), and concerns the production of hybrid cereal crops such as common wheat. In these publications there are described plant lines used for the production of hybrids which have an alien chromosome or chromosome segment bearing a dominant male-fertility allele homoeoallelic to the male-sterility mutant allele and a color marker gene conferring coloration on the progeny seed. The maintenance of the male-sterile (female) parental line is accomplished by physically separating the progeny seeds by color sorting. Such genetically .altered common wheat plants contain a modified chromosome with a dominant normal male-fertility allele from the diploid wheat Triticum monococcum as an addition or substitution for one of the wheat 4B chromosomes. The modified chromosome carries the short arm of chromosome 4A^m of T. monococcum (4A^mS) carrying the Ms-A^m1 allele and a second arm with a proximal segment from the long arm of either chromosome 4A^m of T. monococcum (4A^mL) or chromosome 4E of Agropyron elongatum (4EL) with the coloration allele (C) and a distal segment of wheat chromosome arm 4BL. Part of this modified chromosome is homologous and part of it is homoeologous to the wheat chromosome 4B bearing the recessive male-sterility allele. The homologous part, i.e. the distal region of 4BL can pair with the normal wheat 4BL, thus ensuring regular segregation at meiosis. Another possibility to mark this chromosome carrying the normal dominant male-fertility allele, Ms-A^m1, is by the use of a gene conferring increased plant height on progeny plants.

However, the above hybrid-production system has too a number of drawbacks as regards the efficient maintenance of the parental lines. First, pollination of female plants by the maintainer will yield a larger number of seeds with the recombinant alien/4BL chromosome, which will develop into male-fertile plants. Secondly, the maintenance of the male-sterile female parent involves a complex procedure of progeny selection based on marker genes. Thirdly, the maintainer line for the female (male-sterile) parental line is also a genetically unstable line in that it carries 20 pairs of normal common wheat chromosomes, one 4B chromosome carrying the male-sterility (ms-B1-b) mutant allele (known as 'Probus') (Wilson and Driscoll, 1983) and one recombinant alien group 4/4BL chromosome having the normal, male-fertility Ms-A^m1 allele and the seed coloration allele. Thus, the maintainer line is male-fertile, and upon selfing will yield fertile plants homozygous or heterozygous for the modified chromosome. It will thus be impossible to distinguish between the two genotypes on the basis of the coloration gene and very difficult on the basis of the height gene. Hence, the propagation of the maintainer and its use to provide the male-sterile female line is laborious and not practical for large-scale commercial applications.

To overcome the difficulties of mechanical or other indirect means of selection against the alien chromosome carrying the male fertility Ms allele, T.R. Endo, Kyoto University, Kyoto, Japan, suggested (as cited by Tsujimoto and Tsunewaki 1983) to use the gametocidal gene Gc1 and link it to the male sterility allele ms . The gametocidal allele, originated from Ae. speltoides, brings about abortion of gametes not carrying it (but rather carrying the native recessive gc1 allele). According to Endo's proposal, the male-sterile female line is homozygous for both ms and Gc1, which are tightly linked, while a male-fertile line (the maintainer) isogenic to the female line but having Ms and gc1 alleles, is used to pollinate the female line to yield a double heterozygote msMsGdgd. Due to abortion of gametes carrying gd, all the progeny of such selfed line will be homozygote msmsGclGd and identical to the female line. However, according to their proposal the male line (R line) in the hybrid production system should also be bred to contain the Gel allele otherwise the fertility of the Fi hybrid will be reduced. Moreover, Gc1 causes the abortion of female as well as male gametes and therefore, a cross (between the female and the maintainer) and a self (of the double heterozygote) are required each year to renew the female seed stock. This is a drawback in time and cost. Another disadvantage of Endo's proposal stems from the fact that the male-sterile female parent contains an alien chromosome segment carrying the Gc1 allele that was derived from Ae. speltoides. This segment may carry also alleles with negative effect on the performance of the female, increasing the cost of hybrid seed production, or even affecting the yield of the hybrid.

In a previous disclosure [application No. PCT/IL98/00220 (WO 98/51142 )], a method was provided for the production of hybrid wheat based on the ability to stably maintaining a genie male-sterility female parental line of common and durum wheat. The method was based on the production of a male-sterile female line homozygous for a recessive male-sterility allele and for a dominant pollen-killing allele, and a maintainer line which is readily and stably propagated. The maintainer line is isogenic to the female line but has an alien engineered chromosome carrying a dominant male-fertility allele that restores fertility to the maintainer line, a recessive pollen-killing allele that is susceptible to the killing effect of the native pollen killer thus preventing the transmission of this chromosome to the female line, and one or more selectable markers that facilitate the maintenance of the maintainer itself. However, the engineered chromosome, due to some frequency of transversal division of the centromere, was found to be unstable resulting in male gametes that contain the chromosome arm with the male-fertility allele. These male gametes were transmitted upon pollination of the female plants resulting in a certain degree of male-fertile female plants, which led to reduced hybrid purity. Consequently, this method was not used in commercial practice.

Another method to obtain male-sterile plants based on the use of foreign DNA causing male-sterility has been developed recently in several crop plants such as oilseed rape or maize, aiming to use it also in wheat. The male-sterility gene is a heterologous gene comprising foreign DNA that codes for a cytotoxic protein such as RNase (European Patent Application No. EP 0,412,911) or for a protein that binds an essential cellular factor such as biotin (International PCT Patent Application No. WO 96/40949), and a plant promoter that enables the expression of the foreign DNA only in stamen cells. This method requires the transformation of the male parent too with another heterologous gene comprising antisense DNA that is capable to inhibit the cytotoxic effect by suppressing the transcription activity of the male-sterility DNA or coding for a protein that inhibits the cytotoxic effect, with a plant constitutive promoter. To maintain the male-sterile female line there is a need for additional heterologous gene with an inducible promoter or to treat the male-sterile female plants with an exogenous compound such as biotin, to supply the essential factor. Hence, the method requires breeding of the male parent too rendering the hybrid seed production expensive. In addition, the hybrid plants contain at least two heterologous genes rendering their grains unsuitable for human consumption in the present time. As regards the importance of common wheat hybrid production, it should be noted that different reports on experimental hybrid performance indicate a yield increase of the best wheat hybrids of up to 30% above the leading best cultivars (Wilson and Driscoll, 1983). Further, it is well known that many hybrids exhibit an improved quality and greater tolerance to environmental and biotic stresses than the conventional cultivars.

DEFINITIONS

Throughout the description and the claims, the following terms and abbreviations will be used: Common wheat = bread wheat, Triticum aestivum L. ssp. aestivum MacKey, being an allohexaploid species (2n=42) having the three genomes ABD. Durum wheat = macaroni wheat, Triticum turgidum L. ssp. durum (Defs.) Husn., being an allotetraploid species (2n=28) having the two genomes AB. Aegilops searsii = a diploid species (2n=14), closely related to the donor of the B genome of durum and common wheat having genomes S^s, whose chromosomes are homoeologous (partial homologous) to those of wheat. 4BS = the short arm of chromosome 4B of common and durum wheat. 4S^SS = the short arm of chromosome 4S^S of Aegilops searsii. AA= an alien chromosomal arm 4S^SS of Aegilops searsii, added to the complement of common wheat as a monotelosomic addition. Ms = a dominant allele responsible for male-fertility in wheat. Ms-B1 = a dominant allele for male-fertility in durum and common wheat located on

4BS. ms = a recessive mutant allele of Ms that confers male-sterility. ms-B1 = a recessive mutant allele of Ms-B1, that confers male-sterility in durum and common wheat, when present in homozygous state. ms-B1-a = msla which is the 'Pugsley' mutant ms-B1 allele. ms-B1-b = mslb which is the 'Probus' mutant ms-B1 allele. ms-B1-c = mslc which is the 'Cornerstone' mutant ms-B1 allele.

Ms-S^s1 = a dominant allele for male-fertility, homoeoallelic to Ms-B1, on 4S^SS.

Msu = a heterologous gene capable of killing microspores, microgametophytes or pollen grains carrying it, controlled by a microspore-or microgametophytic-specific promoter. Ski = a heterologous gene capable of killing seedlings carrying it, controlled by a seedling-specific promoter. Ski inhibitor = a heterologous gene capable of inhibiting the expression of Ski, controlled by an inducible promoter. BaRNase = a gene from Bacillus amyloliquefaciens coding for RNase. Barstar = a gene from Bacillus amyloliquefaciens coding for a protein that inhibits

RNase. Hr= a heterologous specific herbicide-resistance gene by which plants having this gene can be selected, controlled by a constitutive promoter. Ba - a dominant glufosinate (Basta)-resistance gene. Sc = a heterologous seed-color gene by which plants having this gene can be selected, controlled by an endosperm-specific promoter, cv. = cultivar. Female line = a male-sterile line of common and durum wheat homozygous for one of the recessive male-sterility alleles of ms-B1. Male line = a male-fertile line of common and durum wheat homozygous for the dominant male-fertility allele Ms-B1. Maintainer line = a male-fertile line isogenic to the male-sterile female line but contains the alien chromosomal arm AA, carrying a dominant male-fertility allele (Ms) linked to a heterologous microspore-suicide gene (Msu) capable of killing microspores or pollen grains carrying it, controlled by a microspore-specific promoter, and a heterologous selectable marker gene, by which plants having this chromosomal arm can be selected, controlled either by a constitutive promoter or a tissue- specific promoter.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for maintaining a genie male-sterile female parental line of a common or durum wheat cultivar, which method provides for a simple means for stably maintaining the male-sterile female parental line.

Yet another object of the present invention is to provide a maintainer line for use in the above method, which maintainer line is easily, rapidly and stably propagated.

It is a still further object of the present invention to provide for the production of hybrid lines of common and durum wheat, and more specifically, to provide a new method for maintaining a male-sterile female parental line for use in the production of hybrid wheat seeds, which female line is homozygous for a recessive male-sterility mutant allele.

It is another object of the invention to provide a new maintainer line for maintaining the female parental line, which is isogenic to the female line, but has an alien chromosomal arm, as a monotelosomic addition, carrying a dominant male-fertility allele homoeoallelic to the recessive male-sterility allele, a heterologous microspore-suicide gene capable of killing the microspores or pollen grains carrying it, and a heterologous selectable marker. These three genes on the alien chromosomal arm are permanently linked due to lack of pairing and recombination between the alien chromosomal arm and the wheat chromosomes. The presence of the microspore-suicide gene on the alien chromosomal arm ensures that all the maintainer viable male gametes (about 80% of the gametes) lack this chromosomal arm, and consequently, the dominant male-fertility allele and the selectable marker allele. Hence, pollination of male-sterile female plants with such a maintainer will yield only male-sterile female plants. On the other hand, since female gametes carrying the alien chromosomal arm are viable, self pollination will yield a mixture of seeds, all of which are homozygous for said male- sterility allele, about 80% of which lack the alien chromosome arm, said seeds, when grown, developing into male-sterile female plants, and about 20%, of which contain the alien chromosome arm, said seeds, when grown, developing into male-fertile maintainer plants.

When the selectable marker is a gene conferring resistance to a specific herbicide, it is possible by way of spraying with this herbicide, to kill all plants lacking the alien chromosomal arm thus allowing the growth of only maintainer plants, all of which contain the alien chromosomal arm and therefore are resistant to said herbicide. On the other hand, when the selectable marker is seed color such as blue grains, it is possible, by way of seed sorting, to separate the maintainer seeds (the blue seeds) from the female seeds (the red or white seeds).

The invention further provides the maintainer line.

The resulting hybrid plants do not contain the heterologous genes and are all heterozygous for the dominant male-fertility allele and for the recessive male-sterility allele and are, therefore, male-fertile.

The present invention makes possible the commercial production of hybrids of common and durum wheat. In one aspect, the invention provides a novel method for the maintenance of a male-sterile female parental line that is homozygous for a recessive male-sterility mutant (ms) allele. The maintainer (Figs. 2 - 5) is isogenic to the female line and has further the alien chromosomal arm 4S^SS of Aegilops searsii as a monotelosomic addition, herein referred to as AA, carrying a dominant male-fertility allele (Ms) linked to a heterologous microspore-suicide gene (Msu) capable of killing microspores, microgametophytes or pollen grains carrying it, controlled by a microspore-specific promoter, and a heterologous selectable marker gene, which is either a specific herbicide-resistance gene (Hr) controlled by a constitutive promoter or a seed-color gene (Sc) controlled by an endosperm-specific promoter (Fig. 1). Pollen grains of the maintainer containing the AA are not functional.

Thus, a simple system has been developed in accordance with the present invention, by which the male-sterile female parental line is maintained by pollinating it with the male-fertile maintainer line, and all of the resulting progeny are male-sterile female plants (Figs. 2 and 3). Similarly, the maintainer line is itself easily maintained by self-pollination, resulting in a mixture of progeny seeds of which about 20% carry the AA alien chromosomal arm, when grown, developing into male-fertile plants, and about 80% lack this alien chromosomal arm, when grown, developing into male-sterile plants. Spraying said progeny plants with the specific herbicide kills the male-sterile plants thereby allowing only the male-fertile maintainer plants to grow (Fig. 2). When the selectable marker is seed color, the red or white seeds, when grown, developing into male-sterile female plants, can be separated by a seed sorter from the blue seeds, when grown, developing into male-fertile maintainer plants (Fig. 3).

The AA alien chromosomal arm of the maintainer line can carry one (Fig. 1b) or two (Fig. 1c) additional heterologous genes facilitating the use of the progeny of the selfed maintainer as a source for male-sterile female plants (Figs. 4a and 5a) or of male-fertile maintainer plants (Figs. 4b and 5b).

The alien chromosomal arm was derived from Ae. searsii. It is the short arm of chromosome 4S^S (4S^SS). Being an alien single telocentric chromosome that was added to the complement of common wheat, it does not pair during meiosis and only enters to about 20% of the gametes, either male or female. Hence, -while it is not transmitted through the male gametes because of the presence of the microspore-suicide gene, the rate of its transmission through the female gametes is about 20%. The Ms-^l gene is homoeoallelic to the Ms-B1 gene and dominant over the ms- B1 alleles of common and durum wheat and is expressed in all tested genetic backgrounds. The heterologous microspore-suicide gene (Msu) is any gene that codes for heterologous protein that is toxic to the microspores, microgametophytes or pollen grains possessing it or disturbs their metabolism. It consists of a foreign DNA sequence coding for a toxic protein, which is toxic to the microspores, microgametophytes or pollen grains, or codes for a protein that significantly disturbs the metabolism and thereby the viability of the microspores, microgametophytes or pollen grains, inserted into an expression cassette under control of a microspore-specific promoter. The heterologous specific herbicide-resistance gene (Hr) codes for a heterologous protein that confers resistance to a specific herbicide under control of a constitutive promoter. The heterologous seed-color gene (Sc) codes for an anthocyanin and thus conferring coloration of the grains containing it, under control of an endosperm-specific promoter.

The above-mentioned heterologous genes are introduced to the AA alien chromosomal arm by standard genetic-engineering manipulations.

In still another aspect, the invention relates to a method for producing a hybrid plant line of common or durum wheat, wherein the male-sterile female parental line is crossed with any cultivar of the same species, which by its nature is male-fertile homozygous for the Ms-B1 allele, to yield Fi hybrid progeny that are all heterozygous

(Ms-B1ms-B1 and therefore, male fertile.

The invention further provides a plant homozygous for a recessive male-sterility allele and having in the nuclear genome of its cells an alien chromosomal arm carrying a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene (Msu) comprising a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, and a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; and a heterologous selectable marker comprising a selectable-marker DNA encoding for a heterologous protein that facilitates the selection of plants carrying it, and a plant promoter, either constitutive or tissue specific, directing the expression of said selectable marker DNA in all plant tissues or in a specific tissue, respectively.

The invention provides a plant homozygous for a recessive male-sterility allele and having in the nuclear genome of its cells an alien chromosomal arm carrying a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene comprising a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable; and a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; a heterologous seedling-killer gene comprising a seedling-killer DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those seedlings that possess it, thus killing the seedlings, and a seedling-specific promoter directing the expression of said seedling-killer DNA selectively in the seedlings that possess it; and a heterologous selectable marker comprising a selectable-marker DNA encoding for a heterologous protein that facilitates the selection of plants carrying it, and a plant promoter, either constitutive or tissue specific, directing the expression of said selectable marker DNA in all plant tissues or in a specific tissue, respectively.

The invention provides a plant homozygous for a recessive male-sterility allele, and having in the nuclear genome of its cells an alien chromosomal arm carrying a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene comprising a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to, or disturbs, the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, and a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; a heterologous seedling-killer gene comprising a seedling-killer DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those seedlings that possess it, and a seedling-specific promoter directing the expression of said seedling-killer DNA selectively in the seedlings that possess it; a heterologous gene counteracting the deleterious effect of the seedling-killer gene comprising a DNA encoding a heterologous protein that either counteracts the deleterious effect of the seedling-killer gene or disturbs its activity; and a plant constitutive or inducible promoter directing the expression of said DNA in the plants that possess it; and a heterologous selectable marker comprising a selectable-marker DNA encoding for a heterologous protein that facilitates the selection of plants carrying it; and a promoter, either constitutive or tissue specific, directing the expression of said selectable marker DNA in all plant tissues or in a specific tissue, respectively.

With respect to the plants noted above, the male-sterility allele can be a heterologous gene. Also, the male-sterility allele can be one of the male-sterility alleles of common wheat, durum wheat or another species of the Triticeae. In particular, the male-sterility allele may be one of the male-sterility alleles of the Ms-B1 locus either ms- B1-a, ms-B1-b, ms-B1-c or any other allele of MS-B1. The dominant male-fertility allele (Ms) can be a heterologous gene. Also, the dominant male-fertility allele (Ms) may be one of the male-fertility alleles of the Triticeae. The dominant male-fertility allele (Ms) may be Ms-^l of chromosome arm 4S^SS of Aegilops searsii.

Further, for the plants noted above, the microspore-suicide gene may be any of the genes affecting the viability of microspores, microgametophytes, pollen grains or male gametes that carry it. In particular, the microspore-suicide gene encodes avidin or sterptavidin of variant thereof. The microspore-suicide gene may encode RNase of variant thereof. The microspore-suicide DNA may encode ribosomal-inhibitor protein (RIP) of variant thereof. For the plants noted above, the microspore-specific promoter may be any promoter that is expressed in plants directing gene expression in the microspore, male- gametophyte or pollen grain. The heterologous selectable marker may be any gene encoding for a heterologous protein that facilitates the selection of plants carrying it. The heterologous selectable marker can be a specific herbicide-resistance gene. The specific herbicide-resistance gene may be Bar conferring resistance to glufosinate (Basta). The constitutive promoter may be of the specific herbicide-resistance gene expressed in plants. The heterologous selectable marker may be a seed-color gene coloring the aleurone layer or the endosperm. The promoter of the seed-color gene may be an endosperm-specific promoter or an aleurone-specific promoter. The heterologous selectable marker may be a seed-size, weight or morphology gene. The promoter of this selectable marker gene may be a seed-specific promoter.

For the plants noted above, the seedling-killer gene may encode a protein that renders the cells containing it inviable. The seedling-killer DNA may encode avidin or sterptavidin of variant thereof. The seedling-killer gene may encode Rnase of variant thereof. The seedling-killer gene may encode ribosomal-inhibitor protein (RIP) of variant thereof. The seedling-specific promoter may be any promoter that is expressed in plants directing gene expression in the seedling.

The DNA counteracting the effect of the seedling-killer gene may encode a protein that neutralizes the effect of the seedling-killer gene of variant thereof. More particularly, the DNA counteracting the effect of the seedling-killer gene may encode biotin of variant thereof. The DNA counteracting the effect of the seedling-killer gene may encode RNase antisense of variant thereof or RIP antisense of variant thereof.

The constitutive promoter may be any promoter that is expressed in plants directing gene expression in the plants. The inducible promoter is any promoter that is expressed in plants directing gene expression in the plants. The inducible promoter can be induced by temperature, light, chemical or any other environmental treatment. The inducible promoter can be induced by tetracyclin.

The alien chromosomal arm may be any arm of a Triticeae species carrying a dominant male-fertility allele. The alien chromosome arm may be chromosomal arm 4S^SS of Aegilops searsii. The plants noted above may be selected from any of the Gramineae species. The plants noted above may be selected from any of wheat, triticale, barely, rye, oat, rice, and maize.

The invention provides a method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat comprising:

(a) crossing a female parent with a male parent, said female parent being a male-sterile plant homozygous for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B (4BS), said male parent being the maintainer line as defined above, and

(b) harvesting from the cross of (a) the progeny seed, all of which are homozygous for the male-sterility allele and lack the alien chromosome arm, the seeds, when grown, developing into the male-sterile female line.

The invention further provides a method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat, said method comprising:

(a) selfing a maintainer line as defined above, and

(b) harvesting from the selfing of (a) the progeny seed, all of which are homozygous for the male-sterility allele, of which 80% lack the alien chromosomal arm, and therefore, they are red or white, said seeds, when grown, developing into male-sterile female plants, and 20% have the alien chromosomal arm, and therefore, they are blue, the seeds, when grown, developing into male-fertile maintainer plants, and separating by a seed sorter the red or white female seeds from the blue maintainer seeds. The invention further provides a method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat comprising:

(a) selfing a maintainer line isogenic to the female line and homozygous for the same ms-B1 allele of the female line, and having the alien chromosomal arm 4S^SS that derived from Aegilops searsii, as a monotelosomic addition, herein referred to as AA, carrying the dominant male-fertility allele Ms-S^s1, a heterologous microspore-suicide gene (Msu) capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound, and thus, causes cell death, controlled by a microspore-specific promoter, and a heterologous seedling-killer gene capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound and thus causes cell death, controlled by a seedling-specific promoter, and a heterologous selectable marker, by which plants having this chromosome arm can be selected, and

(b) harvesting from the selfing of (a) the progeny seed, all of which are homozygous for the male-sterility allele, of which 80% lack the alien chromosomal arm, the seeds, when grown, developing into male-sterile female plants, and 20% have the alien chromosomal arm, the seeds, when grown, developing into male-fertile maintainer plants, but dying as seedlings leaving only the male-sterile plants alive.

The invention further provides a method for the maintenance of a male-fertile maintainer line of common or durum wheat for use in the maintenance of male-sterile female line comprising:

(a) selfing a maintainer line isogenic to the female line and homozygous for the same ms-B1 allele of the female line, and having the alien chromosomal arm 4S^SS that derived from Aegilops searsii, as a monotelosomic addition, herein referred to as AA, carrying the dominant male-fertility allele Ms-S^s1, a heterologous microspore-suicide gene (Msu) capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound and thus causes cell death, controlled by a microspore-specific promoter, and a heterologous selectable marker controlled either by a constitutive promoter or by a tissue-specific promoter, by which plants having this chromosome arm can be selected; and

(b) harvesting the maintainer of (a) its progeny seed, all of which are homozygous for the male-sterility allele, of which 80% lack the alien chromosome arm, the seeds, when grown, developing into male-sterile female plants, and 20% have the alien chromosome arm, the seeds, when grown, developing into male-fertile maintainer plants; and

(c) selecting the maintainer plants by the selectable marker.

The invention providing a method for a maintenance of a male-fertile maintainer line of common or durum wheat for use in the maintenance of male-sterile female line of wheat comprising:

(a) selfing a male-fertile maintainer line homozygous for one of the male-sterility ms-B1 alleles , and having the alien chromosomal arm 4S^SS that derived from Aegilops searsii, as a monotelosomic addition, herein referred to as AA, carrying the dominant male-fertility allele Ms-S^s1, a heterologous microspore- suicide gene (Msu) capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound and thus causes cell death, controlled by a microspore-specific promoter, a heterologous seedling- killer gene capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound and thus causes cell death, controlled by a seedling-specific promoter, and a heterologous selectable marker controlled either by a constitutive promoter or by a tissue-specific promoter, by which plants having this chromosome arm can be selected, and

(b) harvesting from the maintainer of (a) the selfed progeny seed, all of which are homozygous for said male-sterility allele, of which 80% lack the alien chromosome arm, said seeds, when grown, developing into male-sterile plants, and 20% contain the alien chromosome arm, when grown, developing into male-fertile maintainer plants; and

(c) in case of a specific herbicide-resistance gene as a selectable marker, treating the progeny seeds of (a) with biotin, and germinating the progeny seeds of (a) and spraying the plants with the specific herbicide thus killing all progeny plants lacking the alien chromosome arm, and leaving to grow only those plants that carry the alien chromosome arm and therefore, will be the male-fertile maintainer.

(d) in case of a seed-color gene as a selectable marker, the progeny seeds of (a) will be separated by a seed sorter, the blue seeds will be the maintainer seeds and the red or white seeds will be added to the male-sterile female line. The blue seeds will be treated with biotin.

In the method described, the selectable marker gene is a specific herbicide- resistance gene (Hr), controlled by a constitutive promoter. The selectable marker gene may be a seed-color gene (Sc), controlled by an endosperm-specific promoter or a seed-size, weight or morphology gene, controlled by a seed-specific promoter.

The invention further provides a method for producing a hybrid plant line of common or durum wheat comprising :

(a) crossing a male parent with a male-sterile female parent of the same species, wherein the male parent is selected from any desired common or durum wheat cultivar which, by its nature, is homozygous for the dominant wild-type male-fertility (Ms-B1) allele, and said male-sterile female parent is a line of the wheat species being homozygous for both any one of the recessive mutant male-sterility (ms-B1) allele, the male-sterile female parent being maintained by a maintainer line as described above ; and

(b) collecting the progeny seed of the cross of (a), which seeds, when grown, develop into progeny hybrid plants all of which are heterozygous ms-B1Ms-B1 and therefore, male fertile..

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts schematic drawings of the alien chromosomal arm 4S^SS of Aegilops searsii (AA) carrying the dominant male-fertility allele Ms-S^s1, a heterologous microspore-suicide gene (Msu) with a microspore-specific promoter, and a heterologous selectable marker gene (Sm), either specific herbicide resistance (Hr) or seed color (Sc), with a constitutive promoter or an endosperm-specific promoter, respectively (1a); same arm as in (a) with one additional heterologous seedling-killer gene (Ski) with a seedling-specific promoter (1b); and same arm as in (b) with one additional heterologous seedling-killer inhibitor gene (Ski-inhibitor) with an inducible promoter (1c).

Fig. 2 depicts a general scheme for maintaining a male-sterile female parental line by pollinating it by a maintainer line and maintaining the maintainer line by self- pollination. The maintainer line is isogenic to the female line and has further the AA alien chromosomal arm added to the wheat complement, carrying the dominant male- fertility allele Ms-S^s1, a heterologous microspore-suicide gene (Msu) with a microspore- specific promoter, and a heterologous specific herbicide-resistance gene (Hr) with a constitutive promoter.

Fig. 3 depicts a general scheme for maintaining a male-sterile female parental line by pollinating it by a maintainer line as well as by separating the red or white seeds (the female line seeds) by a seed sorter from the progeny of the selfed maintainer, and maintaining the maintainer line by separating the blue seeds (the maintainer seeds) from the progeny of the selfed maintainer. The maintainer line is isogenic to the female line and has further the AA alien chromosomal arm added to the wheat complement, carrying the dominant male-fertility allele Ms^l, a heterologous microspore-suicide gene (Msu) with a microspore-specific promoter, and a heterologous seed-color gene (Sc) with a endosperm-specific promoter.

Fig. 4 depicts a general scheme for maintaining a male-sterile female parental line by selfing the maintainer line and obtaining progeny seeds of which 80%, when grown, developing into the female line, and 20%, when grown, developing into the maintainer line, the latter die in an early seedling stage due to the production of sterptavidin that specifically binds the essential factor biotin (a), and maintaining the maintainer line by treating the mixture of the said progeny seeds with exogenous biotin and spraying the grown seedlings with the specific herbicide, thereby killing all the plants lacking the Hr gene, i.e., the male-sterile female plants, leaving to grow only the male-fertile maintainer plants (b).

Fig. 5 depicts a general scheme for maintaining a male-sterile female parental line by selfing the maintainer line and obtaining progeny seeds of which 80%, when grown, developing into the male-sterile female line, and 20%, when grown, developing into the male-fertile maintainer line, the latter die in an early seedling stage due to the production of sterptavidin that specifically binds the essential factor biotin (a), and maintaining the maintainer line by separating the blue seeds (the maintainer seeds) from the red or white seeds (the female seeds) by a seed sorter and treating the blue seeds with biotin (b). Fig. 6 depicts a general scheme for production of suitable material for transformation.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention a simple system has been developed for common and durum wheat as depicted in Figs. 2 and 3 by which the male-sterile female parental line is maintained by pollinating it with the maintainer line, and all of the resulting progeny are male-sterile female plants. Similarly, the maintainer line is itself easily maintained by self-pollination, resulting in a mixture of seeds of which about 20%, when grown, developing into male-fertile plants identical to the maintainer line and carrying the AA alien chromosomal arm, and about 80%, when grown, developing into male-sterile plants due to the absence of the alien chromosomal arm and consequently, the Ms-S^s1 allele (Figs. 2 - 5). Spraying the progeny plants of the selfed maintainer line with the specific herbicide (Figs. 2 and 4), kills the male-sterile plants while the male- fertile plants carrying the AA with the specific herbicide-resistance gene survive. Alternatively, separating the seeds on the basis of their different colors with a seed sorter (Figs. 3 and 5), yields an additional amount of female seeds.

For hybrid seed production, the male-sterile female parental line is crossed with any common or durum wheat cultivar, which by its nature is male-fertile homozygous for the Ms-B1 allele, to yield Fi hybrid offspring that are all heterozygous Ms-B1 ms-B1 and therefore, male-fertile.

Accordingly, in one aspect, the present invention provides a method for the maintenance of a male-sterile female parental line of common or durum wheat (Figs. 2 and 3) for use in the production of hybrid seed, said method comprising:

42. crossing a female parent with a male parent, said female parent being a male-sterile plant homozygous for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B (4BS) of common wheat, said male parent being the maintainer line and being isogenic to the female parent and homozygous for the same ms-B1 allele of the female parent, and having an alien chromosomal arm, as a monotelosomic addition, herein referred to as AA, consisting of the short arm of chromosome 4S^S (4S^SS) of Ae. searsii, carrying the dominant male-fertility allele Ms-S^s7, linked to a heterologous microspore-suicide gene (Msu) that disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, controlled by a microspore-specific promoter, and a heterologous selectable marker gene by which plants having this chromosomal arm can be selected, controlled by a constitutive or a tissue-specific promoter. Pollen grains of the maintainer containing this alien chromosome arm are not functional, and

(b) harvesting from the cross of (a) the progeny seed, all of which are homozygous for said male-sterility allele and lack the AA alien chromosomal arm, said seeds, when grown, developing into said male-sterile female line.

The present invention further provides an alternative method for the maintenance of a male-sterile female parental line of common or durum wheat (Figs. 4 and 5) for use in the production of hybrid wheat, said method comprising:

(a) selfing a maintainer line isogenic to the female line and homozygous for the same ms-B1 allele of the female line, and having an alien chromosomal arm, as a monotelosomic addition, herein referred to as AA, consisting of the short arm of chromosome 4S^S (4S^SS) of Ae. searsii, carrying the dominant male-fertility allele Ms- S^s1, linked to three heterologous genes, a microspore-suicide gene (Msu) that disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, controlled by a microspore-specific promoter, a seedling-killer gene (Ski) coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, such as streptavidin gene coding for a protein that binds biotin, or a gene that codes for RNase or ribosome-inhibitor protein (RIP) that cause cell death, controlled by a seedling-specific promoter, and a selectable marker gene by which plants having this chromosomal arm can be selected, such as a specific herbicide-resistance gene (Hr) controlled by a constitutive promoter or a seed-color gene (Sc) controlled by an endosperm-specific promoter. Pollen grains of the maintainer containing this alien chromosomal arm are not functional, and

(b) harvesting the seeds from the selfed maintainer of (a), all of which are homozygous for said male-sterility allele, 80% of which lack the AA alien chromosomal arm, said seeds, when grown, developing into said male-sterile female line, and 20% of which contain the AA alien chromosomal arm, said seeds, do not germinate or germinate into plants that die in early seedling stage.

Any male-sterility ms-B1 allele may be used according to the invention such as, for example, the ms-B1-a, ms-B1-b and ms-B1-c alleles or any other allele of this locus or another locus of common or durum wheat inducing male sterility.

The heterologous microspore-suicide gene (Msu) is any gene that codes for heterologous protein which is toxic to the microspores, microgametophytes or pollen grains possessing it, or codes for a protein that disturbs the metabolism of the microspores, microgametophytes or pollen grains possessing it. It consists of a foreign DNA sequence coding for a toxic protein such as barnase that destroys RNA (Mariani et al., 1992), or ribosome-inhibitor protein (RIP) that destroys ribosomes (Logemann et al., 1992; Jach et al., 1995), or codes for a protein that specifically binds an essential factor such as streptavidin which specifically binds the metabolically-essential factor biotin, inserted into an expression cassette under control of a microspore-specific promoter.

The heterologous selectable marker by which maintainer plants or seeds can be selected among the progeny of the selfed maintainer, can be a specific herbicide- resistance gene (Hr) or a gene conferring seed color (Sc). The heterologous specific herbicide-resistance gene (Hr) codes for a heterologous protein that confers resistance to a specific herbicide, such as, glufosinate (De Block et al., 1987; Weeks et al., 1993), under control of a constitutive promoter. The heterologous seed-color gene (Sc), controlled by an endosperm-specific promoter, codes for an anthocyanin that is accumulated in the aleurone layer or in the endosperm resulting in blue coloring of the grain (Dooner et al., 1991).

In another aspect, the present invention provides a male-fertile maintainer line of common or durum wheat which in itself is maintained by selfing (Figs. 2 - 5), said maintainer line being isogenic to the female parent and homozygous for any one of the ms-B1 male-sterility alleles of the female parent, and having an alien chromosomal arm (4S^SS) as a monotelosomic addition, herein referred to as AA, carrying the Ms-S^s1 male-fertility allele, a heterologous microspore-suicide gene (Msu), under control of a microspore- specific promoter. The selectable marker by which plants having this chromosome can be selected is a specific herbicide-resistance gene conferring resistance to specific herbicide such as glufosinate (basta), controlled by a constitutive promoter (Figs. 2 and 4). Being an alien monotelosomic addition, the alien chromosomal arm does not pair during meiosis and consequently, enters only to about 20% of the gametes, either male or female. Hence, since the alien chromosomal arm is not transmitted through the male gametes, the amount of seeds containing the AA chromosomal arm among the progeny of the selfed maintainer is about 20%. These seeds, when grown, developing into plants resistant to the specific herbicide and can be selected by it. Alternatively, the selectable marker is a heterologous seed-color gene (Sc) coding for an anthocyanin in the aleurone layer of the endosperm resulting in blue- grain coloring (Figs. 3 and 5). The blue seeds containing the AA chromosomal arm can be separated from the red or white seeds lacking the AA chromosomal arm by a seed sorter. The blue seeds, when grown, developing into male-fertile maintainer plants, and the red or white seeds, when grown, developing into male-sterile female plants. The present invention further provides an alternative male-fertile maintainer line of common or durum wheat (Figs. 4 and 5) which in itself is maintained by selfing, said maintainer line being isogenic to the female parent and homozygous for any one of the ms-B1 male-sterility alleles of the female parent, and having the AA alien chromosomal arm, as a monotelosomic addition, carrying the Ms-S^s1 male-fertility allele and three heterologous genes, a microspore-suicide gene (Msu), controlled by a microspore- specific promoter, a seedling-killer gene (Ski) coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, such as streptavidin gene coding for a protein that binds biotin, and a selectable marker gene by which plants having this chromosomal arm can be selected, such as a specific herbicide-resistance gene (Hr) controlled by a constitutive promoter or a seed-color gene (Sc) controlled by an endosperm-specific promoter. Treating the mixed progeny seeds of the selfed maintainer with biotin will allow also the seedlings carrying the AA chromosomal arm to develop, resulting in a mixture of plants of which 20% developing into male-fertile maintainer and 80% developing into male- sterile female plants. When the selectable marker, by which plants having this chromosome arm can be selected, is a heterologous herbicide-resistance gene (Hr), spraying the mixture with the specific herbicide will kill all the female plants and leave only the maintainer plants to grow (Fig. 4b). When the selectable marker by which plants having this chromosome arm can be selected is a heterologous seed-color gene (Sc) such as gene coding for an anthocyanin that is accumulated in the aleurone layer resulting in blue coloring of the grain, the mixture of the maintainer progeny seeds will consist of blue and red or white seeds. Separating the seeds by a seed sorter yields 80% red or white seeds, when grown, developing into male-sterile female plants, and 20% blue seeds, when grown, developing into male-fertile maintainer plants (Fig. 5b).

The present invention further provides an additional alternative male-fertile maintainer line of common or durum wheat which in itself is maintained by selfing, said maintainer line being isogenic to the female parent and homozygous for any one of the ms-B1 male-sterility alleles of the female parent, and having the AA alien chromosomal arm, as a monotelosomic addition, carrying the Ms-S^s1 male-fertility allele and four heterologous genes, a microspore-suicide gene (Msu), controlled by a microspore- specific promoter, a seedling-killer gene (Ski) coding for a toxic protein that kills the cells such as a gene that codes for RNase or RIP, controlled by a seedling-specific promoter, an anti seedling-killer gene (Ski inhibitor) coding for an antisense RNA or other types of RNA, protein or peptides that inhibit the Ski gene, or coding for biotin, controlled by an inducible promoter, and a selectable marker gene by which plants having this chromosomal arm can be selected, such as a specific herbicide-resistance gene (Hr) controlled by a constitutive promoter or a seed-color gene (Sc) controlled by an endosperm-specific promoter (Fig. 1c). Treating the mixed progeny of the selfed maintainer with an inducing chemical activates the promoter of the Ski-inhibitor gene resulting in the activity of the Ski-inhibitor gene thus inhibiting the seedling-killer gene or its effect, and consequently, also the seedlings carrying the AA chromosomal arm germinate and grow, resulting in a mixture of plants of which 20% developing into male- fertile maintainer and 80% developing into male-sterile female plants. When the selectable marker, by which plants having this chromosome arm can be selected, is a heterologous herbicide-resistance gene (Hr), spraying the mixture with the specific herbicide will kill all the female plants and leave only the maintainer plants to grow. When the selectable marker by which plants having this chromosome arm can be selected is a heterologous seed-color gene (Sc) such as gene coding for an anthocyanin that is accumulated in the aleurone layer resulting in blue coloring of the grain, the mixture of the maintainer progeny seeds will consist of blue and red or white seeds. Separating the seeds by a seed sorter yields 80% red or white seeds, when grown, developing into male-sterile female plants, and 20% blue seeds, when grown, developing into male-fertile maintainer plants. Yet another aspect of the present invention is a method for producing hybrid plants of common or durum wheat, comprising:

(a) crossing a male parent with a male-sterile female parent of the same species, wherein said male parent is selected from any desired common or durum wheat cultivar, which, by its nature, is homozygous for the dominant wild-type male-fertility (Ms-B1) allele, and said male-sterile female parent is a line of said wheat species being homozygous for any one of the recessive mutant male-sterility (ms-B1) alleles, said male-sterile female parent being maintained by a maintainer line of the invention as noted above; and

(b) collecting the progeny seed of the cross of (a), which seeds, when grown, develop into progeny hybrid plants all of which are heterozygous for the said mutant male-sterility allele, i.e., ms-B1Ms-B1 and therefore, are male fertile.

The present invention will now be described in more detail in the following non- limiting examples and their accompanying drawings.

Example 1 :

The hybrid system

The conditions required for a successful production of hybrid seeds from a male-sterile female parent and a male parent by genetic means are as follows :

1) complete and stable male-sterility of the female parent, called the 'Female line';

2) complete and stable male-fertility restoration by the male parent, called the 'Male line'; and 3) easy propagation of the female line by a male-fertile maintainer line, called the 'Maintainer line'. The Fi hybrid seeds produced in this way are all male-fertile. The female line is propagated either by pollination with the maintainer line or by planting the progeny of the selfed maintainer line, and the maintainer line is itself maintained by selfing, and the desired male-fertile plants among the progeny of the selfed maintainer are selected each generation by the use of a selectable marker characterizing the maintainer.

An example of production of hybrid common or durum wheat using the invention, is described below. A cultivar, herein designated cv. 'One', equipped with genes for male-sterility, i.e. homozygous for one of the male-sterility recessive mutant alleles (ms-B1ms-B1) on the short arm of chromosome 4B, is used as the male-sterile female line. Three such male-sterility alleles have been described (review in Wilson and Driscoll, 1983). These alleles are 'Pugsley' (Pugsley & Oram, 1959 - spontaneous appearance; ms-B1-a), 'Probus' (Fossati & Ingold, 1970 - induced by X rays; ms-B1-b) and 'Cornerstone' (Driscoll, 1977 - induced by gamma-irradiation; ms-B1-c). The alleles ms-B1-b and ms-B1-c are terminal deletions. Several additional mutant alleles were previously induced by the inventors, either by gamma-irradiation or by treatment with ethyl methanesulfonate (EMS) (unpublished data). The EMS-treated mutants can be distinguished from the various deletions of the Ms-B1 locus by the presence in these mutants of a terminal C-band on 4BS. The DNA marker Xpsr921 which is located on the distal region of 4BS, is absent in ms-B1-c and in several of our gamma-irradiated mutants (and possibly also in ms-B1-b) (our unpublished data), i.e., it is located in the deleted segment and its absence can mark homozygosity for the deletion.

The maintainer line is of the same cultivar as the female line, i.e., cv. 'One', is homozygous for the same ms-B1 allele present in the female line but has the AA alien chromosomal arm as a monotelosomic addition (Figs. 2 and 3), consisting of 4S^SS of Aegilops searsii, that carries the dominant male-fertility allele Ms-S^s1, and the heterologous streptavidin gene coding for streptavidin which binds specifically biotin causing biotin depletion and consequently, cell death, controlled by a microspore- specific promoter. Because of the presence of the Sreptavidin gene, all pollen-grains carrying it die and thus, the alien chromosomal arm is not transmitted through the pollen grains. Hence, pollinating the male-sterile female line by the maintainer line yields progeny, all of which are identical to the female line and are male-sterile (Figs. 2 and 3). On the other hand, self pollination of the maintainer line yields a mixture of seeds, of which about 80%, when grown, developing into male-sterile plants, and about 20%, when grown, developing into male-fertile plants (Figs. 2 and 3). Due to the presence of the selectable marker which is a heterologous gene either conferring glufosinate resistance (Fig. 2) or coding for anthocyanin in the aleurone layer (Fig. 3), on the alien chromosomal arm, the male-fertile offspring of the maintainer are either resistant to glufosinate while the male-sterile offspring are susceptible (Fig. 2), or the color of the seeds which, when grown, developing into male-fertile plants, is blue while that of the other seeds, when grown, developing into male-sterile plants, is red or white. This differential reaction to glufosinate or the difference in seed color facilitates the selection of the male-fertile offspring, thus growing only the maintainer line.

The male parent (cv. 'Two') is any normal common or durum wheat cultivar, which by its nature is homozygous for the male-fertility Ms-B1 allele.

Thus, by the described method, hybrid seeds of common or durum wheat are readily and efficiently produced as the Fi progeny, all of which are heterozygous for the male-sterility alleles (Ms-B1ms-B1) and therefore, are male-fertile. So far, all cultivars that were used as male parents were able to fully restore the male fertility of the Fi hybrid.

Example 2

Testing the expression of male-fertility, brought about by one dose of the male- fertility allele Ms-S^s1 of Aegilops searsii, in ms-B1ms-B1 genotype of common wheat

In order to test the feasibility of the above hybrid production system and maintenance of the male-sterile female parental line, it was first necessary to produce an alien monotelosomic addition line in which chromosomal arm 4S^SS of Aegilops searsii, carrying the dominant male-fertility allele Ms-S^s1 , was added to the full complement of common wheat homozygous for the male-sterility allele ms-B1-c. This was carried out by crossing the ditelosomic 4S^SS addition line (derived from crosses of Aegilops searsii ace. TE-10 with the common tall wheat cultivar Chinese Spring and kindly provided by Prof. G. Hart, Soil and Crop Science, Texas A & M University, College Station, Texas) which is homozygous for Ms-B1 and carries two doses of Ms- S^s1, with the ms-B1-cms-B1-c male-sterile genotype of the semi-dwarf common wheat cv. Gamenia (kindly provided by Dr. M. MacKay, curator of the Australian Winter Cereal Collection, RRM 944, Calala Lane, Tamworth, NSW 2340, Australia). The resultant Fi plants were all heterozygous Ms-B1ms-B1-c and carried one additional chromosomal arm of 4S^SS with the Ms-S^s1 allele. These plants were selfed and F2 plants homozygous for ms-B1-c (as was indicated by the absence of the DNA marker Xpsr921) were selected. All euploid homozygous ms-B1-cms-B1-c plants were, as expected, male-sterile, but those homozygous that had an additional 4S^SS chromosomal arm carrying Ms-S^s1, were all male-fertile, indicating the complete dominance of Ms-S^s1 over two doses of ms-B1-c.

Example 3:

Construction of the alien chromosomal arm 4S^SS carrying the Ms-S^s1 allele, the heterologous Streptavidin gene controlled by a microspore-specific promoter, and the heterologous Bar gene controlled by a constitutive promoter

Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S^SS carrying the dominant male-fertility allele Ms-S^s1, are transfected with the heterologous Streptavidin and Bar genes, the first is linked to a microspore-specific promoter and the second to a plant constitutive promoter. The transgenic plants thus obtained are screened and those carrying the two heterologous genes on the alien chromosomal arm are selected and analyzed for their expression. Those plants with the desirable heterologous gene expression are selected. Example 4:

Construction of the alien chromosomal arm 4S^SS carrying the Ms-S^s1 allele, the heterologous Streptavidin gene controlled by a microspore-specific promoter, and the heterologous seed-color gene controlled by an endosperm-specific promoter

Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S^SS carrying the dominant male-fertility allele Ms-S^s1, are transfected with the heterologous Streptavidin and seed-color (Sc) genes, the first is linked to a microspore-specific promoter and the second to an endosperm- specific promoter. The transgenic plants thus obtained are screened and those carrying the two heterologous genes on the alien chromosomal arm are selected and analyzed for heterologous gene expression. Those plants with the desirable heterologous gene expression are selected.

Example 5:

Construction of the alien chromosomal arm 4S^SS carrying the Ms-S^s1 allele, the heterologous BaRNase gene controlled by a microspore-specific promoter, the heterologous Streptavidin gene controlled by a seedling-specific promoter, and the heterologous Bar gene controlled by a constitutive promoter

Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S^SS carrying the dominant male-fertility allele Ms-S^s1, are transfected with the three heterologous genes, BaRNase, controlled by a microspore-specific promoter, Streptavidin, controlled by a seedling-specific promoter, and Bar, controlled by a plant constitutive promoter. The transgenic plants thus obtained are screened and those carrying the three heterologous genes on the alien chromosomal arm are selected and analyzed for gene expression. Those plants with the desirable heterologous gene expression are selected.

Example 6:

Construction of the alien chromosomal arm 4S^SS carrying the Ms-S^s1 allele, the heterologous BaRNase gene controlled by a microspore-specific promoter, the heterologous Streptavidin gene controlled by a seedling-specific promoter, and the heterologous seed-color gene (Sc) controlled by an endosperm-specific promoter

Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S^SS carrying the dominant male-fertility allele Ms-S^s1, are transfected with the three heterologous genes, BaRNase, controlled by a microspore-specific promoter, Streptavidin, controlled by a seedling-specific promoter, and seed-color (Sc) gene controlled by an endosperm-specific promoter. The transgenic plants thus obtained are screened and those carrying the three heterologous genes on the alien chromosomal arm are selected and analyzed for gene expression. Those plants with the desirable heterologous gene expression are selected.

Example 7:

Construction of the alien chromosomal arm 4S^SS carrying the Ms-S^s1 allele, the heterologous Streptavidin gene controlled by a microspore-specific promoter, the heterologous BaRNase gene controlled by a seedling-specific promoter, the heterologous Barstar gene controlled by an inducible promoter, and the heterologous BAR gene controlled by a constitutive promoter

Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S^SS carrying the dominant male-fertility allele Ms-S^s1, are transfected with the four heterologous genes, Streptavidin, controlled by a microspore-specific promoter, BaRNase, controlled by a seedling-specific promoter, Barstar, controlled by an inducible promoter, and Bar, controlled by a plant constitutive promoter. The transgenic plants thus obtained are screened and those carrying the four heterologous genes on the alien chromosomal arm are selected and analyzed for gene expression. Those plants with the desirable heterologous gene expression are selected.

Example 8:

Construction of the alien chromosomal arm 4S^SS carrying the Ms-S^s1 allele, the heterologous Streptavidin gene controlled by a microspore-specific promoter, the heterologous BaRNase gene controlled by a seedling-specific promoter, the heterologous Barstar gene controlled by an inducible promoter, and the heterologous seed-color gene (Sc) controlled by an endosperm-specific promoter Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S^SS carrying the dominant male-fertility allele Ms-S^s1, are transfected with the four heterologous genes, Streptavidin, controlled by a microspore-specific promoter, BaRNase, controlled by a seedling-specific promoter, Barstar, controlled by an inducible promoter, and seed-color gene (Sc), controlled by a plant constitutive promoter. The transgenic plants thus obtained are screened and those carrying the four heterologous genes on the alien chromosomal arm are selected and analyzed for gene expression. Those plants with the desirable heterologous gene expression are selected.

Example 9

Production of Suitable Material for Transformation

Regarding the method to maintain genie male-sterile female parental lines for the production of hybrid wheat, the production of suitable material for transformation is described below. Construction of the material for transformation (see Figure 6).

A female parent, homozygous for the recessive ms-B1-c male-sterility allele, and therefore, male sterile, was crossed with a male parent, isogenic to the female parent, but homozygous for the dominant male-fertility allele (Ms-B1), and having an alien chromosome arm, as ditelosomic addition, consisting of the short arm of chromosome 4S^S (4S^SS) of Aegilops searsii carrying the dominant male-fertility allele Ms-S^s1, and therefore, male fertile.

The progeny seed from the cross were all heterozygous ms-B1-c Ms-B1 and had the alien chromosome arm as monotelosomic addition. The seeds were germinated and the plants developed from them were male fertile. Selfing of these plants yielded, among other combinations, about 1% of plants homozygous for the male-sterility ms-B1-c allele and the alien chromosome arm as ditelosomic addition line. This is the suitable material for transformation.

Transmission of the different alleles and the alien chromosome arm

The transmission of the male-sterility and male-fertility alleles was studied in several genetic combinations during several years and under greenhouse and field conditions. While the transmission of the ms-B1-c allele through the female gametes is as expected, i.e., 50%, the transmission through the male gametes is somewhat lower, i.e., 40-45%. This difference stems from the fact that the ms-B1-c allele is located within a small deletion.

The transmission of the alien chromosome arm 4S^SS when present in two doses, as in the ditelosomic line, is almost normal, i.e., 95-98%. On the other hand, the transmission of this arm when present in a single dose, as in monotelosomic line, is only around 20%.

Fertility data

During four years, 3000 plants were screened, all progeny of the cross of male-sterile female (homozygous for the male-sterility ms-B1-c allele) with male-fertile male (isogenic to the female but contained the alien chromosome arm 4S^SS that carries the male-fertility Ms-^S1 allele). It was found that all plants lacking the Ms-S?1 allele were male sterile, and those carrying it were male fertile. Evidently, the male fertility Ms-S^s1 allele is dominant over two doses of the male sterility ms-B1-c allele.

Although the invention has been shown and described in terms of specific embodiments and examples, changes and modifications will be evident to those skilled in the art. Such changes and modifications, which do not depart from the teachings herein, are deemed to fall within the purview of the invention as claimed.

REFERENCES

Bruns, R. and Peterson, C. J. (1998) Euphytica 100:1-5.

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Driscoll, C.J. (1972) Crop Sci. 12:516-517.

Driscoll, C.J., (1985) Crop Sci. 25:1115-1116.

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199. Jach, G., Gornhardt, B., Mundy, J., et al., (1995) Plant J. 8: 97-109. Jordaan, J. P., Engelbrecht, S. A., Malan, J. H. and Knobel, H. A. (1999) The Genetics and Exploitation of Heterosis in Crops, Madison, WI, USA, pp. 411-421. Kihara, H. (1951) Cytologia 16:117-193.

Logemann, J., Jach, G., Tommerup, H., et al. (1992) Bio/Tech. 10: 305-308. Mariani, C, Gossele, V., Debeuckeleer, M., et al. (1992) Nature 357: 384-387. Mclntosh, R.A. (1998) Proc. 9th Int. Wheat Genet. Symp., Saskatoon, Canada., Vol. 5, pp. 1-235. Pickett, A. A. (1993) Plant Breed. 15, Berlin. Tsujimoto, H. and Tsunewaki, K. (1983) Proc. 6th Int. Wheat Genet. Symp.,

Kyoto.Japan. pp. 1077-1081. Weeks, J.T., Anderson, O.D., Blechl, A.E., (1993) Plant Pphysiol. 102: 1077-1084. Wilson, P. and Driscoll, C.J. (1983) in Frenkel, R. (ed.) Monographs on Theoretical and

Applied Genetics, Vol. 6, Heterosis, Spripger-Verlag, pp. 94-123. [For Pugsley and Oram, 1959; Fossatti and Ingoid, 1970; and Driscoll, 1977 see above review by Wilson and Driscoll, 1983].

Claims

1. A plant homozygous for a recessive male-sterility allele and having in the nuclear genome of its cells an alien chromosomal arm carrying: a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene (Msu) comprising: i. a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those microspores carrying it thus rendering them inviable; and ii. a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; a heterologous selectable marker comprising: i. a selectable-marker DNA encoding for a heterologous protein that facilitates the selection of plants carrying it; and ii. a plant promoter, either constitutive or tissue specific, directing the expression of said selectable marker DNA in all plant tissues or in a specific tissue, respectively.

2. A plant homozygous for a recessive male-sterility allele and having in the nuclear genome of its cells an alien chromosomal arm carrying: a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene comprising: i. a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those microspores carrying it thus rendering them inviable; and ii. a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; a heterologous seedling-killer gene comprising: i. a seedling-killer DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those seedlings that possess it, thus killing the seedlings; ii. a seedling-specific promoter directing the expression of said seedling- killer DNA selectively in the seedlings that possess it; a heterologous selectable marker comprising: a selectable-marker DNA encoding for a heterologous protein that facilitates the selection of plants carrying it; and a plant promoter, either constitutive or tissue specific, directing the expression of said selectable marker DNA in all plant tissues or in a specific tissue, respectively.

3. A plant homozygous for a recessive male-sterility allele and having in the nuclear genome of its cells an alien chromosomal arm carrying: a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene comprising: i. a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those microspores carrying it thus rendering them inviable; and ii. a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; a heterologous seedling-killer gene comprising: i. a seedling-killer DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those seedlings that possess it; ii. a seedling-specific promoter directing the expression of said seedling- killer DNA selectively in the seedlings that possess it; a heterologous gene counteracting the deleterious effect of the seedling- killer gene comprising: i. a DNA encoding a heterologous protein that either counteracts the deleterious effect of the seedling-killer gene or disturbs its activity; and ii.' a plant constitutive or inducible promoter directing the expression of said DNA in the plants that possess it; a heterologous selectable marker comprising: i. a selectable-marker DNA encoding for a heterologous protein that facilitates the selection of plants carrying it; and ii. a promoter, either constitutive or tissue specific, directing the expression of said selectable marker DNA in all plant tissues or in a specific tissue, respectively.

4. The plants of claims 1-3, wherein said male-sterility allele is a heterologous gene.

5. The plants of claims 1-3, wherein said male-sterility allele is one of the male-sterility alleles of common wheat, durum wheat or another species of the Triticeae.

6. The plants of claims 1-3, wherein said male-sterility allele is one of the male-sterility alleles of the Ms~B1 locus either ms-B1-a, ms-B1-b, ms-B1-c or any other allele of MS-B1.

7. The plants of claims 1-3, wherein said dominant male-fertility allele (Ms) is a heterologous gene

8. The plants of claims 1 -3, wherein said dominant male-fertility allele (Ms) is one of the male-fertility alleles of the Triticeae.

9. The plants of claims 1-3 , wherein said dominant male-fertility allele (Ms) is Ms-S^s1 of chromosome arm 4S^SS of Aegilops searsii.

10. The plants of claims 1-3, wherein the said microspore-suicide gene is any of the genes affecting the viability of microspores, microgametophytes, pollen grains or male gametes that carry it.

11. The plants of claims 1-3, wherein the said microspore-suicide gene encodes avidin ' or sterptavidin of variant thereof.

12. The plants of claims 1-3, wherein the said microspore-suicide gene encodes RNase of variant thereof.

13. The plants of claims 1-3, wherein the said microspore-suicide DNA encodes ribosomal-inhibitor protein (RIP) of variant thereof.

14. The plants of claims 1-3, wherein the said microspore-specific promoter is any promoter that is expressed in plants directing gene expression in the microspore, male-gametophyte or pollen grain.

15. The plants of claims 1-3, wherein the heterologous selectable marker is any gene encoding for a heterologous protein that facilitates the selection of plants carrying it.

16. The plants of claims 1-3, wherein the heterologous selectable marker is a specific herbicide-resistance gene.

17. The plants of claims 1-3; wherein the specific herbicide-resistance gene is Bar conferring resistance to glufosinate (Basta).

18. The plants of claims 1-3, wherein the constitutive promoter of the specific herbicide- resistance gene expressed in plants.

19. The plants of claims 1-3, wherein the heterologous selectable marker is a seed-trait gene affecting the size, weight or morphology of the seeds.

20. The plants of claims 1-3, wherein the promoter of the seed-trait gene is a seed- specific promoter.

21.The plants of claims 1-3, wherein the heterologous selectable marker is a seed-color gene coloring the aleurone layer or the endosperm.

22. The plants of claims 1-3, wherein the promoter of the seed-color gene is an endosperm-specific promoter.

23. The plants of claims 1-3, wherein the promoter of the seed-color gene is an aleurone-specific promoter.

24. The plants of claims 2 and 3. wherein the said seedling-killer gene encodes a protein that renders the cells containing it inviable.

25. The plants of claims 2 and 3, wherein the said seedling-killer DNA encodes avidin or streptavidin of variant thereof.

26. The plants of claims 2 and 3, wherein the said seedling-killer DNA encodes Rnase of variant thereof.

27. The plants of claims 2 and 3, wherein the said seedling-killer DNA encodes ribosomal-inhibitor protein (RIP) of variant thereof .

28. The plants of claims 2 and 3, wherein the said seedling-specific promoter is any promoter that is expressed in plants directing gene expression in the seedling.

29. The plant of claim 3. wherein the said DNA counteracting the effect of the seedling- killer gene encodes a protein that neutralizes the effect of the seedling-killer gene of variant thereof.

30. The plant of claim 3, wherein the said DNA counteracting the effect of the seedling- killer gene encodes biotin of variant thereof.

31.The plant of claim 3, wherein the said DNA counteracting the effect of the seedling- killer gene encodes RNase antisense of variant thereof.

32. The plant of claim 3, wherein the said DNA counteracting the effect of the seedling- killer gene encodes RIP antisense of variant thereof.

33. The plant of claim 3, wherein the said constitutive promoter is any promoter that is expressed in plants directing gene expression in the plants.

34. The plant of claim 3, wherein the said inducible promoter is any promoter that is expressed in plants directing gene expression in the plants.

35. The plant of claim 3, wherein the said inducible promoter is induced by temperature, light, chemical or any other environmental treatment.

36. The plant of claim 3, wherein the said inducible promoter is induced by tetracyclin.

37. The plants of claims 1-3, wherein the alien chromosomal arm is any arm of a Triticeae species carrying a dominant male-fertility allele.

38. The plants of claims 1-3, wherein the alien chromosome arm is chromosomal arm 4S^SS of Aegilops searsii.

39. The plants of claims 1-3, which is selected from any of the Gramineae species.

40. The plants of claims 1-3, which is selected from wheat, triticale, barely, rye, oat, rice, and maize.

41. A method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat, said method comprising:

(a) crossing a female parent with a male parent, said female parent being a male-sterile plant homozygous for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B (4BS), said male parent being the maintainer line as defined in claims 1-3, and

(b) harvesting from the cross of (a) the progeny seed, all of which are homozygous for said male-sterility allele and lack the alien chromosome arm, said seeds, when grown, developing into said male-sterile female line.

42. A method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat, said method comprising:

(a) selfing a maintainer line as defined in claims 2-3, and

(b) harvesting from the selfing of (a) the progeny seed, all of which are homozygous for said male-sterility allele, of which 80% lack the alien chromosomal arm and therefore they are red or white, said seeds, when grown, developing into said male-sterile female plants, and 20% have the alien chromosomal arm and therefore they are blue, said seeds, when grown, developing into male-fertile maintainer plants, and separating by a seed sorter the red or white female seeds from the blue maintainer seeds.

43. A method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat, said method comprising: (a) selfing a maintainer line isogenic to the female line and homozygous for the same ms-B1 allele of the female line, and having the alien chromosomal arm 4S^SS that derived from Aegilops searsii, as a monotelosomic addition, herein referred to as AA, carrying the dominant male-fertility allele Ms-S^s1, a heterologous microspore-suicide gene (Msu) capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound and thus causes cell death, controlled by a microspore-specific promoter, and a heterologous seedling-killer gene capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound and thus causes cell death, controlled by a seedling-specific promoter, and a heterologous selectable marker, by which plants having this chromosome arm can be selected, and

(b) harvesting from the selfing of (a) the progeny seed, all of which are homozygous for said male-sterility allele, of which 80% lack the alien chromosomal arm, said seeds, when grown, developing into said male-sterile female plants, and 20% have the alien chromosomal arm, said seeds, when grown, developing into male-fertile maintainer plants, but dying as seedlings leaving only the male-sterile plants alive.

44. A method for the maintenance of a male-fertile maintainer line of common or durum wheat for use in the maintenance of male-sterile female line, said method comprising:

(b) harvesting the maintainer of (a) its progeny seed, all of which are homozygous for said male-sterility allele, of which 80% lack the alien chromosome arm, said seeds, when grown, developing into said male-sterile female plants, and 20% have the alien chromosome arm, said seeds, when grown, developing into male-fertile maintainer plants; and (c) selecting the maintainer plants by the selectable marker.

45. A method for a maintenance of a male-fertile maintainer line of common or durum wheat for use in the maintenance of male-sterile female line of wheat, said method comprising:

(a) selfing a male-fertile maintainer line homozygous for one of the male- sterility ms-B1 alleles , and having the alien chromosomal arm 4S^SS that derived from Aegilops searsii, as a monotelosomic addition, herein referred to as AA, carrying the dominant male-fertility allele Ms-S^l, a heterologous microspore- suicide gene (Msu) capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, or destroys an essential compound and thus causes cell death, controlled by a microspore-specific promoter, a heterologous seedling-killer gene capable of coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the ceil containing it, or destroys an essential compound and thus causes cell death, controlled by a seedling-specific promoter, and a heterologous selectable marker controlled either by a constitutive promoter or by a tissue-specific promoter, by which plants having this chromosome arm can be selected, and

(b) harvesting from the maintainer of (a) the selfed progeny seed, all of which are homozygous for said male-sterility allele, of which 80% lack the alien chromosome arm, said seeds, when grown, developing into male-sterile plants, and 20% contain the alien chromosome arm, when grown, developing into male- fertile maintainer plants; and

(d) in case of a seed-color gene as a selectable marker, the progeny seeds of (a) will be separated by a seed sorter, the blue seeds will be the maintainer seeds and the red or white seeds will be added to the male-sterile female line. The blue seeds will be treated with biotin to allow their germination and growth.

46. A method for a maintenance of a male-fertile maintainer line of common or durum wheat for use in the maintenance of male-sterile female line of wheat, said method comprising:

(a) selfing a male-fertile maintainer line as defined in claim 3,

(c) in case of a specific herbicide-resistance gene as a selectable marker, germinating the progeny seeds of (a) and treating the seedlings with the chemical that induces activity of the inducible promoter of the anti seedling-killer gene that counteracts the expression of the seedling-killer gene and spraying the plants with the specific herbicide thus killing all progeny plants lacking the alien chromosome arm, and leaving to grow only those plants that carry the alien chromosome arm and therefore, will be the male-fertile maintainer.

(d) in case of a seed-color gene as a selectable marker, the progeny seeds of (a) will be separated by a seed sorter, the blue seeds will be the maintainer seeds and the red or white seeds will be added to the male-sterile female line. The blue seeds will be treated with the chemical that induces activity of the inducible promoter of the anti-seedling-killer gene that counteracts the expression of the seedling-killer gene to allow their germination and growth.

47.A method according to claim 44-46, wherein the selectable marker gene is a specific herbicide-resistance gene (Hr), controlled by a constitutive promoter.

48.A method according to claim 44-46, wherein the selectable marker gene is a seed- color gene (Sc), controlled by an endosperm-specific promoter.

49. A method according to claims 44-46, wherein the selectable marker gene is a seed- trait gene affecting seed size, weight or morphology, controlled by a seed-specific promoter.

50.A method for producing a hybrid plant line of common or durum wheat, comprising :

(a) crossing a male parent with a male-sterile female parent of the same species, wherein said male parent is selected from any desired common or durum wheat cultivar which, by its nature, is homozygous for the dominant wild-type male-fertility (Ms-B1) allele, and said male-sterile female parent is a line of said wheat species being homozygous for both any one of the recessive mutant male-sterility (ms-B1) allele, said male-sterile female parent being maintained by a maintainer line according to any one of claims 44-46; and

(b) collecting the progeny seed of the cross of (a), which seeds, when grown, develop into progeny hybrid plants all of which are heterozygous ms-B1 Ms-B1 and therefore, male fertile.