WO2002059269A2

WO2002059269A2 - Maintenance of genic male-sterile female parental lines using a maintainer line with a stable engineered chromosome

Info

Publication number: WO2002059269A2
Application number: PCT/US2002/000176
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: 2002-08-01
Also published as: WO2002059269A3; AU2002246937A1

Abstract

The present invention provides a method for stably maintaining a genic malesterile 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 malesterility allele and for a dominant pollen-killer allele, and a maintainer line that is readily and stably propagated. The maintainer line isogenic to the female line but has a stable alien engineered chromosomal arm, added to the wheat complement, carrying a dominant male-fertilizy allele that restores fertility to the maintainer line, a rece ssive pollen-killer allele which is sensitive to the dominant polen-killer allele thus causing abortion of pollen grains carrying it and thus preventing the transmission of this chromosome arm to the femal 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

MAINTENANCE OF GENIC MALE-STERILE FEMALE PARENTAL LINES USING A MAINTAINER LINE WITH A STABLE ENGINEERED

CHROMOSOME

BACKGROUND OF THE INVENTION

Field Of The Invention

The present invention relates to the production of hybrid lines of common and durum wheat and to a novel plant. More specifically, the present invention relates to a new method for maintaining a male-sterile female parental line for use in the production of hybrid wheat plants, which female line is homozygous for a recessive male-sterility mutant allele and for a dominant pollen-killer allele, and a new maintainer line for maintaining the female parental line which is isogenic to the female line, but has an additional stable alien engineered chromosomal arm (SEC), as a monotelosomic addition, carrying a dominant male-fertility allele, a recessive pollen-killer allele that is susceptible to the action of said dominant pollen-killer allele that induces abortion of pollen grains carrying it, and a heterologous selectable marker. The three genes on the SEC are permanently linked due to lack of pairing and recombination between the SEC and the wheat chromosomes. The presence of the recessive pollen-killer allele on the SEC ensures that all the maintainer viable pollen grains (about 80% of the pollen grains) will lack this chromosomal arm and consequently, the dominant male-fertility allele and the selectable marker allele. Hence, pollination of male-sterile female plants by such a maintainer will yield only male-sterile female plants. On the other hand, since female gametes carrying the SEC are viable, self-pollination will yield a mixture of seeds, about 80% that, when grown, developing into male-sterile female plants and about 20%, 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 only the maintainer plants to grow. On the other hand, when the selectable marker is seed color such as blue seeds, 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 gene and all are 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) critical to male-fertility in common wheat. Whereas in many commercial crops it is the GMS that prevails, this type has not yet been fully exploited in common or durum wheat and 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 grain yield is negatively affected by the interaction between the wheat nuclear genome and the alien cytoplasm, as opposed to GMS which involves a native cytoplasm that allows for full yielding capacity of the hybrid. In addition, it has been difficult to find stable fertility restoration genes for such 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. Hence, no cytoplasm-induced deleterious effects on plant performance are expected. 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 F 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.

Of the chromosome arms that have been described in common wheat to carry genes affecting male-fertility, the short arm of chromosome 4B (4BS) was most used. Male-sterility is due to mutations in the Ms-B1 locus, on the distal region of chromosome arm 4BS. The three recessive alleles found or induced that cause male-sterility are ms-B1-a, ms-B1-b and ms-B1-c (often also called msla, mslb and mslc, respectively), and 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). This system however, 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. 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), which 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 gd 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 msmsGc1Gc1 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 Gc1 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 patent [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-sterile 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-killer allele, and a maintainer line which is readily and stably propagated. The maintainer line is isogenic to the female line but has an alien stable 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 stable engineered chromosome, due to some frequency of transversal division of the centromere, was found to be unstable, resulting in pollen grains that contain the chromosome arm with the male-fertility allele. These pollen grains were transmitted upon pollination of the female plants resulting in several percentages of male-fertile female plants, which led to reduced hybrid purity. Consequently, this method was not used in commercial practice.

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 longissima = a diploid species (2n=14), closely related to the donor of the

B genome of durum and common wheat having genome S', whose chromosomes are homoeologous (partial homologous) to those of wheat. Aegilops searsii = a diploid species (2n=14), closely related to the donor of the B genome of durum and common wheat having genome 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.

6S'L = the long arm of chromosome 6S¹ of Aegilops longissima.

Ki-B1= a dominant pollen-killer allele on 6BL of common wheat, inducing the killing of pollen carrying ki-B1-a or ki-SH-a. ki-B1-a- a recessive pollen-killer allele on 6BL of common wheat; pollen carrying it is killed in plants possessing KI-B1. ki-B1-n- a neutral pollen-killer allele on 6BL of common wheat; pollen carrying it is neither killed in plants possessing KI-B1 nor it induces killing of pollen carrying ki-B1-a or ki-S -a. ki-SH-a= a pollen-killer allele on 6S'L of Aegilops longissima; pollen carrying it is killed in plants possessing Ki-B1. 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.

Hr = a heterologous specific herbicide resistance gene by which plants having this gene can be selected, controlled by a constitutive promoter. Bar= 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. SEC= a stable alien engineered chromosomal arm 6S'L/4S^SS, carrying the Ms-S^s1 male-fertility allele, a ki-S'1-a pollen-killer allele, and a selectable marker by which plants having this stable engineered chromosomal arm can be selected, cv. = cultivar. Maintainer line = a male-fertile line isogenic to the male-sterile female line homozygous for both the ms and Ki alleles, and contains the alien chromosomal arm SEC.

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.

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-B1) allele on 4BS and for a dominant pollen-killer allele KI-B1 allele on 6BL (Figs. 2 and 3). The maintainer is isogenic to the female line, and has further a stable alien engineered chromosomal arm (SEC) as a monotelosomic addition (Fig. 1), carrying a dominant male-fertility allele (Ms-S^s1) linked to the recessive ki-S'l-a allele which causes abortion of male gametes carrying it in the presence of Ki-B1, and a selectable marker by which progeny of the selfed maintainer carrying the SEC can be selected. The selectable marker is a heterologous selectable marker gene, which is either a specific herbicide resistance gene (Hr) controlled by a constitutive promoter (Fig. 2) or a seed color gene (Sc), i.e., blue seeds, controlled by an endosperm-specific promoter (Fig. 3). Pollen grains of the maintainer containing the SEC 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 seeds in which about 20% carry the SEC and, when grown, developing into male-fertile plants, and about 80% lack this chromosomal arm and, when grown, developing into male-sterile plants. Spraying the progeny plants with the specific herbicide kills the male-sterile plants allowing the growth of only the male-fertile maintainer plants (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 stable alien engineered chromosomal arm is a translocated arm of chromosome arms 6S'L of Aegilops longissima and 4S^SS of Ae. searsii. Being an alien single telocentric chromosome, which 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 recessive pollen-killer allele, the rate of its transmission through the female gametes is about 20%.

The Ms-S^s1 gene is dominant over the ms-B1 alleles of common and durum wheat and is expressed in all tested genetic backgrounds. The pollen-killer allele (ki- ^1-a) causes abortion to the pollen grain possessing it in the presence of the native Ki-B1. The specific herbicide resistance gene (Hr) is any heterologous gene that codes for a heterologous protein conferring resistance to a specific herbicide under control of a constitutive promoter. The seed color gene (Sc) is a heterologous color gene that codes for an anthocyanin or another pigment, under control of an endosperm-specific promoter.

The above mentioned heterologous genes are introduced to the SEC by standard genetic-engineering manipulations.

According to the invention it is a principal object to provide a plant homozygous for a recessive male-sterility (ms) allele and for a dominant pollen-killer (Ki) allele and having in the nuclear genome of its cells an alien engineered chromosomal arm carrying a male-fertility dominant allele (Ms); a recessive pollen- killer allele (ki) which is susceptible to the killing action of the dominant pollen-killer (Ki) allele, causing abortion of pollen grains carrying it; and a selectable marker that facilitates the selection of plants carrying it. In a preferred form, the plant is homozygous for one of the male-sterility alleles of one of the Triticeae species.

It is a still further object of the present invention to provide a plant homozygous for one of the male-sterility alleles of the Ms-B1 either ms-B1-a, ms-B1- b, ms-B1-c or any other allele of MS-B1.

It is a still further object of the present invention to provide a plant, wherein the dominant pollen-killer allele is from any Triticeae species, and more particularly, wherein the dominant pollen-killer allele is Ki-B1 on chromosomal arm 6BL of common and durum wheat.

It is a still further object of the present invention to provide a plant, wherein said dominant male-fertility allele (Ms) is an heterologous gene.

It is a still further object of the present invention to provide a plant, wherein said dominant male-fertility allele (Ms) is one the male-fertility alleles of the Triticeae. Even more specifically, wherein said dominant male-fertility allele (Ms) is Ms-S^s1 of chromosome arm 4S^SS of Aegilops searsii. It is a still further object of the present invention to provide a plant, wherein said recessive pollen-killer allele (ki) is selected from any pollen-killer alleles of the Triticeae, and more particularly, wherein said recessive pollen-killer allele is ki-^1-a on 6S'L of Aegilops longissima. Also, said recessive pollen-killer allele can be ki-B1- a on 6BL of bread wheat. The recessive pollen-killer allele can be a heterologous gene.

It is a still further object of the present invention to provide a plant, wherein the said selectable marker gene is a specific herbicide resistance gene. Further, the specific herbicide resistance gene can be a native gene for which differential response prevails among genotypes.

It is a still further object of the present invention to provide a plant, wherein the said selectable marker gene is a seed specific gene. Also, the seed specific gene can be a seed coloration gene that was transferred from one of the species of the Triticeae. Further, the seed color gene is selected from one of the Ba blue aleurone genes, Ba-A^m1 on 4A L of Triticum monococcum and Ba-E1 on 4EL of Agropyron elongatum.

It is a still further object of the present invention to provide a plant, wherein the selectable marker is a heterologous gene encoding for a heterologous protein that facilitates the selection of plants carrying it and having 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. The heterologous gene can be a specific herbicide-resistance gene expressed by a constitutive promoter, and more particularly, wherein the specific herbicide-resistance gene can be Bar conferring resistance to the glufosinate (Basta). Further, the constitutive promoter of the specific herbicide-resistance gene can be expressed in plants. Also, the heterologous gene can be a seed color gene, and the promoter of the seed color gene can be an endosperm-specific promoter. The promoter of the seed color gene can also be an aleurone-specific promotβr.

It is a still further object of the present invention to provide a plant, wherein the engineered chromosomal arm consists of any two arms of Triticeae species carrying a dominant male-fertility allele and a recessive pollen-killer (ki) allele. More particularly, the engineered chromosomal arm can be the chromosomal arms 6S'L/4S^SS.

It is a still further object of the present invention to provide a plant, having the engineered chromosomal arm 6S'L/4S^SS, as a monotelosomic addition, herein referred to as SEC, carrying the dominant male-fertility allele Ms-S^s1, a pollen-killer allele (ki-S'l-a) which is susceptible to the killing action of the dominant pollen-killer allele Ki-B1, causing abortion of male gametes carrying it, and a selectable marker by which plants having this chromosome arm can be selected. The plant can be selected from a Triticeae species, including one of wheat, triticale, barely, rye, oat, rice, and maize.

It is a still further object of the present invention to provide 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 both for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B (4BS), and for the dominant pollen-killer KI-B1 allele on the long arm of chromosome 6B (6BL), said male parent being the maintainer line and being isogenic to the female parent and homozygous for the same ms-B1 and Ki-B1 alleles of the female parent, and having an alien engineered chromosomal arm, as a monotelosomic additon, herein referred to as SEC, consisting of the long arm of chromosome 6S¹ and the short arm of chromosome 4S^S, carrying the dominant male-fertility allele Ms-S^s1, linked to a recessive allele ki-Sll-a which is susceptible to the killing action of the native pollen-killer allele Ki-B1, and a selectable marker either heterologous gene or native allele, by which plants having this chromosomal arm can be selected. 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 SEC stable engineered chromosomal arm, said seeds, when grown, developing into said male-sterile female line. It is a still further object of the present invention to provide 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 the maintainer line being isogenic to the female parent being a male-sterile plant homozygous both for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B, and for the dominant pollen-killer Ki-B1 allele on the long arm of chromosome 6B, and having an alien engineered chromosomal arm, as a monotelosomic additon, herein referred to as SEC, consisting of the long arm of chromosome 6S¹ and the short arm of chromosome 4S^S, carrying the dominant male-fertility allele Ms- S^s1, linked to a recessive allele ki-S'l-a which is susceptible to the killing action of the native pollen-killer allele KI-B1 and a selectable marker either heterologous gene or native allele, by which plants having this chromosomal arm can be selected, and

(b) selecting from the progeny, by using the selectable marker those about 80% of the progeny lacking the stable alien engineered chromosomal arm, said selected progeny being the female line.

It is a still further object of the present invention to provide a method for the maintenance of a male-fertile maintainer line of common or durum wheat, said method comprising:

(b) selecting from the progeny, by using the selectable marker those about 20%of the progeny having the stable alien engineered chromosomal arm, said selected progeny being the maintainer line.

It is a still further object of the present invention to provide a method as described above,, wherein said alien chromosome arm, herein designated SEC, carries as a selectable marker the heterologous Bar gene conferring resistance to the herbicide glufosinate (Basta), controlled by a constitutive promoter, and/or wherein said alien chromosome arm, herein designated SEC, carries as a selectable marker a heterologous seed color gene (Sc) coding for anthocyanin in the aleurone layer of the endosperm, controlled by aleuron-specific promoter.

The present invention provides a method for stably maintaining a genie 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 for a dominant pollen-killer allele, and a maintainer line that is readily and stably propagated. The maintainer line is isogenic to the female line but has a stable alien engineered chromosomal arm, added to the wheat complement, carrying a dominant male-fertility allele that restores fertility to the maintainer line, a recessive pollen-killer allele which is sensitive to the dominant pollen-killer allele thus causing abortion of pollen grains carrying it and thus 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.

Other and further objects of the invention will become readily apparent from the following detailed description of the invention and preferred embodiments when taken in conjunction with the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts a schematic drawing of the stable alien engineered chromosomal arm 6S'L/4S^SS derived from a translocation between chromosomes 6S¹ of Ae. longissima and 4S^SS of Aegilops searsii, herein referred to as SEC, carrying the dominant male-fertility allele Ms-S^s1, a pollen-killer allele (ki-S'l-a), 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.

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. SEC = the stable alien engineered chromosome arm 6S'L/4S^SS added to the wheat complement, carrying the dominant male-fertility allele Ms-S^s1, a pollen- killer allele (ki-S'1-a), 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. SEC = the stable alien engineered chromosome arm 6S'L/4S^SS added to the wheat complement, carrying the dominant male-fertility allele Ms-S^s1, a pollen-killer allele (ki-S'l-a), and a heterologous seed color gene (Sc) with an endosperm-specific promoter.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a simple system has been developed for common or 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. Alternatively, when the selectable marker is a seed color (Sc) allele, the male-sterile female parental line is maintained by selfing the maintainer and selecting from the progeny seeds, the red or white seeds, said seeds when grown, developing into male-sterile 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 stable engineered chromosomal arm (SEC), and about 80%, when grown, developing into male-sterile plants due to the absence of the SEC and consequently, the Ms-S^s1 allele (Figs. 2 and 3). Spraying the progeny plants of the selfed maintainer line with the specific herbicide (Fig. 2), causes death of the male-sterile plants while the male-fertile plants carrying the SEC with the specific herbicide resistance gene survive. Alternatively, separating the seeds of the selfed maintainer line on the basis of their different color with a seed sorter (Fig. 3), allows selection of seeds which are colored by the expression of Sc allele, said seeds, when grown, developing into maintainer plants.

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 wheat, said method comprising:

(a) crossing a female parent with a male parent, said female parent being a male-sterile plant homozygous both for any one of the recessive ms-B1 male-sterility alleles, and for the dominant pollen-killer Ki-B1 allele, said male parent being the maintainer line and being isogenic to the female parent and homozygous for the same ms-B1 and KI-B1 alleles of the female parent, and having a stable alien engineered chromosomal arm, as a monotelosomic addition, herein referred to as SEC, consisting of a translocated arm of chromosome arms 6S'L of Aegilops longissima and 4S^SS of Ae. searsii, carrying the dominant male-fertility allele Ms-S^s1, linked to a recessive allele ki-S'l-a which is susceptible to the killing action of the native pollen-killer allele Ki-B1 and a heterologous selectable marker with a plant constitutive promoter, by which plants having this chromosomal arm can be selected. Pollen grains of the maintainer containing this chromosomal 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 SEC, said seeds, when grown, developing into said male-sterile female line.

The present invention provides an alternative method for the maintenance of a male-sterile female parental line of common or durum wheat (Fig. 3) 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 and Ki-B1 alleles of the female line, and having a stable alien engineered chromosomal arm, as a monotelosomic addition, herein referred to as SEC, consisting of a translocated arm of chromosome arms 6S^!L of Aegilops longissima and 4S^SS of Ae. searsii, carrying the dominant male-fertility allele Ms-S^s1, linked to a recessive allele ki-S'l-a which is susceptible to the killing action of the native pollen-killer allele Ki- B1 on 6BL and a heterologous seed-color gene (Sc) controlled by an endosperm-specific promoter, by which seeds lacking the SEC (the red or white seeds) can be selected. Pollen grains of the maintainer containing the SEC are not functional, and (b) harvesting the seeds from the selfed maintainer of (a), all of which are homozygous for said male-sterility and pollen-killer alleles, and selecting by a seed sorter said red or white seeds lacking the SEC, said seeds, when grown, developing into said male-sterile female line.

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. Any ki allele of one of the species of the Triticeae, susceptible to the killing effect of KI-B1 or any other pollen-killer gene may be used according to the invention.

The selectable marker by which maintainer plants can be selected among the progeny of the selfed maintainer, can be a heterologous specific herbicide resistance gene (Hr) that codes for a heterologous protein that confers resistance to said specific herbicide, such as, the Bar gene that confers resistance to glufosinate (De Block et al 1987, Weeks et al 1993), under control of a constitutive promoter.

Alternatively, the selectable marker is a seed marker that allows for separation of the maintainer seed from the female seeds by their different color or by any other seed characteristics. The grain coloration allele may be any Ba allele of one of the species of the Triticeae according to the invention, such as, for example, the Ba-

A^m1 of T. monococcum, the Ba-E1 of A. elongatum, the Ba-R1 of rye and the Ba-H1 of barley, or any heterologous seed color gene (Sc) controlled by an endosperm- specific promoter, that codes for an anthocyanin that is accumulated in the aleurone layer 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 and 3), said maintainer line being isogenic to the female parent being a male-sterile plant homozygous both for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B (4BS), and for the dominant pollen-killer KI-B1 allele on the long arm of chromosome 6B (6BL), and having a stable alien engineered chromosomal arm, as a monotelosomic addition, herein referred to as SEC, consisting of a translocated arm of chromosome arms 6S'L of Aegilops longissima and 4S^SS of Ae. searsii, carrying the dominant male-fertility allele Ms-S^s1, linked to a recessive allele ki-S'l-a which is susceptible to the killing action of the native pollen- killer allele Ki-B1, and a heterologous selectable marker controlled by a plant constitutive promoter, by which plants having this chromosomal arm can be selected. Being an alien monotelosomic addition, the SEC does not pair during meiosis, and consequently, enters only to about 20% of the gametes, either male or female. Since male gametes having the SEC are not functional, the amount of seeds containing the SEC among the progeny of the selfed maintainer is about 20%. These seeds when grown, developing into male-fertile plants resistant to the specific herbicide and can be selected by it. Alternatively, the selectable marker can be a seed color gene (Sc) controlled by an endosperm-specific promoter, coding for an anthocyanin in the aleurone layer of the endosperm resulting in blue seed coloring (Fig. 3). The blue seeds containing the SEC can be separated from the red or white seeds lacking the SEC 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 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 separating the seed progeny of the selfed maintainer line according to their different color, and the maintainer line is itself maintained by selfing, and the desired male-fertile plants among the selfed progeny of the 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 this method 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-B1) on the short arm of chromosome 4B and homozygous for the dominant pollen-killer allele (Ki-B1) on the long arm of 6B, is used as the male-sterile female.

The maintainer line is of the same cultivar as the female line, i.e., cv. 'One', is homozygous for the same ms-B1 and Ki-B1 alleles present in the female line, but has the SEC as a monotelosomic addition (Figs. 2 and 3), consisting of a translocated arms of chromosome arms 6S'L of Aegilops longissima and 4S^SS of Ae. searsii, carrying the dominant male-fertility allele Ms-S^s1 and the recessive pollen- killer allele ki-SH-a, rendering pollen-grains carrying it amenable to killing by the Ki- B1 allele of common or durum wheat, and a heterologous gene, either the Bar gene conferring glufosinate resistance or a gene inducing blue coloration of the seed.

Because of the presence of ki-SH-a, the SEC 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 80% male-sterile and 20% male-fertile offspring (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 resistant to glufosinate while the male-sterile offspring are susceptible (Fig. 2) or the color of the seeds, which, when grow, developing into male-fertile plants, is blue while that of the other seeds, when grow, developing into male-sterile plants, is red or white (Fig. 3). This different reaction to glufosinate or the difference in seed color facilitates the selection of the male-fertile offspring, thus maintaining 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 rapidly 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 parent 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-c ms-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 stable alien engineered chromosomal arm 6S'L/4S^SS carrying the Ms-S^s1 allele, the ki-Sh-a allele.

Plants homozygous for the male-sterility recessive allele ms-B1-c and for the dominant pollen-killer allele Ki-B1, and double ditelosomic addition both for the alien chromosomal arms 4S^SS of Ae. searsii, carrying the dominant male-fertility allele

Ms-S^s1, and 6S'L of Ae. longissima, carrying the recessive pollen-killer allele ki-SH- a, were exposed to x ray irradiation before flowering and used to pollinate plants of similar background constitution but without the alien additional chromosomal arms (and hence are male-sterile). Offspring with the desired translocated telocentric chromosomal arm (6S'L/4S^SS) are selected.

Example 4: Addition of the heterologous herbicide resistance gene Bar, controlled by a constitutive promoter, to the stable alien engineered chromosomal arm 6S'L/4S^SS carrying the Ms-S^s1 allele, the ki-S -a allele.

Plants homozygous for the male-sterility recessive allele ms-B1-c and for the dominant pollen-killer allele Ki-B1, and two doses of the stable alien chromosomal arm 6S'L/4S^SS carrying the dominant male-fertility allele Ms-S^s1, and the recessive pollen-killer allele ki-sH-a, are used to pollinate plants of similar background constitution but without the alien additional chromosomes (and hence are male- sterile). All the resultant seeds containing the stable alien chromosomal arm are transfected with the heterologous Bar gene activated by a constitutive promoter. The transgenic plants thus obtained are screened and those carrying the heterologous gene on the stable engineered chromosomal arm are selected and analyzed for heterologous gene expression. Those plants with the desirable gene expression are selected. Example 5:

Addition of the heterologous seed color gene (Sc) controlled by an endosperm-specific promoter, to the stable alien engineered chromosomal arm 6S'L/4S^SS carrying the Ms-S^s1 allele. the ki-Sh-a.

Plants homozygous for the male-sterility recessive allele ms-B1-c and for the dominant pollen-killer allele Ki-B1, and two doses of the stable alien chromosomal arm 6S'L/4S^SS carrying the dominant male-fertility allele Ms-S^s1, and the recessive pollen-killer allele ki-SH-a, are used to pollinate plants of similar background constitution but without the alien additional chromosomes (and hence are male- sterile). All the resultant seeds containing the stable alien chromosomal arm are transfected with the heterologous seed color (Sc) gene linked to an endosperm- specific promoter. The transgenic plants thus obtained are screened and those carrying the heterologous gene on the stable engineered chromosomal arm are selected and analyzed for heterologous gene expression. Those plants with the desirable gene expression are selected.

Although the present invention has been described in terms of specific and preferred embodiments and examples, nevertheless, changes and modifications are possible which do not depart from the teachings herein. Such changes and modifications, as are apparent to one or ordinary skill in the art, are deemed to fall within the purview of the invention as claimed.

LITERATURE CITATIONS

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

De Block et al., (1987) EMBO J. 6: 2513

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

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

Dooner, H.K., Robbins, T.P., Jorgensen, R.A. et al., (1991) Ann. Rev. Genet. 25:

179-199. Jordaan, J. P., Engelbrecht, S. a., Malan, J. H. and Knobel, H. A. (1999) The

Genetics and exploitation of Heterosis in Crops, Madison, Wl, USA, pp.411-

421. Kihara, H. (1951) Cytologia 16:117-193.

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, Springer-Verlag, pp. 94-

123. [For Pugsley and Oram, 1959; Fossatti and Ingold, 1970; and Driscoll, 1977 see above review by Wilson and Driscoll, 1983].

Claims

1. A plant homozygous for a recessive male-sterility (ms) allele and for a dominant pollen-killer (Ki) allele and having in the nuclear genome of its cells an alien engineered chromosomal arm carrying: a male-fertility dominant allele (Ms); a recessive pollen-killer allele (ki) which is susceptible to the killing action of the dominant pollen-killer (Ki) allele, causing abortion of pollen grains carrying it; and a selectable marker that facilitates the selection of plants carrying it;

2. The plant of claim 1, which is homozygous for one of the male-sterility alleles of one of the Triticeae species.

3. The plant of claim 2, which is homozygous for one of the male-sterility alleles of common or durum wheat.

4. The plant of claim 2, which is homozygous for 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.

5. The plant of claim 1 , wherein the dominant pollen-killer allele is from any Triticeae species.

6. The plant of claim 5, wherein the dominant pollen-killer allele is KI-B1 on chromosomal arm 6BL of common and durum wheat.

7. The plant of claim 1, wherein said dominant male-fertility allele (Ms) is an heterologous gene.

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

9. The plant of claim 8, wherein said dominant male-fertility allele (Ms) is Ms-S^s1 of chromosome arm 4S^SS of Aegilops searsii.

10. The plant of claim 1, wherein said recessive pollen-killer allele (ki) is selected from any pollen-killer alleles of the Triticeae.

11. The plant of claim 10, wherein said recessive pollen-killer allele is ki-S'l-a on 6S'L of Aegilops longissima.

12. The plant of claim 10, wherein said recessive pollen-killer allele is ki-B1-a on 6BL of bread wheat.

13. The plant of claim 1 , wherein said recessive pollen-killer allele is a heterologous gene.

14. The plant of claim 1 , wherein the said selectable marker gene is a specific herbicide resistance gene.

15. The plant of claim 14, wherein the specific herbicide resistance gene is a native gene for which differential response prevails among genotypes.

16. The plant of claim 1 , wherein the said selectable marker gene is a seed specific gene.

7. The plant of claim 16, wherein the seed specific gene is a seed coloration gene that was transferred from one of the species of the Triticeae.

18. The plant of claim 17, wherein the seed color gene is selected from one of the Ba blue aleurone genes, Ba-A^m1 on 4A^mL of Triticum monococcum and Ba-E1 on 4EL of Agropyron elongatum.

19. The plant of claim 1 , wherein the selectable marker is a heterologous gene encoding for a heterologous protein that facilitates the selection of plants carrying it and having 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.

20. The plant of claim 19, wherein the heterologous gene is a specific herbicide- resistance gene expressed by a constitutive promoter.

21. The plant of claim 20, wherein the specific herbicide-resistance gene is Bar conferring resistance to the glufosinate (Basta).

22. The plant of claim 19, wherein the constitutive promoter of the specific herbicide- resistance gene is expressed in plants.

23. The plant of claim 19, wherein the heterologous gene is a seed color gene.

24. The plant of claim 23, wherein the promoter of the seed color gene is an endosperm-specific promoter.

25. The plant of claim 23, wherein the promoter of the seed color gene is an aleurone-specific promoter.

26. The plant of claim 1 , wherein the engineered chromosomal arm consists of any two arms of Triticeae species carrying a dominant male-fertility allele and a recessive pollen-killer (ki) allele.

27. The plant of claim 26, wherein the engineered chromosomal arm is the chromosomal arms 6S'L/4S^SS.

28. The plant of claim 1, having the engineered chromosomal arm 6S'L/4S^SS, as a monotelosomic addition, herein referred to as SEC, carrying the dominant male- fertility allele Ms-S^s1, a pollen-killer allele (ki-S'l-a) which is susceptible to the killing action of the dominant pollen-killer allele KI-B1, causing abortion of male gametes carrying it, and a selectable marker by which plants having this chromosome arm can be selected.

29. The plant of claim 28 selected from a Triticeae species.

30. The plant of claim 28, which is one of wheat, triticale, barely, rye, oat, rice, and maize.

31. 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 both for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B (4BS), and for the dominant pollen-killer KI-B1 allele on the long arm of chromosome 6B (6BL), said male parent being the maintainer line and being isogenic to the female parent and homozygous for the same ms-B1 and KI-B1 alleles of the female parent, and having an alien engineered chromosomal arm, as a monotelosomic addition, herein referred to as SEC, consisting of the long arm of chromosome 6S¹ and the short arm of chromosome 4S^S, carrying the dominant male-fertility allele Ms-S^s1, linked to a recessive allele ki-S'l-a which is susceptible to the killing action of the native pollen-killer allele KI-B1, and a selectable marker either heterologous gene or native allele, by which plants having this chromosomal arm can be selected. 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 SEC stable engineered chromosomal arm, said seeds, when grown, developing into said male-sterile female line.

32. 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 the maintainer line being isogenic to the female parent being a male-sterile plant homozygous both for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B, and for the dominant pollen-killer KI-B1 allele on the long arm of chromosome 6B, and having an alien engineered chromosomal arm, as a monotelosomic addition, herein referred to as SEC, consisting of the long arm of chromosome 6S¹ and the short arm of chromosome 4S^S, carrying the dominant male-fertility allele Ms- S^s1, linked to a recessive allele ki-S'l-a which is susceptible to the killing action of the native pollen-killer allele KI-B1 and a selectable marker either heterologous gene or native allele, by which plants having this chromosomal arm can be selected, and (b) selecting from the progeny, by using the selectable marker those about 80% of the progeny lacking the stable alien engineered chromosomal arm, said selected progeny being the female line.

33. A method according to claims 31 or 32, wherein said alien chromosome arm, herein designated SEC, carries as a selectable marker the heterologous Bar gene conferring resistance to the herbicide glufosinate (Basta), controlled by a constitutive promoter.

34. A method according to claim 31 or 32, wherein said alien chromosome arm, hereindesignated SEC, carries as a selectable marker a heterologous seed color gene (Sc) coding for anthocyanin in the aleurone layer of the endosperm, controlled by aleuron-specific promoter.

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

(a) selfing the maintainer line being isogenic to the female parent being a male-sterile plant homozygous both for any one of the recessive ms-B1 male-sterility alleles on the short arm of chromosome 4B, and for the dominant pollen-killer KΪ-B1 allele on the long arm of chromosome 6B, and having an alien engineered chromosomal arm, as a monotelosomic addition, herein referred to as SEC, consisting of the long arm of chromosome 6S¹ and the short arm of chromosome 4S^S, carrying the dominant male-fertility allele Ms-S^s1, linked to a recessive allele ki-S'l-a which is susceptible to the killing action of the native pollen-killer allele Ki- B1 and a selectable marker either heterologous gene or native allele, by which plants having this chromosomal arm can be selected, and (b) selecting from the progeny, by using the selectable marker those about 20% of the progeny having the stable alien engineered chromosomal arm, said selected progeny being the maintainer line.

36. A method according to claims 35, wherein said alien chromosome arm, herein designated SEC, carries as a selectable marker the heterologous Bar gene conferring resistance to the herbicide glufosinate (Basta), controlled by a constitutive promoter.

37. A method according to claim 35, wherein said alien chromosome arm, herein designated SEC, carries as a selectable marker a heterologous seed color gene (Sc) coding for anthocyanin in the aleurone layer of the endosperm, controlled by aleuron-specific promoter.