WO1993013649A1

WO1993013649A1 - Genetically modified wheat plants and progeny and method for production of hybrid wheat

Info

Publication number: WO1993013649A1
Application number: PCT/AU1993/000017
Authority: WO
Inventors: Geoffrey Frank Smart
Original assignee: Pacific Seeds Pty. Ltd.
Priority date: 1992-01-16
Filing date: 1993-01-18
Publication date: 1993-07-22
Also published as: CA2108666A1; EP0577794A1; EP0577794A4

Abstract

The invention concerns genetically modified wheat plants and the progeny arising these plants, and also a method of producing hybrid wheat using these plants. The genetically altered wheat utilizes homoeologous pairing to add a fertility restorer chromosome which contains telomeric chromatin which is homologous to another wheat chromosome which bears a recessive male sterility gene. The fertility restorer chromosome contains a major portion of alien chromatin from other wheat species, and also contains a male fertility restorer gene, and one or more marker genes. The genetic makeup of the wheat plant in one preferred form can be described as: 20'' + 4B(ms) + [4m (or 4th) - 4EL - 4BL]. These plants possess meiotic stability, and are used to produce hybrid wheat strains.

Description

GENETICALLY MODIFIED WHEAT PLANTS AND PROGENY AND METHOD FOR PRODUCTION OF HYBRID WHEAT

TECHNICAL FIELD

The present invention is directed generally to genetically modified wheat plants and to progeny arising from these plants, and also to a method of producing hybrid wheat using these plants. The genetically altered wheat plant utilize homoeologous pairing to add a fertility restorer chromosome which contains telomeric chromatin which is homologous to another wheat chromosome which bears a recessive male sterility gene. The fertility restorer chromosome contains a major portion of alien chromatin from other wheat species, and also contains a male fertility restorer gene, and one or more marker genes. These plants possess meiotiσ stability, and are used to produce hybrid wheat strains.

BACKGROUND ART

The production of hybrid crops carrying selected desirable characteristics is of enormous commercial and economical importance to both individual farmers and to the agricultural industry as a whole. One way of potentially obtaining hybrid crops expressing both "X" and "Y" characteristics ("XY" ) is to grow rows of "X" plants interspersed by rows of "Y" plants and attempt to ensure that only pollen from "X" plants pollinated "Y" plants, or vice versa. Such a procedure does not permit the production of "XY" hybrids with any degree of certainty due mainly to the bisexual nature of crop plants which generally favours self-pollination.

Over the years, this problem has been addressed with varying success by the use of plants rendered male sterile. Traditionally, this manner of producing hybrid plants involves manual emasculation of an intended female parent, to prevent self-pollination and which is planted proximate the fertile male parent. This procedure is only practical when the pollen bearing structures are readily removable. In many species, however, the flowers are so insignificant in size that manual emasculation is impractical. This is particularly so with small-grained cereals such as wheat, barley, rice and grass species.

Male sterility may also be induced by treatment of the intended female parent plants with a chemical hybridising agent (CHA) which inhibits synthesis of viable pollen. Compared with physical emasculation, CHA treatment is somewhat inefficient and a certain amount of self-pollination still occurs. The seed hybrids which are produced using CHAs therefore tend to be contaminated with seed of the female parent with separation of the contaminant, if not impossible, being very costly.

An alternative procedure available to plant breeders utilises the phenomenon of cytoplasmic male sterility (CMS). This type of male sterility arises from genetic material present in the cytoplasm of plant cells. It is rare for genetic information from the cytoplasm to be transferred via the pollen to the zygote during pollination, as the cytoplasm of the zygote arises almost exclusively from the female parent. When a plant carrying CMS is used as a female parent in a cross, the progeny all possess the CMS trait. In hybrid production, CMS inbred lines are crossed with pollinators which possess a nuclear encoded "restorer" gene which inhibits expression of the male sterility characteristic encoded in the cytoplasm and, therefore, yields male fertile progeny. Therefore, the progeny still retain the male sterility genetic material in the cytoplasm; expression is suppressed by the dominant male fertility gene in the nucleus. An example of CMS system will be found in United States Patent Number 2, 753, 663 which describes the production of hybrid maize by this method.

Another procedure which is available to plant breeders

utilises the phenomenon of "nuclear male sterility" occasioned by the presence in plant cell nuclei of a gene directing expression of the sterility trait. The nuclear male sterility genes are generally of the recessive type, referred to as " s". The presence of the normal dominant male fertility gene is referred to as "+Ms". Accordingly, the possible genotypes of a male fertile plant are +Ms/+Ms and +Ms/ms whereas a male sterile plant can only have a ms/ms genotype.

Nuclear male sterility has been exploited in production of hybrid crops. One system, described by Driscoll, C. J. , Crop Science 12; 516-517, 1972, is known as the "XYZ system".

International Patent Application No. PCT/AU91/00319 (WO 92/01366) describes an improvement to the XYZ system by providing a method for the maintenance of a male sterile parental plant comprising crossing a homozygous male sterile plant, representing the female parent, with a male parent which is isogeniσ to the female but having a chromosome bearing a dominant male fertility gene and a marker gene which confers a characteristic colouration of the progeny seed, harvesting from that cross a population of progeny seed consisting of a mixture of the two parental lines and physically separating the progeny seed on the basis of the colour marker. The invention of PCT/AU91/00319 describes a genetically modified plant for use in this method which is created using spontaneous or induced translocations between homologous chromosomes. More precisely these plant were modified by means of centric fusion or translocated chromosome which combined a male fertility gene and a colour marker, for example, a blue aleurone marker. The translocation was produced when alien addition chromosomes or substitution lines were combined.

In work leading to the present invention, a high pairing mutant was used to induce homoeologous recombination to facilitate the production of a translocation chromosome. Furthermore, a previously uncharacterised alien chromosome has been identified carrying both a male fertility gene and a marker. - -

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows two chromosomes of the wheat in accordance with the invention.

Figure 2 shows a procedure by which genetically modified plants may be produced.

Figure 3 shows another procedure by which genetically modified plants may be produced.

Figure 4 shows another procedure by which genetically modified plants may be produced.

Figure 5 shows another procedure by which genetically modified plants may be produced.

DISCLOSURE OF INVENTION

Accordingly, one aspect of the present invention provides a genetically modified plant and/or its progeny being monosomic for a wheat chromosome having a recessive male sterility gene. The plant has an additional homoeologous chromosome in which homologous chromatin is present on the long arm telomere, the rest of the chromatin arising from alien wheat chromatin, said homoeologous chromosome having a dominant male fertility restorer gene and one or more selectable marker genes, and which has been created using induced homoeologous pairing.

The genetic makeup of the wheat plant in one preferred form can be described as:

20" + 4B(ms) + [4m (or 4th) - 4Eg₁ - 4-B^] Another aspect of the invention is a genetically modified precursor plant (and its progeny) for creating the previous plant which is characterised by containing a chromosome bearing a suppressor of pairing (Ph) gene which is not functioning, and which carries one or more genes for male sterility (ms), male fertility restorer genes (+Ms) or marker genes such as blue seed colouring or increased plant height.

A further aspect of the invention concerns the use of the genetically modified wheat plant to maintain a male sterile parental plant line, as generally described in PCT/AU91/00319. More particularly, this aspect concerns a method for the maintenance of a male sterile parental plant line for use in the production of hybrids, which comprises crossing a female parent with a male parent, the female parent being a homozygous male sterile plant, and the male parent being isogenic to the female but having a fertility maintainer chromosome bearing a dominant male fertility restorer gene and a marker gene which confers a selectable characteristic on progeny. The method further includes harvesting from that cross a population of progeny consisting of a mixture of the two parental lines; and then physically separating the progeny on the basis of the presence of the marker. In accordance with this invention this method is characterised by the fertility maintainer chromosome being a homoeologous chromosome in which wheat chromatin is present on the long arm telomere, the rest of the chromatin arising from alien wheat chromatin, whereby the homoeologous chromosome has a dominant male fertility restorer gene and one or more selectable marker genes, and the homoeologous chromosome having been created using induced homoeologous pairing as described in more detail hereafter.

It is preferred that the wheat be tetraploid or hexaploid wheat, especially bread wheat. Any suitable wheat strain can be used as the starting wheat plant for the genetic alterations described in the invention.

The monosomic wheat chromosome is preferable chromosome 4B, although other chromosomes may be used instead, for example in mutant strains of wheat or in other wheats with different chromosome numbers, other chromosomes may be suitable for use in accordance with the present invention.

The alien wheat chromatin preferably comes from chromosomes in Agropyron elongatum, Ag. trichophorum, Triticum thaoudar or T^ monococcum. The Agropyron lines may contain a blue aleurone marker, and the T^ thaoudar and T. monococcum lines may contain a blue marker gene, perhaps a height marker gene, and a male fertility restore gene, as generally described in PCT/AU91/00319. A combination or mixture of chromatin from T^ thaoudar and Ag. elongatum can also be used, if desired. However, other wheat strains can be constructed or selected to contain the genes required in the invention.

The preferred marker genes are the blue aleurone gene that confers a blue colouring to seed, and a plant height gene that confers extra height to progeny plants.

The more preferred ways to ensure that the supressor of pairing gene(s) (Ph) in wheat is not functioning is to use the high pairing mutant gene (phlb), or else an apparent high pairing supressor gene found in ^ speltoides, for example. However, other ways can be used instead. The invention is now described in more detail. In the late 1950' s it was discovered how chromosome pairing is genetically controlled in wheat. Wheat, which is of the genus Tritiσum is (at least for commercially significant species) hexaploid or tetraploid. Hexaploid wheat species normally have a chromosome number of 42, designated as chromosomes (1-7)AABBDD, tetraploid wheat species normally have a chromosome number of 28, designated (1-7)AABB, and diploid wheat species have a chromosome number usually of 14, which are chromosomes 1-7 of any of A, B or D. Hexaploid or tetraploid species have evolved special genetic mechanisms to control pairing among homoeologous chromosomes, to prevent chromosome 4A pairing with chromosome 4B, instead of with 4A, its homologous chromosome, for instance. It is possible to interfere with the pairing suppressor genes in hexaploid wheat for example, to prevent them functioning normally, and to cause homoeologous pairing. This is used in the present invention to produce wheat that has more meiotic stability, and which has been genetically modified to serve in the system for producing hybrid wheat described in PCT/AU91/00319, for example.

Homoeologous pairing is known to be controlled by the interaction of genes on several chromosomes, and in wheat the major supressor or pairing (Phi) is located on chromosome 5B, and another suppressor of pairing (Ph2) occurs on chromosome 3D. In terms of the present invention, the suppressor of pairing gene (Ph) is non-functioning by being (a) mutated, so that it no longer suppresses homoeologous pairing such as with the p_h mutant phlb, or (b) by itself being suppressed, such as with strains of Ae. or (T. ) speltoides, or Ae. or ( ^) utica, which apparently contain genes that inhibit pairing, or (c) by being lacking in the plants (although such plants tend to be more difficult to work with). Preferably the invention utilizes the high pairing mutation (phlb), or genes that suppress the suppressor of pairing, such as from T^ speltoides, for example.

In one preferred aspect of the invention, the selectable marker is a colour marker (such as the blue aleurone gene), or else may be a height marker, or a combination of a colour and height markers. However other markers may be used, especially those producing an easily visible physiological variation on the plant. The present invention in one preferred form utilizes the blue aleurone marker gene available on chromosome 4 of Agropyron elongatum, Ag. trichophoru , Triticum thaoudar or T. monococcum. Agropyron elongatum is also sometimes known as Thinopyron elongatum. A range of other suitable markers could also be used affecting colour, texture, size, weight or other physically identifiable characteristics. All such markers are encompassed by the present invention.

Some of the advantages of the instant invention concern the rapidity with which a male sterile population can be produced because cross-pollination is not desired and the advantage that no special male fertility restorer is required for the male parent in the final hybrid cross.

The present invention provides improved plant lines for use in producing hybrid crops, over the plant lines described in PCT/AU91/00319, which are essentially created by Robertsonian translocation or centric fusion. Homoeologous recombinants in accordance with the present invention, will be more stable. In the homozygous condition pairing and recombination will take place between wheat chromosomes and their alien homoeologues.

In general, homoeologous recombination is a very rare event within the wheat genome. However, in the presence of the high pairing mutant (phlb), or with T_;_ speltoides, for example, recombination between wheat homoeologues and alien homoeologues does occur at much higher frequencies permitting the opportunity of detecting recombination between alien homoeologues carrying the blue aleurone marker(s) and the male fertility restorer gene.

The high pairing mutant, phlb, is known to be a recessive mutant which occurs on the long arm of chromosome 5B (designated "5BL"). In the normal situation, pairing occurs only between homologues. Therefore, at meiosis, chromosome 5B will only pair and recombine with chromosome 5B; similarly, chromosome 3A will only recombine with σhromosome 3A.

Wheat is a diploid species and a segmented hexaploid and when chromosomes are absent, compensation occurs. In accordance with the present invention, two methods exist whereby the high pairing mutant may be used to combine the desired marker(s) and male fertility restorer gene.

Firstly, when the high pairing mutant is in the homozygous condition (ie, phlb.phlb), homoeologous pairing will occur. Thus, as an example, chromosome 3A can pair and therefore can recombine with either chromosome 3B or 3D. When alien chromosomes are present, recombination can occur between an alien chromosome and wheat homoeologues (or possibly even between an alien chromosome and alien homoeologues).

Secondly, when a chromosome is absent from the wheat genome, the plant is described as monosomic for that chromosome, having 41 chromosomes instead of the usual 42. When both chromosomes from a pair are absent, the plant is described as nullisomic (i.e. carrying 40 chromosomes). When a nullisomic plant is self-pollinated, the progeny only carry 40 chromosomes. However, when a monosomic plant self-pollinates the progeny are of 3 types, with predictable frequencies:

TABLE 1

The above table represents average frequencies. However, the important factor is that a monosomic plant yields approximately 73% monosomiσs and 3% nullisomics; also of importance is the transmission rate of the monosomic through the female gamete (75%).

This means that when a plant that is phlb homozygous is crossed as a male to a female plant monosomic for chromosome 5B, 75% of the progeny are monosomic for chromosome 5B and therefore hemizygous for phlb (ie, 1 dose). The plant may be used as a male parent in future crosses as a donor of phlb.

Therefore, in accordance with the present invention, if the male parent of a cross is known to carry the phlb mutant and the female parent is monosomic for chromosome 5B, 75% of the progeny would have the opportunity to exhibit homoeologous recombination at meiosis.

Figure 1 shows in general form the homoeologous chromosomes in the plant according to the invention. By using a non-functioning suppressor of pairing (Ph), such as mutant phlb on chromosome 5B, or T^ speltoides which carries a suppressor of Phi, a wheat plant can be created which has a wheat 4B chromosome having a (recessive) male sterility gene (ms). Its homoeologue, of which the majority of chromatin is from an alien species, and which the 4B chromosome will pair with at meiosis, carries two marker genes, for blue seed colouring and for increased height, as well as a (dominant) male fertility restorer gene (+Ms). The alien chromosome however has some native 4B chromatin on its long arm telomere, which will cause the chromosome to pair with its homoeologue 4B chromosome.

Various methods for producing wheat having the general features shown in Figure 1 are now described.

In the Examples and Figures that follow, m=monosomic (ie, lacking one chromosome), and n=nullisomiσ (ie, lacking a pair of chromosomes).

MODES FOR CARRYING OUT THE INVENTION

EXAMPLE 1 This example describes homoeologous recombination applied to the nuclear male sterile hybrid wheat system Ag. elongatum. This contains the blue aleurone marker gene on chromosome 4E. The steps in creating the desired plant are set out in Figure 2 of the drawings.

In step 1 of Figure 2, a plant monosomic in chromosomes 4B and 5B is crossed with an addition line containing chromosome 4 of of Ag. elongatum, this being an alien chromosome the chromatin of which (except for the 4B long arm telomere) is to incorporated in the resulting wheat strain. These strains are both readily available. The progeny arising from this step is then crossed with a wheat plant with two doses of the phlb high pairing mutant. The progeny of this cross is then crossed again with the phlb mutant, the resulting progeny ending up with two doses of phlb. Alternatively, the plant from step 3 can be used directly in the next step, as the 5B(phlb) chromosome, while being recessive, is present in the plant as a singular chromosome, and will therefore be able to cause homoeologous recombination. In the next step, the wheat with either one or two άoses of the high pairing mutant is crossed with a plant being male sterile. Due to the presence of the high pairing mutant, homoeologous recombination occurs, and plants having blue seed and which are male sterile are selected, being 4B(ms) + 4E - 4B + 20" plants. These are male sterile, because while the male sterility (ms) gene is recessive, the homoeologous chromosome (4E - 4B ) lacks the dominant gene on 4B (+Ms).

The procedure can then continue with further crosses with plants which carry the male sterility gene (ms), for example, to get a desired product.

EXAMPLE 2

This example concerns Homoeologous recombination applied to the nuclear male sterile hybrid wheat system.

The steps in creating the desired plant are set out in Figure 3 of the drawings.

The components and steps required to create an alien recombinant chromosome are presented below. The critical steps are 3 and 4.

Step 3 creates material which is homozygous for phlb. Therefore, these plants will exhibit homoeologous recombination. Thus, recombination between chromosome 4th (or 4m) and the telomeric region of chromosome 4BL is expected to occur.

The cross made in step 4 combines, or repeats step 3 and introduces chromosome 4 from Agropyron elongatum, which carries a very effective blue aleurone marker. When the progeny from step 4 are screened, blue seed, which produce fertile plants are expected to carry the desired recombinant chromosome.

In the Figure, "4th" is the 4thaoudar homoeologue from a T^ thaoudar substitution line containing a blue aleurone marker, and a height marker. This strain is obtained in the manner described in PCT/AU91/00319. It also has a dominant male fertility restorer gene (+Ms). This strain lacks a normal 4B chromosome, and in place has an alien chromosome containing the marker genes and the +Ms gene. It is possible to utilize 4monococcum with the same characteristics in place of 4thaoudar.

In step 1, the substitution 4thaoudar is the male parent (having the fertility restorer gene +Ms as well as the marker genes), which is readily available in seed banks, and is crossed with a strain^'monosomic in chromosomes 4B and 5B. The progeny strain, m4th. n4B. m5B, is selected by its marker characteristics, namely height and blue seed, and has one alien 4th chromosome, one 4B chromosome and one 5B chromosome. This chromosome configuration arises from the nature of chromosome transmission in monosomics, as described in Table 1 above.

In step 2, the progeny from step 1 is crossed with a strain that is monosomic for chromosomes 4B and 5B, but its remaining 5B chromosome has the high pairing mutation (phlb). The resulting marked progeny contain one chromosome with the high pairing mutant.

In step 3, this is crossed with a strain that has two doses of the high pairing mutant (phlb), and which is male sterile; this strain obviously being the female parent. The progeny of this cross, has two doses of the (recessive) high pairing mutant phlb, and when this plant is crossed with a strain of wheat with the high pairing mutant and an extra chromosome(s) of 4 Ag. elongatum, homoeologous recombination occurs to give the desired genetically stable wheat, which can be used in hybrid production. Blue fertile seed are selected to verify the desired genes are linked.

EXAMPLE 3

This example describes an alternate method for producing the homoeologous recombined wheat plant, using the markers from T_;_ monococcum.

The steps in creating the desired plant are set out in Figure 4 of the drawings. Step 3 creates material which is homozygous for phlb. Therefore, these plants will exhibit homoeologous recombination. Thus, recombination between chromosome 4m and the telomeric region of chromosome 4BL is expected to occur.

In the Figure, "4m" is 4monococcum, which is a substitution line containing a blue aleurone marker, and a height marker. This strain is obtained in the manner described in PCT/AU91/00319. It also has a dominant male fertility restorer gene (+Ms). This strain lacks a normal 4B chromosome, and in place has an alien chromosome containing the marker genes and the +Ms gene. It is possible to utilize 4thaoudar with the same characteristics in place of 4monococcum.

In step 1, the substitution 4monococcum is the male parent (having the fertility restorer gene +Ms as well as the marker genes), which is readily available in seed banks, and is crossed with a strain monosomic in chromosomes 4B and 5B. The progeny strain, m4m. n4B. m5B, is selected by its marker characteristics, namely height and blue seed, and has one alien 4m chromosome, usually one 4B chromosome and one 5B chromosome. This chromosome configuration arises from the nature of chromosome transmission in monosomics, as described in Table 1 above.

In step 3, this is crossed with a strain that has one dose, of the high pairing mutant (phlb), and which is male sterile as the plant is monosomic for 4B(ms) so that the male sterility gene is expressed; this strain being the female parent. The progeny of this cross, has two doses of the (recessive) high pairing mutant phlb, and when this plant is crossed with a strain of wheat with the high pairing mutant and an extra two chromosome of 4 Ag. elongatum, homoeologous recombination occurs to give the desired genetically stable wheat, which can be used in hybrid production. Further crosses can be made to obtain a desired plant line for carrying out the procedure described in PCT/AU91/00319, if necessary, and if desired.

EXAMPLE 4 This involves the utilisation of an alien chromosome which appears to carry both the blue aleurone marker and male fertility gene.

Cermeno and Zeller (1986) found an alien chromosome substitution for chromosome 4B in the European wheat σultivars "Brunn" and "Moskau". The alien chromosome also carried a blue aleurone gene. The fact that this substitutes for chromosome 4B indicates that it also carries the male fertility gene. Cermeno and Zeller (1988) also found that this alien chromosome does not pair with chromosome 4B. In order to utilise this alien chromosome it is therefore necessary to recombine the telomeric region of chromosome 4BL onto it. This will enhance the regular transmission of this alien chromosome through the gametes. Without the telomeric 4BL, the alien chromosome would not pair with normal 4B, which carries the male sterile allele on its short arm. The presence of telomeric 4BL on the fertility restorer chromosome ensures that pairing will occur at meiosis. A rod bivalent forms at meiosis as only homologous regions pair. This contrasts with other homologues within the cell which form ring bivalents. Without regular pairing, both the alien chromosome and chromosome 4B are not regularly transmitted through the gametes. Unfortunately Cermeno and Zeller were unable to identify the alien chromosome. It is non-homologous with an Agropyron elongatum substitution. This suggests that the alien chromosome may be a different Agropyron species, or a diploid wheat. If the former is true then the transfer of telomeric 4BL to this alien chromosome would produce the necessary recombinant.

EXAMPLE 5 Although the method described in Example 4 will produce the desired recombinant alien chromosome, a more efficient method does exist. This method involves crossing the critical chromosomes into high pairing Aegilops speltoides. High pairing Aegilops speltoides induces homoeologous recombination, and thus will induce recombination between the chromosomes 4 Agropyron elongatum / 4T. monococcum or T. thaoudar / telomeric region of 4BL.

This will produce a recombinant chromosome: 20" + 4B(ms) + [4m (or 4th) - 4Eg₁ - ^_± ] The 4E , and 4B-. chromatin each carry a blue aleurone marker gene (Bl).

More details of the production of a recombinant chromosome for the nuclear male sterile hybrid wheat system by this method follows.

Techniques have been described in PCT/AU91/00319 which will produce alien recombinant chromosomes which carry a blue aleurone gene(s) and a male fertility restorer gene, which compensates for the terminal deletion on chromosome 4BS in the cornerstone mutant and probus mutant.

Irrespective of the method used the final product must be a chromosome which combines the abovementioned genetic factors.

So far, induced centric fusions have been described (see International Patent Application No. PCT/AU91/00319). In accordance with the present invention, the phlb mutant or an equivalent method is used to induce homoeologues recombination. A further technique designed to induce homoeologous recombination involves the use of the alien wheat species Triticum speltoides (Aegilops speltoides). When high pairing strains of T^ speltoides are crossed (as the male parent) to wheat, the Phi gene is inhibited. This is the same affect as with the phlb mutant, ie, homoeologous recombination is expected to occur.

The following described crosses are required, which are set ou in Figure 5 in the drawings. The procedure begins with 4thaoudar (4th), and in place of this other strains may be used, such as 4monococcum (4m).

In step 1, 4thaoudar (or 4monocoσcum) is crossed with an addition line of Ag. elongatum, which has 43 chromosomes. The progeny (F₁) is then crossed with the high pairing species T^ speltoides, which is a monoploid species, and homoeologous pairing occurs in the offspring. The critical F. is 20 + 4th +4E +4B +7S. It is expected that this genotype will occur at low frequencies.

In step 3 the critical F₁ (20 + 4th + 4E + 4B) is crossed with +MS. ms (or ms.ms+i) which is male fertile; note that ms.ms+i is a line homologous for male sterility carrying an isochromosome which confers male fertility. This line is used as a male donor of the ms gene because the isochromosome is transmitted through pollen at very low frequency.

In step 4 the blue seed and fertile plants are selected to give the desired wheat.

This procedure may be repeated using Aegilops mutica (Triticum tripsacoides).

Another factor is the survival of the critical F. and the initial interspecific hybrid ie, endosperm failure and abortion may occur. To circumvent this problem embryo rescue may be required. This is described in more detail in Example 9. EXAMPLE 6 In accordance with the previously described Examples the following plant lines were created.

Reference No. Cross/Pedi ree

91SU132 m4B. m5B/4thsub

91SU134 m4B. m5B/4thsub

91SU136 m4B. m5B/4thsub

91SU138 N5B. T5D/4msub

91SU150 m4B. m5B/4thsub

91SU153 m4B. m5B/4msub

92SU1029 N5B. T5D/91SU1// phlb

Blue seed of this cross has 40 chromosomes

92SU1204 m4B. πι5B/4Ag tricyh//phlb

Blue seed has 41 chromosomes, another seed had only 39 seed

92SU1197 m4B. m5B/91SUl//92SU598

Dark blue seed has 41 chromosomes

EXAMPLE 7 The chromosome 4 substitution from Triticum monococcum also carries a height marker (gene for tallness). This is demonstrated in the results shown of the following table.

Table 2 Plant Height of Different Chromosome 4B Substitutions

Post-Anthesis

% Marin a Tall

The short arm of 4m is known to carry the critical fertility restorer, if the height gene is present, also on this arm, male fertility will be initially identified by blue seed, and secondly by taller plants.

EXAMPLE 8 Crosses between chromosome 4 of Agropyron elongatum and chromosome 4 of T^ monococcum and T_;_ thaoudar are listed below:

Reference No. Pedigree

X91.58: 91SU69/91SU28 X91.134: 91SU50/91SU1 X91.166: 91SU28/91SU22 X91.133: 91SU17/91SU10 X91.172: BLUE2/91SU50 X91.175: 91SU28/91SU22 X91.175: 91SU28/91SU22

X91.75: BLUE3/4thsub Reference No. Pedigree

X91.15: 378.002/91SU9

S91.18: 378. , 006/91SU26

X91.79: 4thsub/91SU34

X91.16: 378.001/91SU9

X91.78: 4thsub/91SUl

X91.116: 91SU1/91SU10

X91.56: 91SU22/378.006

X91.91: 91SU1/378.006

X91.123: 91SU31/91SU23

X91.100A: 91SU34/BLUE3

X91.77: BLUE3/91SU34

X91.15: 378.002/91SU9

X91.50: 378.001/91SU53

X91.49: 378.001/BLUE3

X91.113: 91SU34/91SU1

X91.87: 91SU1/91SU34

X91.74: 91SU1/91SU53

X91.17: 378.006/91SU26

X91.85: 378.007/91SU34

X91.135: 91SU50(ster)/91SU28

X91.115: 9lSUl/4thsub

X91.93: BLUE3/91SU1

X91.157: 91SU69/91SU22

X91.160: 91SU69/91SSU52

X91.167: 91SU28/91SU34

X91.159: 91SU 69/91SU1

X91.161: 91SU69/BLUE3

X91.163: BLUE2/91SU69

X91.15: BLUE2/91SU50

X91.73: 91SUl/4thsub

Note: 91SU1 is a 4Ag blue addition line

4th sub is a 4T. thaoudar substitution line

Blue 2 and 3 are 4Ag trichophorum blue substitutions EXAMPLE 9 The haploid hybrids, 20 + 4E + 4B + 4th(or E or m) + 7S have been created, these are sterile; and are being backσrossed, as females to common wheat. The operations involved with this that are often difficult to perform are:

(i) producing the above entioned hybrid, and then (ii) producing seed on the haploid hybrids by backcrossing with common wheat pollen.

To overcome these difficulties, the following methods can be used.

Embryos produced through the described procedures are routinely rescued by σulturing the 15-20 day old embryos in special media, until a plantlet is produced. A brief summary of the procedure is described below.

The embryo culture methodology adopted involves the following steps: Step 1 : 15-20 days after crosspollination, excise seed from the inflorescence; Step 2 : wash seed 2 times in 6% sodium hypochlorite and sterile distilled water, and excise embryo from the endosperm; Step 3 : transfer embryo to a jar containing suitable media; Step 4 : wrap jar in foil, and leave at room temperature for 14 days; and Step 5 : transfer 2 leaf plantlet to plot. Plantlets derived from embryo rescue grow successfully.

The embryo rescue media used in this procedure is prepared as follows:

1. Autoclave: a. 100ml flasks/containers b. water (distilled) c. or media preparation:

(i) regeneration containers (ii) filter sterilizer (liquid) d. petri dishes in groups of 4 e. 1L flasks 2. MS media preparation a. 4.48g/L MS powder b. 30g/L sucrose c. 2.7g/L Agarose d. 2mg/L glycine (stock) e. supplementary vitamins (stock) f. pH=5.5

Procedure

1. Pour 500 ml of sterile distilled H₂0(st. dH,0) into beaker. Weigh 4.4g of MS powder, and slowly pour into

„ _Λ and mix by stirring. Powder must be completely dissolved;

2. weigh 30g of sucrose and dissolve in 150ml H^O;

3. weigh 2.7g of agarose and add H.O to make up to 300ml. Autoclave agarose before adding to medium;

4. add stock solutions (glycine and vitamins) to the media;

5. mix all solutions by stirring them. Ensure precipitation does not occur;

6. adjust pH to 5.5 using NaOH;

7. filter-sterilize the liquid media in laminar flow cabinet before mixing it with hot agarose solution;

8. pour media into individual containers and allow to cool until solid; and

9. close container tightly. Wait 2 days to check contamination.

Claims

THE CLAIMS:

1. A genetically modified wheat plant and/or its progeny being monosomic for a wheat chromosome having a recessive male sterility gene, and having an additional homoeologous chromosome in which homologous chromatin is present on the long arm telomere, the rest of the chromatin arising from alien wheat chromatin, said homoeologous chromosome having a dominant male fertility restorer gene and one or more selectable marker genes, and which has been created using induced homoeologous pairing.

2. The modified plant according to claim 1, which is tetraploid or hexaploid wheat.

3. The modified plant according to claim 1, wherein said monosomic chromosome is chromosome 4B(ms).

4. The modified plant according to claim 1, wherein said alien wheat chromatin is from chromosome 4Ag, 4E, 4m, or 4th (as herein defined).

5. The modified plant according to claim 1, wherein the selectable marker gene is a blue aleurone gene that confers a blue colouring to progeny seed, and/or a plant height gene that confers extra height to progeny plants.

6. The modified plant according to claim 1, wherein the homoeologous pairing has been induced by a non-functioning suppressor of pairing gene (Ph).

7. The modified plant according to claim 6, wherein the homoeologous pairing has been induced by the presence of a high pairing mutant gene (phlb), or by the presence of a gene which suppresses the functioning of the suppressor of pairing gene (phi).

8. A genetically modified precursor wheat plant (and its progeny) for creating the genitically modified plant defined in claim 1, said precursor plant characterised by containing at least one chromosome bearing a suppressor of pairing (Ph) gene which is not functioning, and further characterised in that at least one gene is present in said precursor plant that is selected from any one or more of: male sterility gene, male fertility restorer gene, marker gene.

9. The genetically modified precursor wheat plant according to claim 8, wherein said supressor of pairing gene which is not functioning is the high pairing mutant (phlb).

9. The genetically modified precursor wheat plant according to claim 8, wherein said supressor of pairing gene which is not functioning is not functioning because of the presence of a supressor or similar gene from T_;_ speltoides.

10. A method for the maintenance of a male sterile parental plant line for use in the production of hybrids, which comprises crossing a female parent with a male parent, said female parent being a homozygous male sterile plant, said male parent being isogeniσ to the female but having a fertility maintainer chromosome bearing a dominant male fertility restorer gene and a marker gene which confers a selectable characteristic on progeny; harvesting from that cross a population of progeny consisting of a mixture of the two parental lines; and physically separating the progeny on the basis of the presence of the marker; characterised on that said fertility maintainer chromosome is a homoeologous chromosome in which wheat chromatin is present on the long arm telomere, the rest of the chromatin arising from alien wheat chromatin, said homoeologous chromosome having a dominant male fertility restorer gene and one or more selectable marker genes, and said homoeologous chromosome having been created using induced homoeologous pairing.