CA2108666A1 - Genetically modified wheat plants and progeny and method for production of hybrid wheat - Google Patents

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

2 ~ ~86fi6 WO 93/13649 P~/AU93/00017 GENETI CALLY MODI FI ED WHEA~ PLANq~S AND PROGENY
;~ND METHOD FOR PRODUCTI ON OF HYBRI D WHEAT

TECHNI CAL FI ELD
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 hy~rid wheat using the~e plants. The genetiaally al~ered wheat plant utilize homoeologous pairing to add a fertility restorer chromosome which contains telomeric chxomatin which is homologous to another wheat chromosome which bears a recessive male sterility gene. ~he fertility restorer chromosome contains a major portion of alien chromatin from other wheat species, and also contalns a male fertility restorer gene, and one or mors marker genes. These plants possess meiotic 6tability, an; are used to produce hybrid wheat ~trains.

BACRGROUND ART
The production of hybrid crops carrying ~elacted desirable ch~racteristics is o enormous commercial and economical importance to both individual farmer and to the agricultural industry as a whole. One way of potentially obtaining hybrid crops expre6sing both "X" and "Y"
characteristics ("XY") is to grow rows of "X" plants intersper~ed by rows of "Y" plants and attempt to ~nsure that only pollen from "X" plants pollinated "Y" plant6, or vice vsrsa. Such a procedure does not p~rmit the production of "XY" hybrids with any degree of certainty due mainly to the bisexual nature of crop plants which generally favours 6 elf-pollination.
Over the years, this problem ha~ been addres6ed with varying success by the u6e of plants rendered male ~terile.
Traditionally, this manner of producing hybrid plants involves manual emasculation of ~n intend~d female parent, to prevent 6elf-pollination and which is planted proximate the fertile male parent. ~his procedure is only practical 21 08~6~
Wo93/13~9 PCT/AU93/0001 when the pollen bearing structures are readily remo~able.
In many species, however, the flowers are so insigni~icant 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 6ynthesis of viable pollen.
Compared with physical ema~culation, CHA treatment is somewhat inefficient and a certain amount of self-pollination still occurs. ~he 5 eed hybri ds whi ch are produced using CHAs therefore tend to be contaminated with seed o~ the emale parent with separation of th~
contaminant, if no~ imposæible, being very costly.
An alternative procedure available to plant breeders utilises the phenomenon of cytoplasmic male sterility (CMS). Thi6 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 expre6sion of the ma}e sterility characteri~tic encoded in the cytoplasm and, therefore, yield6 mala fartile progeny. Therefore, the progeny ~till retain the ~ale 6te.rility 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 S~ates Patent Number 2,753,663 which descxibes 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 presenoe in plant cell nuclei of a gene directing expxession of the 6terility trait. The nuclear 21 08~

male sterility genes are generally o the rece6sive type, referred to as ~ms~ he presence o~ 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 c:an only have a ms/ms genotype.
Nuclear male sterility has been exploited in production of hybrid crops. One ~ystem, described by Driscoll, C.J.~
Crop Science 12; 516-517, 1972, i8 known as the "XYZ system".
In~ernational Patent Application ~o. PCT/AU91/00319 ~WO 92/01366) describes an im~3rovement to the X~Z system by providing a method for the maintenance of a male sterils parental plant comprising crossing a homozygous male sterile plant, representing the fPmale parent~ with a male parent which is isogenic to the female but having a chromosome bearing a domina~t male fertility gene and a marker gene which oonfers a characteristic colouration of the progeny seed, harvestlng from that cross a population of progeny seed con6isti~g of a mixture o~ the two parental lines and physically separating the progeny seed on the basis of the colour marker. The invention of PCT/AU91/0031g describes a genetically modified plant for use in this method which is created using spontaneous or i~duced translocations between homologous chromosomes. More precisely these plant were modified by means o~ centric fusion or tra~slocated chromosome which combin0d a ~ale fertility gene and a colour marker, for example, a blue aleurone marker. The translocation was produced when alien addition chromosomes or substitution linos were combined.
In work leading to the present in~ention, a high pairing ~utant was u~ed 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.

21~66 BRIEF DESCRIPTION OF ~HE DRAWINGS
Figure l shows two chromosomes of the wheat in accordance with the invention.
Figure 2 shows a pxocedure by which ~enetically modified plants may ~e 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.
Pigure 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 or 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 ahromatin, said homoeologous chromosome having a dominant male fertility restorer gene and one or more selectable marker genes, ~nd 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) - 4EBl - 4BBl]
Another aspect of the invention is a genetically modified precursor plant (and its progeny) for creating the prsviou~ plant which is characterised by containing a chromosome bearing a suppressor of pairing (Ph) gene whioh i6 not functioning, and which carries one or more genes for male sterility (ms), male ~ertility restorer genes (~Ms) or marker gene~ such as blue seed colouring or increased plant height.
A further aspect of the invention concerns the use of the genetically mod~fied wheat plant to maintain a male st~rile parental plant line, as gensrally described in ~1 08~fi~

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 terile 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 6eparating 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 chromo60me has a dominant male fertility restorer gene and one or more selectable marker genes, and the homoeologous chromosome having bee~ 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 ctarting wheat plant for the genetic alterations described in the invention.
The monosomic wheat chromosome i 5 preferable chromosome 4B, although other chromosome6 may be used instead, for example in mutant straius of wheat or in other wheats ~ith di f f erent chromosome nu~bers, other chromosomes may be suitable for use in accordance with the present in~ention.
The alien wheat chromatin pre~erably comes from chromosome~ in ~gE3a~E~ lonqatum, ~ trichoPhorum, Triticum thaoudar or T. monococcum. The ~E~Y~ 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 WO93Jl3~9 PCT/AU93~0001 generally described in PCT/AU91/00319. A oombination or mixture of chromatin from T. thaoudar and Ag. elon~atum oan 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 ~he b:Lue aleurone gene that confers a blue colouring to seed, and a plant height gene that confers extra height to progeny plants.
The more prefsrred ways to ensure th~t the supressor of pairing gene(s) (Ph) in wheat is not functioning ls to use the high pairing mutant gene (~hlb), or else an apparent high pairi~g supressor gene found in T. ~E~ for example. However, other ways can be used instead.
The invention is now described in more detail.
In the late 1950~s it ~as discovered how chro~osome pairing is genetically controlled in wheat. Wheat, which is of the genus Triticum is (at least for commercially significant species) hexaploid or tetraploid. Hexaploid wheat species normally have a chromosome number ot~ 42, designated as chromosomes tl-7)AABBDD, tetraploid wheat species normally have a ohromosome number of 28, designated (1-7)AABB, and diploid wheat species have a ohromosome number usually of 14, ~hich 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 prsvent chromosome 4A pairing with chromosome 4B, instead of with 4A, its homologous chromosome, for in~tance. It i8 possible to interfere with the pairing suppressor genss in hexaploid wheat for exam~le, to prevent them functioning normally, and to aause homoeologous pairing. This i8 used in the present invention to produce ~heat 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 ~everal ohromosomes, and in wheat .' ~ U ~
W093/13~9 PCT/AU93/00017 the major 6upressor or pairing (Phl) is located on chromosome 5B, and another suppressor of pairing (Ph2) occurs on chromosome 3D. In terms of the pressnt invention, the suppressor of pairing gene (Ph) is no~-functioning by being (a) mutated, so that it no longer suppresses homoeologous pairing such as with the ~ mutant phlb, or (b) by itself being suppressed, such as with strains of Ae. or (T.) speltoides, or Ae. or (T.) mutica, ~hich 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 uppress the 6uppressor o pairing, such as from T. peltoides, for exampl e.
In one preferred a6pect of the invention, the selectable marker is a colour marker (such as the blue aleurone gene), or el B e may be a height marker, or a oombination o~ a colour and height markers. However other markers may be used, especially those producing an easily visible phy~iological variation on the plant. The presen~
invention in one pre~erred form utilizes the blue aleuron3 marker gene available on chromosome 4 of A~ pyron elonqatum, Ag. trichophorum, Triticum thaoudar or T.
monococcum ~9Es~æ~EZ~ elonqatum is also sometimes known as Thinopyron elonqatum. A range o~ other suitable markers could aleo be used affecting colour, texture, ~ize, weight or other physically identifiable characteristics. All such markers are encompassed by the pre~ent invention.
Some o~ the advantages o~ the instant invention concern the rapidity with which a male sterile population can be produced because cross-pollination i~ 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 PC~/AU9l/003l9, which are essentially created by Robertsonian translocation or ce~tric fusion.

~ l V~
W093/13649 : PCT/AU93/0001 Homoeologous recombinants in accordanae 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 wAeat genome. However, in the presence of the high pairing mutant (~hlb), or with T. speltoldes, 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 ~ertility restorer gene.
The high pairing mutant, ~ , is known to be a recessive mutant which occurs on the long arm of chromosome SB (designated " 5BL" ). In the normal situation, pairing occurs only between homologues. Therefore, at meiosis, chromosome 5B will only pair a~d recombine with chromosome 5B; æimilarly, chromosome 3A will only recombine with chromosome 3A.
Wheat i8 a dlploid species and a segmented hexap].oid 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 homozygou~ condition tie, ~hlb.~ ), 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 pr~sent, 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 4~.
When both chromosomes from a pair are ab~ent, the plant is described as nullisomic (i.e. carrying 40 chromosomes~.

WO 93tl3649 ;~ I ~ 8 ~ ~ ~ Pcr/Au93/000l7 g When a nullisomic plant ~s self-pollinated, the proge~y only carry 40 chromosomes. However, when a monosomic plant self-pollinates the progsny are of 3 types, with predictable frequencies:

Male Parent n=21 n=20 56% 4%

Female n=21 2n=42 2n=41 Parent 25% ~4% 1%

n=20 2n=41 2n=40 75~ 72% 3%

The above table represents average frequencies.
However, the important faotor i8 that a monosomlc plant yields approximately 73% monosomic 9 and 3 % nulli 8 omic 8;
also of importance is the transmission rate of the monosomic through the female gamete t75%).
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 monosomlc for chromosome 5B and therefore hemizygous for Phlb (ie, 1 dose). ~he plant may be used as a male parent in future crosses as a donor of phlb.
Therefore, in aocordance with the preæent invention, if the male parent of a crocs is known to carry the ~hlb mutant arld the female parent is mono80mic for ohromosome 5B, 75% of the progeny ~ould have the opportunity to exhibi~
homoeologoug recombinatlon at meiosis.
Figure 1 shows in general form the homoeologous chromosomes in the plant according to the invention. By using a non-func~ionlng supprassor of pairing ~Ph), uch as mutant ~hlb on chromoæome 5B, sr T_ s~eltoides which caxries a suppressor of Phl, a wheat plant can be created which has ~l VO ~U V
, ..

a wheat 4B chromosome having a (reces~ive) male sterility gene (ms). Its homoeologue, of which the majority of chromatin is from an alien species, and wpich 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).
~he 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=nullisomic (ie, lacking a pair of chromosomes).

MODES FOR CARRYING OUT THE INVENTION
EXAMP~E 1 This example describes homoeologous recombination applied to the nuclear male sterile hybrid wheat system A~.
elongatum. This contains the blue aleurone marker gene on chromosome 4E. The steps in creat~ng the desired plant are ~et 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 Aq. ~ , this being an alien chromosome the chromatin o~ which (except for the 4B long arm telomere) is to incorporated in the resulting wheat strain. These strai~s are both readily available. The progeny arising from this step is then crossed with a wheat plant with two doses of the ~lb high pairing mutant. The progeny of this cro R i8 then cros~ed again with the phlb mutant, the resulting progeny ending up with two doses of phlb. Alternatively, the pla~t from step 3 aan 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.

5 ~
WO93/13649 PCT~AU93/00017 In the next step, the wheat with either one or two doses 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 ~3elected, being 4B(ms) ~ 4E - 4B ~ 20~l plants. These are male sterile, becauæe while the male sterility (ms) gene is recessive, the homoeologous chromosome (4EL - 4BL) lacks the dominant gene on 4BS(+Ms).
The procedure can then continue with further cros~es with plants which carry the male sterility gene (ms), Por example, to get a desired product.

~XAMP~B 2 This example concerns Homoeologou6 recombination applied to the nuclear male 8 terile hybrid wheat system.
The steps in creating the desired plant are ~et out in Figure 3 of the drawings.
The components and steps required to create an alien recombinant chromosome are pre~ented below. The critical steps are 3 and 4.
Step 3 creates material which is homoæygous for phlb.
Therefore, these plants will exhibit homoeologous recombination. ~hus, recombination between chromosome 4th (or 4m) and the telomeric region of chromosome 4BL is expected to occur.
~ he cross made in step 4 combines, or rapeat~ step 3 and introduces chromo~ome 4 from A~roPy-ron elongatum, which carries a very e~ective blue aleurone marker. When the pr~geny from step 4 are screened, blue seed, which produce fertile plants are expected to carry the desired recombinant chromosome.
In the Figure, "4th" ie the 4thaoudar homoeologue from a ~. thaoudar ~ubstitution line containing a blue aleurone marker, and a height marker. This strain i6 obtai~ed in th~
man~er described in PCT/AU91/00319. It al80 has a dominant male fertility restorer gene (~Ms~. This strain lacks a 2~ ~86~6 Wo93/13649 - 12 - PCT/AU93/000 normal 4B chromosome, and in place has an alien chromo~ome 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 i6 readily available in seed banks, and is crossed with a strain monosomic in chromosomes 4B and 5B. The progeny strain, m4th.n4B.m5B, i5 selected by its marker characteristics, namely height and blue seed, and has one alien 4th chromosome, one 4B chromosome and one SB
chromosome. This chromosome configuration arises from the nature of chromosome transmission in monosomics, as described in Table l above.
In 6tep 2, the progeny from step l is crossed with a strain that is monosomic for chromosomes 4B and SB, but its remaining SB chromosome has the high pairing mutation ~phlb). The resulting marked progeny contain one ohromosome with the high pairing mutant.
In step 3, this is crossed with a strain that has two doses of the high pairing mutant (~ ), and which is male sterile; this strain obviously bæing the female parent. The progeny of this cross, has two doses of the (recessive) high pairing mutant phlb, and when this plant is cros~ed with a ~train of wheat with the high pairing mutant and an extra chromosome(s) of 4 ~q. elonqatum, homoeologous recombination occurs to giv2 the desired genetically stable wheat, which can be used in hybrid production. Blue fertile seed are selected to verify the desired genes are linked.

EXAMP~E 3 ~ his 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.

2 ~ 5 6 W093/1~649 PCT/AU93/00017 Step 3 creates material which i6 homozygous for phlb.
Therefore, these plants will exhibit homoeologous recombination. Thus, recombination between chromo~ome 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 Aqro~yron ~ , which carries a very ef~ective blue aleurone marker. When the progeny from step 4 are screened, blue seed, which produce fertile plants are expected to caxry the desired recombinant chromosome.
In the Figure, ~4m~' is 4monococcum, which is a substitution line containing a blue aleurone marker, and a height marker. This strain i8 obtained in the manner described in PCT/AU9l/003l9. It also has a dominant male fertility restorer gene (+Ms). ~his strain lacks a normal 4B chromosome, and in plaae has an alien chromosome containing the marker gene6 and the ~Ms ge~e. It is possible to utilize 4thaoudar with the same characteristics in place of 4monococcum.
In step l, the substitution 4monococcum is the male parent (having the fertility restorer gene IMs 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 6~ed, and has one alien 4m chromosome, usually one 4B chromosome and one SB chromosome. This chromosome configuration arises from the nature of chromosQme transmission in monosomics, as described in Table l above.
In step 2, the progeny from step l is crossed with a strai~ that is monosomi~ for chromosomes 4B and SB, but its remainl~g 5B chromosome has the high pairing mutation ~phlb). The resulting ~arked progeny contain one chromosome with the high pairing mutant.
In step 3, this is crossed with a strain that has one dose of the high pairing mutant (phlb), and which is male : , ,, 2108fi6~ `.
W093/13~9 PCT/~U93tOOOl sterile as the plant is monosomic for 4B(ms) ~o that the male sterility gene is expressed; this strain being the female parent. ~he 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 chromosomle of 4 A~
elonqatum, homoeologou recombination occurs to give the desired genetically stable wheat, which can be used in hybrid production. Further cross~s can ble made to obtain a aesired plant line for carrying out the procedure described in PCT/AU91/00319, if nece~sary, and if desired.

~XAMP~E 4 This involves the utilisation of an alien chromo~ome which appears to carry both the blue aleurone marker and male fertility gene.
Cermeno and Zeller (1986) found an alien chromosome sub6titution for chromosome 4B in the European wheat cultivars "Brunn" and "Moskau". The alien chromosome also carried a blue aleurone gene. The faat that this ~ubstitute6 for chromosome 4B indicates that it also carries the male fertility gene. Cermeno and Zeller tl988) also found that this alien chromosome does not pair with chromosome 4B. In order to utilise this alien chromosome it i8 therefore necessary to recombinQ the telomeric region of chromosome 4BL onto it. ~his will enhanae 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 49L on the fertility restorer chromo ome ensures that pairing will occur at meiosis. A rod bivalent forms at meiosis as only homologous regions pair. ~his contrasts with other homologues within ths cell which form ring bivalents.
Without regular pairing, both the alien chromo ome and ohromo~ome 4B are not regularly transmittsd through the gametes.

2~85~6 W093tl3649 PCT/AU93/~0017 Unfortunately Cermeno and Zeller were unable to identify the alien chromosome. It i6 non-homologous with an Agxopyron ~5~ s ubs ti tuti on. This suggests that the alien chromosome may be a different Aqro~y~ species, or a diploid wheat. If the former is true then the transer of telomeric 4BL to this alien chromosome would produce the necessary recombinant.

~XANP~E 5 Although the method described in Example 4 will produce the des~red recombinant alien chromosome, a more e~icient method does exist. This method involves crossing the critical chromosomes into high pairing Aeqilops speltoides.
High pairing Ae~ilops speltoides induces homoeologous recombination, and thus will induce recombination between the chromoRomes 4 Aqro~yron elon~atum / 4T. monococcum or _ thaoudar / telomeric region of 4BL.
This will produce a recombinant chromosome:
20" ~ 4B(ms) ~ ~4m (or 4th) - 4EBl - 4BBl]
The 4ELl and 4BLl chromatin each carry a blue aleurone marker gene (Bl).
More details of the production of a recombinant chromosome for the nuclear male ~terile hybrid wheat system by this method follows.
Techniques have been described in PCT/AU9l/00319 which will produce alien reco~binant chromosomes which carry a blue aleurone genetS) and a male fertility restorer gene, which compensate~ 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 de~cribed (see International Patent Application No. PCT/AU9l/00319).
In accordance with the present invention, the phlb mutant or an equivalent method is u6ed to induce homoeologues recombination.

2108fi~ `
WO93/13649 - l6 - PCT/AU93/0001 A further technique designed to induce homoeologous recombination involves the use of the alien wheat species Triticum s~eltoides (Aegilops speltoides). Nhen high pairing strains of T. ~eltoides are cros~ed (as the male parent) to wheat, the Phl gene is inhibit~ed. 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 pxocedure beyins with 4thaoudar (4th), and in place of this other strains may be used, such as 4monococcum (4m).
In step l, 4thaoudar (or 4monococcum) is crossed with an addition line of Aq. elonqatum, which has 43 chromosomes. The progeny (Fl) is then crossed with the high pairing species T. speltoides, which is a monoploid species, and homoeologous pairing occurs in the offspring.
~he critical Fl is 20I + 4th ~4E +4B ~7S. It is expected that this genotype will occur at low fre~uencies.
In step 3 the critical Fl (20I ~ 4th + 4E + 4Æ) is crossed with +MS.ms (or ms.msli) which is male fertile; note that ms.ms+i i~ 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 Ae~ilops mutica (Triticum trip6acoides~.
Another factor is the survival of the critical F1 and the initial interspecific hybrid ie, endosperm failure and abortion may occur. To circumvent this problem embryo rescue may be required. ~his is described in more detail in Example 9.

~, l qU ~ b b W093/13~9 PCT/AU93/OOOt7 In accordance with the previously described Examples the followi~g plant lines were created.

Reference No. _ _ Cross~Pediqree 9lSUl32 m4B.m5B/4thsub 9lSUl34 m4B.m5B/4thsub 91SU136 m4B. m5B/4thsub 91SU138 N5B. T5D/4msub 9lSUl50 m4B. m5B/4thsub 9lSUl53 m4B. m5B/ 4ms ub 92SU1029 N5B. T5D/9lSU1// phlb Blue seed o~ this cross has 40 chromosomes 92SUl204 m4B. m5B/4Ag tricyh//phlb Blue seed has 4l chromosomes, another seed had only 39 seed 92SU1 l97 m4B.mSB/9lSUl//92SU598 Dark blue s eed ha~ 41 chromosomes .. : ., : .

fi ~ 6 W093/13649 PCTtAU93tOOO~

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

Table 2 Plant Heiqht of Different Chromosome 4B_Substitutions Post-A t_~sls Ide~tiflcatlon Heiqht (cm) % Marin~ Tall 1. Maringa Rht3 53 50 2. Maringa Rht1 87 84 3. Maringa Tall 104 100 4. 92SU729~ 135 130 5. 92SU730B 135 130 6. ISR388(4m(4B) sub) 130 127 The short arm of 4m i5 known to carry the critical fertility restorer, if the height gene is present, also on this arm, male fertility wlll be initially identified by blue seed, and secondly by taller plants.

Crosses between chromosome 4 of Aqropyron elo~qatum and chromo ome 4 of T. monococcum and T. thaoudar are listed below:

Reerence No. Pediqree , 91SU 188 GFS X91.58:91SU69/9lSU28 91SU 189 GFS X91.134:91SU50/9lSU1 91SU 190 GFS X91.166:91SU28/9lSU22 91SU 191 GFS X91.133:91SU17/9lSU10 91SU 192 GFS X91.172:BLUE2/9lSU50 91SU 193 GFS X91.175:g1SU28/9lSU22 91SU 193 GFS X91.175:91SU28/9lSU22 91SU 194 GFS X91.75:BLUE3/4thsub W093/13649 ~ Q 8 fi 6 6 PCT/AU93/00017 Reference No. Pedi~ree 91SU 195 GFS X91.15:378.002/9lSU9 91SU 196 GFS 891.18:378.,006/9lSU26 91SU 197 GFS X91.79:4t:hsub~91SU34 91SU 198 GFS X91.16:378.00~/9lSU9 91SU 199 GFS X91.78:4t,hsub/9lSUl 91SU 200 GFS X91.116:91SUl/9lSU10 91SU 201 GFS X91.56:9:LSU22/378.006 91SU 202 GFS X91.91:9LSUl/378.006 91SU 203 GFS X91.123:91SU31/91SU23 91SU 204 GFS X91.lOOA:9lSU34/BLUE3 91SU 205 GFS X91.77:BBUE3/9lSU34 91SU 206 GFS X91.15:378.002/9lSU9 91SU 207 GFS X91.50:378.001/9lSU53 91SU 208 GFS X91.49:378.001/BLUE3 91SU 209 GFS X91.113:91SU34/9lSUl 91SU 210 GFS X91.87:91SUl/9lSU34 91SU 211 GFS X91.87:91SUl/9lSU34 91SU 212 GFS X91.74:91SUl/9lSU53 91SU 213 GFS X91.17:378.006/9lSU26 91SU 214 ~PS X91.85:378.007/9lSU34 91SU 215 GPS X91.135:91SU50~fiter)/9lSU28 91SU 216 GFS X91.115:91SUl/4thsub 91SU 217 GFS X91.93:BLUE3/9lSUl 91SU 218 GFS X91.157:91SU69/9lSU22 91SU 219 GFS X91.160:91SU69/9lSSU52 91SU 220 GFS X91.167:91SU28/9lSU34 91SU 221 GFS X91.159:91SU~69/9lSUl 91SU 222 GFS X91.161:91SU69/BLUE3 91SU 223 GFS X91.163:B~UE2/9lSU6g 91SU 224 GFS X91.15:BLU~2/9lSU50 915U 225 GFS X91.73:91SUl/4thsub No~e: 91SUl is a 4A~ blue addition line 4th sub is a 4T. thaoudar substitutio~ e Blue 2 and 3 are 4A~ ~ blue sub6titutions 2 1 ~ ' 6 Wo93/13649 PCT/AU93/0001 ~XAMP~ 9 The haploid hybrids, 20I I 4E ~ 4B + 4th(or E or m) ~ 7S have been created, these are sterile; and are being backcrossed, as females to common wheat. ~he operations invol~ed with this that are often difficult to perform are:
(i) producing the abovementioned hybrid, and then (ii) producing seed on the haploid hybrids by backcrossing with common wheat pollen.
To overcome these difficultie 6, ~he following methods can be used.
Embryos produced through the described procedur~s are routinely rescued by culturing the 15-20 day old ~mbryos in special media, until a plantlet is produced. A brief ~ummary 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 endosper~;
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 smbryo rescue grow successfully.
The embryo rescue media used in this procedure is prepared as follows:

1. Autoclave:
a. lOOml fla~ks/container~
b. w~ter ~distilled) c. or media preparation:
(i) regeneration containers (ii) filter sterilizer (liquid) d. petri dishes in groups of 4 e. lL ~lasks WO93/l3~9 210 ~ ~ 5 ~ PCT/AU93/000l7 2. MS med_a preparatlon a. 4.48g/L MS powder b. 30g/L sucrose c. 2.7g/L Agarose d. 2mg/L glycine (stock) e. supplementary vitamins (s toc k ) f. pH=5.5 Procedure 1. Pour 500 ml of sterile distilled H2O(st.dH20) into beaker. Weigh 4.4g of MS powder, and 810wly pour into H o and mix by stirring. Powder must be completely dlssolved;
2. weigh 30g of ~ucrose and dissolve in 150ml H20;
3. weigh 2.7g of agarose and add H20 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 preeipitation doec not occur;
6. adjust pH to 5.5 u ing 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 oool until solid; and 9. close container tightly. Wait 2 days to oheck contamination.

Claims (10)

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 l, 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 (ph1b), or by the presence of a gene which suppresses the functioning of the suppressor of pairing gene (ph1).

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 (ph1b).

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 suppressor 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 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;
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.