Note: Descriptions are shown in the official language in which they were submitted.
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~
WO93/13649 PCT/AU93/00017
-- 3
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
W093/13649 4 PCT/AU93/OU01
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~
W093/13649 PCT/AU93/00017
-- 5 --
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
-- 7 --
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
-- 8
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:
TA LE 1
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
, ..
WO 93/13649 PCI /AU93/000
-- 10 --
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
-- 11 --
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
- 13 -
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
- 15 -
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
- 17 -
EXAMPLE 6
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~
- 18 -
~ 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.
EXAMPLE 8
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
-- 19 --
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
- 20 -
~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
- 21 -
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.
