Note: Descriptions are shown in the official language in which they were submitted.
WO92/01~6 2 ~ 8 7 ~ 2 7 PCT/AU91/~319
PRODUCTION OF HYBRID CEREAL CROPS
T~CHNICAL FIELD
This invention relates to the production of hybrids of
cereal crops, particularly, but not eYclusively, small
grained cereals such as wheat and barley. In addition, the
invention concerns new genetically transformed plants, for
use with this method.
BACKGROUND ART
The traditional manner of producing hybrid plants
involves manual emasculation of the female parent, so that
self-pollination is not posible, which is planted proximate
the fertile male parent. This procedure is only practical
when it is possible to remove the pollen bearing structures
from the female parent. In many species, however, the
flowers are so insignificant in size that manual
emasculation of the female parent of an intended cross is
simply impracticable. This is particularly so with
small-grained cereals such as wheat, barley, rice and grass
species.
When physical removal of the anthers from the femzle
parent is impracticable, sterility may be induced by
treatment of the plant with a chemical hybridising agent
~CHA) which inhibits synthesis of viable pollen. Compared
with physical emasculation, CHA treatment is somehwat
inefficient and a certain amount of self-pollination occurs
which results in the harvested hybrid seed containing some
seed of the female parent. The male parent is normally
grown in clearly indentifiable rows or bloc~s and are simply
not harvested: the male parents thus present no problem to
hybrid purity. As a generalisation then, the seed hybrids
which are produced using CHAs t ad to be contaminated with
seed of the female parent an. separation of this rogue
contaminant is virtually impossible.
A useful alternative procedure which is available to
plant breeders utilises the phenomenon of cytoplasmic male
sterility (CMS). This type of male sterility arises from
WO92/0l~ PCT~AU91/00319
2087~27 - 2 -
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 espression 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.
An alternative to the CMS system has been proposed
[Dris~oll, G.J.; Crop Science 12, 516-517 (1972)~ and is
known as the XYZ system,. This system avoids the
introduction of alien genetic or cytoplasmic material in the
final product seed. The XYZ system is itself an extension
of an even earlier proposal described by Ramage R.T.; Crop
Science 177-178 (1965). The XYZ system is illustrated in
Figure 1 of the accompanying drawings. The system employes
(1) a homozygous genically sterile female parent (the Z
line);
(2) a Y line which is isogenic with æ line but
possesses an additional unpaired chromosome carrying a male
fertility restorer gene; and
(3) an X line which is identical with the Y line but
has two doses of the restorer gene.
Initially the Z and X lines are crossed to produce the
Y line, Then the Y line is crossed with the Z line giving
progeny of which 80% are of the Z line genotype and 20% of
the Y-type genotype. This mixed population is then used in
a cross with any desirable male fertile line to produce the
commercial Fl hybrids. The principal advantage of the XYZ
2087~27
O92/01~6 PCT/AU91/00319
-- 3
system is that the alien restorer chromosome is eliminated
by physical separation and does not apper in the commercial
hybrid.
In Driscoll's original proposal for hybrid wheat
production by the XYZ system, the alien restorer chromosome
which was selected was chromosome 5R from cereal rye, Secale
cereal L.. Chromose 5R from some rye cultivars was
demonstrated to produce normal male fertility when
substituted for chromosome 4A in bread wheat, Triticum
aestivum ~.. Male sterility in the XYz system is the
product of simply inherited recessive mutation on the short
arm of chromosome 4A (4As). The advantage of using
chromosome 5R of a gene restorer chromosome was the presence
on SR of a gene producing a hairy peduncle (Hp) which allows
identification of any plants carrying the alien restorer
chromosome to be identified visually. However, as will be
appreciated, this is only a suitable marker when working
with small populations.
Driscoll subseguently proposed a modified XYZ system
for wheat ~Modified XYZ System o Producing Hybrid Wheat~
Crop Science 25, 1115-1116 (1985)]. In this system it was
stipulated that the alien chromosome was to be an
isochromosome. For the purpose of the present invention
this modification is not of any particular relevance.
An object of the present invention is to obviate or
mitigate the aforesaid disadvantages.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the scheme of the Driscoll xYz system,
as discussed previously.
Figure 2 shows an aspect of the system of the present
invention using an addition plant line.
Figure 3 shows another ~spect of the system of the
present invention using a substitution line and a 3-way
crG~, system.
Figure 4 shows another aspect of the system of the
present invention using a substitution line and a single
cross.
WO92/01366 2 0 ~ 7 ~ 2 7 Pcr/Augl/003l9
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DISCLOSURE OF INVENTION
According to the present invention there is provided a
method for the maintenance of a male sterile parental plant
line or use in the production of hybrids, comprising
crossing a homozygous male sterile plant, representing the
female parent, with a male parent which is isogenic to the
female but having a chromosome bearing a dominant male
fertility gene and a marker gene which confers a
characteristic colouration on 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 chromosome bearing the dominant male fertility
gene, may be an additional gene, so that for example, in
wheat the female has 43 chromosomes, or else may be a
substitution, whereby the female has the usual number of
chromosomes; for esample, 42 for wheat.
Separation of the mised population of progeny seed may
there~ore be effected using commercially available
seed-sorting machinery which is capable of colour
discrimination.
The present invention is particularly applicable to the
production of hybrid wheat and barley. However, it will be
appreciated that the method of the invention may be applied
to any plant species for which the appropriate starting
materials exist or may be created. Other such species
include rice, maize and grass species.
It is preferred that the colour marker be the blue
aleurone marker gene available on chromosome 4 of A~roDYron
elonaatum (4Ag). The male parent used in this invention is
suitably a translocation of 4Ag to the restorer arm of
chromosome 4 of Triticum thaoudar (4th) and chromosome 4 of
Tricicum monococcum (4m), both of which are diploid wheats
or to the restorer arm of chromosome 4 of Triticum urartu.
Blue markers are known also to e~ist within barley
germplasm, as do the necessary genetic male sterility
genes. United States Patent Number 3,710,511 (Patterson)
shows that a range of suitable stocks are known to be
WO92/01~ 2 ~ ~ 7 ~ ~ 7 PCT/AU91/00319
-- 5 --
available for other crop species and are described in the
literature.
The present invention may be viewed as a modification
of the XYZ system of Driscoll. Driscoll's proposal to use
rye chromosome SR effectively limited the size of the Z
populations. This is because of the need for intense
roguing to remove plants carrying the hairy peduncle:
similar roguing would be necessary in the commercial
produstion phase.
In comparison, the present invention utilises a
distinctive colour marker such as the blue aleurone gene
from Aaro~vron elonaatum L. (4Ag). ~owever, it has
previously been shown that chromosome 4Ag substitutions were
vigorous but male sterile. This indicates that 4Ag lacks the
gene for male fertility restoration, necessary to restore
the Z line in Driscoll's XYZ system, which has been
confirmed in recent tests.
However, a translocation between 4Ag and the restorer
arm of alien restorers Triticum monococcum (4m), ~riticum
thaouda~ ~4th) or Triticum urartu ~4u) have been located and
have been ound to carry both the blue aleurone marker (BL)
and a male fertility restorer (IMS). Crossing of the
translocation into a male sterile line provides a suitable
material for use in the present invention.
In addition, propagation of the addition plant lines
can give rise to a substitution line, particularly after
self-pollination for several generations. The substitution
possibly occurs by centric fusion or Robertsonian
t ~nslocation, for e~ample, by the substitution of one arm
f om a A3ro~vron elonaatum L. chromosome with one arm from
T. monococcum or T. thaoudar.
The substitution plant line has some further advantages
in comparison to the addition line. In the first system
described above, the nuclear ma' sterile (NMS) system is
based on an addition line (43 chromosomes, while bread wheat
normally carries 42 chromosomes). However, an addition line
may have some disadvantages particularly with seed
production. When an addition line (in which the 43rd
W092/01~ PCT/AU91/~319
2B~7 ~27 6 -
chromosome carries the blue aleurone marker(s) and the male
fertility restorer) self-pollinates the progeny will be
approximately 72% non-blue and male sterile. Of the remnant
27% will be a monosomic addition, ie, 43 chromosomes (one
do~e of blue), and 1% will be homozygous blue, ie, 44
chromosomes. Through colour sorting, the 28% blue progeny
can be separated. In spite of this, the rate of increase in
seed volumes is quite slow when using an addition line.
However, while this is still satisfactory, it can be
improved upon, since calculations using a substitute line
show a more favourable result. A substitution line consists
of the normal 42 chromosome complement o bread wheat, for
esample, in which a wheat chromosome is replaced by a
non-bread wheat chromosome. This means that the segregation
ratio will approYimate the Mendelian ration of 3:1, and so
approsimately 75% will be blue, and 2S% non-blue.
MODES FOR CARRYING OUT THE INVENTION
The present invention will now be described, by way of
illustration, with reference to the following E~amples.
~a~ Production o Z and Y Lines
A nuclear male sterile line of variety PROBUS was
backcrossed into a variety of cultivars to introduce the
male sterile recessive genotype thereto. This provided a
series of lines constituting the Z line for use in the
breeding scheme of this invention. The cultivars were:
HARRIER, SUNECA, VASCO, TORRES, SUNSTAR, BANKS, S~UA,
AROONA, W LCAN AND TORDO.
The Y line required for this esample was created as
follows: Chinese Spring Wheat was crossed with Aaro~vron
elonaatum or another alien species such as Triticum
thaoudar, Triticum monococcum or Triticum urartu and haploid
plants selected from amongst the progeny. The haploids were
treated with colchicine to induce chromosome doubling. 8y
this method there were obtained the following addition lines:
Chromosome 4 - Triticum urartu in male sterile Chinese
Spring Wheat;
Chromosome 4 - Triticum monococcum in male sterile
Chinese Spring Wheat; and,
WO92/01~6 2 0 8 7 ~ 2 7 PCT/AU91/00319
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Chromosome 4 - Triticum thaoudar in male sterile
Chinese Spring Wheat.
These doubled haploid plants were then self-pollinated
or, alternatively, backcrossed to one of the Z line wheats
described above and plants bearing single alien additions
wère selected.
These lines were then crossed with Aqro~vron containing
a blue aleurone gene in order to create centric fusions
which carry both the male fertility gene and the blue marker.
These then provide the Y lines which are made up of the
Z lines plus the restorer/blue chromosomes.
(b~ Production of Hvbrids
The breeding scheme of the present invention is
illustrated schematically in Figure 2 of the drawings
herewith.
The nuclear male sterile recessive Z-line of wheat,
having the genotype ms.ms was crossed with a fertile
maintainer line (designated the Y line), isogenic with the
sterile line (ms.ms) but containing one alien chromosome
capable of restoring fertility and carrying a blue aleurone
colour marker derived from ~gropyron elonaatum (genotype
Ms.c).
- What was obtained from that cross was a misture of
normal (white) and blue coloured seed which had resulted
from imperfect transmission of the alien addition
chromosome, The normally coloured seed was genetically
identical with the Z line and the blue colour served as a
marker of those seeds containing the alien addition
chromosome, that is, the Y line.
The harvested seed was delivered to a commercial seed
sorting machine which was tuned to discriminate between the
differently coloured seed and to separate them physically.
Using a Sortes 5000 (Trade Mark) seed sorting apparatus, the
seed was separated into 35% white and 65~ blue seed.
The separated Y line was effectively recycled to the
first step of production and the bulk of the product, the Z
line, was used in production of fertile Fl hybrids by
crossing with any selected normal female parent.
WO92/01~6 PCT/AU91/00319
2~87~27 - 8 -
EXAMP~E 2.
TRANSLOCATION INVOLVING CHROMOSOME 4 OF AGROPYRON
ELONGATUM AND CHROMOSOME 4B OF BREAD WHEAT
A translocation (4Ag/4B) was crossed into a male
sterile Vulcan background. Vulcan is an Australian wheat
~ultivar, and was converted to male sterility by
backcrossing the recessive male sterile gene PROBUS into it.
From this point 2 different procedures were followed:
l. The progeny of blue seed was bulked and sent to
be colour sorted. The result of this mechanical sorting
(using a SORTEX 5000 sorter) was 65% blue seed and 35% white
or non-blue seed. The purity of the non-blue seed was quite
satisfactory.
2. In a separate experiment, single plants were
identified as heterozygous at the blue locus. These were
harvested and blue and white seed were sorted by hand to
determine the segregation ratio. In individual heads, the
proportion of blue seeds ranged from 45~ to 85%. However,
the average proportion of blue seed per plant was
appro~imately 65~.
~ XAMP~E 3.
TRANSMISSION RATES INVOLVING THE TRANSLOCATION CF24.
The translocation, CF24 was produced by crossing a
wheat line carrying chromosome 4 of Aaro~vron elonaatum
(blue seed) to a wheat line carrying chromosome 4 of
Triticum monococcum (blue seed). Progeny were selected for
male fertility and intensity of the blue aleurone. Fertile
plants were crossed to male sterile Vulcan and male sterile
Skua (Skua is an Australian wheat cultivar, male sterility
was produced as described above.)
Segregation ratios in small populations indicated that
plants heterozygous at the blue locus would produce
approximately 70% blue seed and 30% non-blue seed. In
classical Mendelian studies, the dominant character - in
this case the blue aleurone - is e~pected to be espressed in
75% of progeny (hence a 3:l ratio of dominant to
recessive). However, the observed ratio of 7:3 and 13:7
(65:35) in E~ample 2, simply indicate that the
W092/0l~6 2 0 ~ 7 ~ 2 7 PCT/AU9l/00319
g
translocations are not normally transmitted, through the
gametes.
EXAMPLE 4,
TRANSMISSION RATES INVOLVING THE TRANSLOCATION CF22.
The translocation CF22, was produced using the same
p~ocess as described in E~ample 3. However, while chromosome
4 of Aaro~vron elonaatum is included, male fertility
restoration is provided by the short arm of chromosome 4 of
Triticum monococcum (in the initial cross only the short arm
of chromosome 4 of T. monococcum was present, ie, it was a
telocentric addition).
In small populations, the transmission rate of the
translocation chromosome CF22 appears to be 70%. A line
designated 89-130(I) is a cross between male sterile Skua
and CF22. ~his line has constantly produce. very good blue
seed and white seed in a 3:1 ratio in small populations.
EXAMP~E 5.
TRANSMISSION RATES INVOLVING THE TRANSLOCATION CF30.
The translocation CF30 was produced in the same manner
as CF22 and carries the same components; i.e. blue aleurone
is provided by chromosome 4 of Agro~Yron elonaatum and male
fertility is provided by the short arm of chromosome 4 of
T. monococcum. So the translocation is espected to be
4Ag/4mt, as in CF22.
In small populations, the blue marker was transmitted
through 33% of progeny from a self pollinated plant,
heterozygous for CF30. This result can be e~plained by the
different components of the translocation. The amount of
chromatin present in the translocation from 4Ag and 4mt will
effect the transmission of the translocation through both
male and female gametes.
EXAMPL~ 6.
COMPARISON OF ADDITION AND SUBSTITUTION PLANT LINES
A calculation can be made that compares the seed
production in accordance with this invention, using the
addition and substitution schemes. The calculation assumes
that 1 plant produces 100 seeds, that 4000 seeds weiqh 1 kg,
and that 35kg/ha of seed produces 1 tonne of wheat grain
wo 92/0l~2 ~ 8 7 ~ 2 ~ - 1 o PCT/AU91/00319
l. Using an addition plant line, and the system
shown in Figure 2, if it is assumed that the male
transmission rate is 10%, and the female transmission rate
is 20%, then after 6 generations of an addition line of
hybrid wheat, the result will be about 77 tonne of
heterozygous blue seed, and 213 tonne of white seed.
2. In comparison, using a substitution plant line,
and the system shown in Figure 3 or 4, for e2ample, if it is
assumed that the male transmission rate is 40%, and the
female transmission rate is 50~, then after 6 generations of
a substitution line of hybrid wheat, the result will be
about 3906 tonne of heterozygous blue seed, and 2343 tonne
of white seed.
EXAMPLE 7.
PRODUCTION OF A SU8STITUTION LINE
A substitution line was created by the following
procedure. A dark blue 43 chromosome line was created
following the procedure described in part ~a) of E~ample l.
These plants were self poliinated for 2 generations. Then
they were crossed with male-sterile plants. The fertility
of the resulting progeny and the segregation ratios for the
blue allerone were checked. Those that had a classic
Mendelian ratio were selected, as indicating a substitution
line. Most likely, a centric fusion or Robertsonian
translocation was responsible.
