Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR PRODUCING APOMICTIC PLANTS
CROSS-REFERENCE TO REhATED APPLICATIONS
This application claims the benefit of U.S.
Provisional Application No. E0/037,211, filed February
5, 1997 .
BACKGROUND OF Tl-iE INVENTION
This invention relates to methods for producing
plants that genetically clone themselves through their
own seed (gametophytic apomicts) from plants that
LO normally reproduce sexually. More particularly, the
invention relates to processes that include (a)
selection of two or more sexual lines that express
reproductive phenotypes divergent from each other, which
may in some cases require plant breeding and selection
to obtain sufficient degrees of divergence, (b)
hybridization among plants divergent in reproductive
phenotype, and (c) amphip~.oidization (doubling of
chromosomes) either before or after hybridization.
It is likely that apomix:is has a greater potential
for increasing yields of food, feed, and fiber than any
other plant mechanism. Apomixis occurs in about 0.30 of
flowering plant species. The present patent application
describes methods for making sexual plants apomictic
without crossing them to wi:Ld apomictic relatives or
using mutagenic procedures, both of which have been
attempted but with disappointing results. The
procedures described herein mimic how apomixis evolved
in nature (J.G. Carman, A~;ynchronous Expression of
Duplicate Genes in Angiosperms May Cause Apomixis,
Bispory, Tetraspory, and Polyembryony, 61 Biol. J.
Linnean 5oc. 51-94 (1997) (incorporated herein by
reference; hereinafter, "Linnean"), and enable persons
skilled in the art of plant breeding and genetics to
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2
convert inbred crops, including wheat, barley, and rice,
into apomictic hybrid crops with potential yield
increases of 10 to 30% over currently used inbred
varieties. Crops currently used as hybrids, such as
S maize, may also be made apomictic. Apomictic hybrids of
either inbred or typically hybrid crops will behave as
hybrids only in terms of their superior yields. The
seed of apomictic hybrids are genetic clones of the
mother hybrid, i.e. genetic segregation does not occur.
0 Thus, farmers could use a small fraction of their
harvest for seed and expect high yields and uniformity
year after year. This would allow hybrids, the seed of
which is typically very expensive, to be used in
impoverished nations for the first time, which could
contribute substantially to another "green revolution."
Gregor Mendel conducted, unknowingly, the first
genetic experiments with gametophytic apomicts (plants
that produce seed asexually). He reported the
successful crossing of different Hieracium lines but
0 commented on the extreme difficulty of preventing self
fertilization. What he thought was high frequency
accidental selfing (in his facultatively-apomictic
Hieracium lines) was actually high frequency apomictic
seed formation. To add to his frustration, Mendel
5 failed to observe segregation among the FZs of the few
Fls he managed to produce. His FZS were actually
apomictic clones of his Fls, and they invariably
expressed their respective F~ phenotypes (S.E. Asker & L.
Jerling, Apomixis in Plants (CRC Press, 1992) (hereby
incorporated by reference; hereinafter, "Asker &
Jerling").
Several thousand species of Hieracium had been
described by the time Mendel hybridized members of this
agamic complex. This pronounced polymorphy, and that
5 observed in other agamic complexes (Antennar.ia,
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Erigeron, Taraxacum, Potentilla) , coupled with Mendel's
results in producing new polymorphs by crossing
facultative apomicts, led early geneticists to conclude
that hybridization in agamic complexes is a major
mechanism of speciation. Evi~3ence for this conclusion
is replete, e.g. in Amelanchier and Crataegus (C. S.
Campbell & T.A. Dickinson, Apomixis, Patterns of
Morphological Variation, and Species Concepts in Subfam.
Maloideae (Rosaceae), 15 Systematic Bot. I24-25 (1990)
.0 (incorporated herein by reference), in Antennaria (Bayer
et al., Phylogenetic Inferences in . Antennaria
(Asteraceae: Gnaphalieae) Based on Sequences from the
Nuclear Ribosomal DNA Internal Transcribed Spacers
(ITS), 83 Amer. J. Bot. 516-527 (1996) (incorporated
.5 herein by reference), in numerous agamic grass complexes
(E.A. Kellogg, Variation and Species Limits in
Agamospermous Grasses, 15 Systematic Bot. 112-23 (1990)
(incorporated herein by reference), in Rebus (Nybom,
Evaluation of Interspecific Crossing Experiments in
'0 Facultatively Apomictic Blackberries (Rebus Subgen.
Rubes) Using DNA Fingerprinting, 122 Hereditas 57-65
(1995) (incorporated herein by reference), in Taraxacum
(Richards, The origin of Taraxacum agamospecies, 66
Biol. J. Linnean Soc. 189-211 (1973) (incorporated
?5 herein by reference), and others. In contrast, two
conflicting opinions soon developed among early
geneticists regarding the role of hybridization in the
origins of apomixis. Str~iusburger, Zeitpunkt der
Bestimmung des Geschlechtes, Apogamie, Parthenogenesis
30 and Reduktionsteilung, 7 Hist. Beitr. 1-124 (1909)
(incorporated herein by reference), Ostenfeld,
Experiments on the Origin of Species in the Genus
Hieracium (Apogamy and Hybridi;sm), 11 New Phytol. 347-54
(1912) (incorporated herein by reference), and Holmgren,
35 Zytologische Studien fiber die Fortpflanzung bei den
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Gattungen Erigeron and Eupatorium, 59 Kgl. Sven
Vetenskapsakad. Ak. Handl. No. 7, 1-118 (1919)
(incorporated herein by reference) believed apomixis is
controlled by genetic factors (genes) specific to
apomixis and is not a consequence of hybridization. In
contrast, A. Ernst, Bastardierung als Ursache der
Apogamie im Pflanzenreich, Fischer, Jena (1918)
(incorporated herein by reference), believed that the
cytological anomalies of reproduction responsible for
!0 apomixis are extensions of the genomic disturbances
observed in wide hybrids.
Ernst amassed much evidence to support his
hybridization hypothesis, which included the facts that
apomicts have high chromosome numbers (they are
L5 generally polyploid), that agamic complexes are highly
polymorphic, and that the sex cells of apomicts often
degenerate in a manner observed in interspecific
hybrids. A major tenet of Ernst's hypothesis, and the
one which soon caused its widespread dismissal (and
~0 continues to cause its legitimate dismissal today), was
that hybrids form a continuum from fully functional
sexual reproduction, to apomixis, and finally to
vegetative reproduction. Where a hybrid fit on the
continuum depended on how closely related the parent
~5 species are, e.g. if the parents are closely related,
the hybrid will reproduce sexually, if the parents are
moderately related, the hybrids may tend to be
apomictic, if the parents are distantly related, the
hybrids may tend to reproduce by vegetative propagation.
30 Thus, according to Ernst, apomixis arises only in wide
hybrids. Ernst did not identify mechanisms to support
a hybrid origin for apomixis other than the wideness of
the cross.
Ernst's hypothesis received support in the 1920s
35 and 1930s (Harrison, The Inheritance of Melanism in
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Hybrids Between Continental Tephrosia crepuscularia and
Britisht bistortata, with Some Remarks on the Origin of
Parthenogenesis in Interspecific Crosses, 9 Genetika
4467 (1927) (incorporated herein by reference); G.L.
5 Stebbins, Cytology of Antenna.ria. II. Parthenogenetic
Species, 94 Bot. Gaz. 322-45 (1.932) (incorporated herein
by reference)), but most geneticists had rejected it by
the time ~lke Gustafsson published his comprehensive
treatise, ~1 Gustafsson, Apomixis in Higher Plants, I.
LO The Mechanism of Apomixis, 42 Lunds Universitets
.~rsskrift 1-67 (1946); ~ Gustafsson, Apomixis in Higher
Plants, II. The Causal Aspect of Apomixis, 43 Lunds
Universitets Arsskrift 69-17~~, (1947); A Gustafsson,
Apomixis in Higher Plants, :CII. Biotype and Species
!5 Formation, 43 Lunds Universitets ~lrsskrift 181-370
(1947) (incorporated herein by reference). In this
treatise, Gustafsson concluded: "In no case is it proved
that hybridization itself has been able to produce
apomixis. On the contrary, it is certain that the
?0~ apomictic method of reproducaion has in many cases
arisen. within a species populat:ion." The fact that some
apomicts are autopolyploid, which was well documented by
1946, legitimately squelched any perceived requirement
for wide hybridization. Hf~nce, Ernst's hypothesis
35 collapsed because it claimeed that the cytological
mechanisms of apomixis are extensions of the cytological
abnormalities observed during gamete formation in wide
hybrids, which, by definition, contain grossly divergent
genomes that prevent normal chromosome pairing during
30 meiosis. We know today that this is not the case, i.e.
chromosome pairing in many apomicts is normal. Since
Gustafsson's treatise, few gE~neticists have suggested
that the role of hybridization in agamic complexes
exceeds that of speciation among taxa already containing
35 a genetically-determined predisposition for apomixis.
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6
Dissecting this genetic predisposition is being
attempted but is proving to be a formidable task.
Few genetic analyses of apomixis were conducted
prior to Gustafsson's treatise, and these lacked the
numbers of progeny required to draw specific conclusions
(Asker & Jerling). Nevertheless, they suggested to
Gustafsson that apomixis is caused by interbalanced
systems of recessive genes. Gustafsson defended this
view by citing examples in (a) Parthenium, Poa, and
Potent~lla, where embryo sac formation and
parthenogenesis are under independent genetic control,
and (b) Poa, Potentilla, and Rubus, where hybrids
between two apomicts or between an apomict and a sexual
parent are either sexual or apomictic with no clear
pattern as to the outcome (suggestive of many recessive
genes ) . In Rubus, sexual Fls had been produced from
apomictic parents, and these Fls produced a low
percentage of apomictic Fzs, which again suggested
segregation for multiple recessive factors.
Another realm of apomixis research that has
produced ambiguities involves the effects of
artificially changing the ploidy of apomicts. The
general trend is for apomixis to intensify when the
ploidy of an apomict is artificially increased.
However, exceptions in Potentilla, Taraxacum, Paspalum,
and Poa have been found in which artificially-induced
increases in ploidy cause (a) haploparthenogenesis, in
which reduced eggs form and develop without
fertilization, (b) BIII hybridization, in which unreduced
eggs are fertilized, and (c) complete restoration of
sexuality. Sexuality has also been restored in
apomictic Poa by haploidization. Concerning such
ambiguities, Asker and Jerling concluded: "Our
difficulties in explaining the 'breakdown of apomixis'
remain connected with our ignorance concerning [its]
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regulation . . . ." Such ambiguities persuaded
Gustaffson to reject simple dominance models for the
inheritance of apomixis. He saw little evidence for
them and was unconvinced by such claims in Dryopteris,
Hi eracium, Hypericum, Potenti.lla, and Sorbus. In each
case, Gustaffson provided reasonable alternatives for
the published claims.
Distorted segregation ratios can also hinder
genetic analyses of apomixi:~. Certain apomicts in
LO Dicanthium and Themeda tend to be sexual when grown in
long days and apomictic when grown in short days (Asker
& Jerling). Nevertheless, replicated studies with
consistent segregation ratios nave now been conducted in
Panicum (Asker & Jerling), Tripsacum (O. Leblanc et al.,
L5 Detection of the Apomictic Mode of Reproduction in
Maize-Tripsacum Hybrids Usin<~ Maize RFLP Markers, 90
Theor. Appl. Genet. 1198-1203 (1995) (incorporated
herein by reference), and Brachiaria (Valle & Miles,
Breeding of Apomictic Species, in Y. Savidan & J.G.
?0 Carman, Advances in Apomixis Research (FAO, Rome, in
press) (incorporated herein by reference), and each
study suggested that apomeiosis (detected cytologically)
is controlled by a single dominant allele. However,
other recent studies challenge this conclusion, e.g. the
35 apomeiosis "allele" in the Tripsacum accession studied
by O. Leblanc et al., 90 Theor. Appl. Genet. 1198-1203
(1995), is part of a large linkage group in which
recombination is suppressed, and a similar linkage group
appears to exist in apomictic Pennisetum (Grimanelli et
30 al., Molecular Genetics in .Apomixis Research, 'fin Y.
Savidan, J.G. Carman, Advances in Apomixis Research
(FAO, Rome, 1998) (in press, incorporated herein by
reference)). These linkage groups may contain multiple
genes required for apomeiosis (Grimanelli et al.,
35 Mapping Diplosporous Apomixis in Tetraploid Tripsacum:
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One Gene or Several Genes?, Heredity (1998) (in press,
incorporated herein by reference)).
Two research groups are presently attempting to
introgress apomixis into maize from Tripsacum, and
neither has reported its expression in addition lines
with less than nine Tripsacum chromosomes. In one
group, two apomictic maize triploids containing nine
Tripsacum chromosomes (3x+9) were produced. Cytogenetic
and molecular studies indicated that the nine Tripsacum
_0 chromosomes, in each line were probably the same (B.
Kindiger et al., Evaluation of Apomictic Reproduction in
a Set of 39 Chromosome Maize-Tripsacum Backcross
Hybrids, 36 Crop Sci. 1108-13 (1996) (incorporated
herein by reference)). A third triploid addition line,
_5 again with nine Tripsacum chromosomes (3x+9), was
produced by the same group. However, many of the nine
chromosomes in this line differed from the nine
chromosomes of the two former lines. The maize
chromosomes were the same for all three lines. The
'0. latter 3x+9 plant was also apomictic, but the frequency
of apomixis was only 10 to 150, compared with 95 to 1000
for the two former lines (5okolov et al, Perspectives of
Developing Apomixis in Maize, Priority Directions of
Genetics, Russia (1997) (progress report; incorporated
'S herein by reference)). These data, and unpublished
findings from the other group attempting to transfer
apomixis to maize (Grimanelli et al., Molecular Genetics
in Apomixis Research, ~n Y. Savidan, J.G. Carman,
Advances in Apomixis Research (FAO, Rome, 1998) (in
30 press)), suggest a complex mode of inheritance for
apomixis. In another study, sexual T. dactyloides
diploids were crossed with highly apomictic T.
dactyloides triploids to produce aneuploids. All but
three of 46 Fls showed tendencies for apomeiosis
35 (determined cytologically). However, the highly
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apomeiotic Fls contained seven or more additional
chromosomes (above the diploid level), and all Fls with
chromosome numbers near the diploid level were sexual
(Sherman et al., Apomixis in Diploid X Triploid
Tripsacum dactyloides Hybrids, 34 Genome 528-32 (1991)
(incorporated herein by reference)), which suggests
complex inheritance. Finally, apomixis in artificially
produced Tripsacum triploids cosegregated with five
Tripsacum linkage groups syntenic with regions from
three maize chromosomes (Blake:y et al, Co-segregation of
DNA Markers with Tripsacum Fei:tility, 42 Maydica 363-69
(1997) (incorporated herein by reference)), which
further discredits a simple inheritance mechanism for
the control of apomixis (at least when attempting to
transfer the apomixis mechanism to other species or
other lines within a specie:). It is reasonable to
assume that a major gene (a regulatory or controlling
gene) could prevent apomixis i=rom occurring when in the
recessive condition, thus making apomixis appear to be
under simple genetic control. However, such genes)
belong to many genes required for the expression of
apomixis and will not confer apomixis to other species
by themselves (Linnean). The studies just reviewed
infer: (a) multiple genes are required for apomixis, (b)
genes affecting facultativeness may behave additively,
(c) some Tripsacum chromosomes affect facultativeness
more than others, and (d) alleles from at least three
maize chromosomes fail to substitute for their
homeologous (syntenic) counterparts from Tripsacum in
conferring apomixis.
Meiotic mutants are central to the simple
inheritance hypotheses (Linnean). Recent mutation
hypotheses suggest apomixis is not expressed unless
appropriate meiotic mutations are combined with an
appropriate polygenic predisposition (Mogie, The
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Evolution of Asexual Reproduction in Plants (1992)
(incorporated herein by reference); Grimanelli et al,
Molecular Genetics in Apomixis Research, in Y. Savidan,
J.G. Carman, Advances in Apomixis Research (FAO, Rome,
5 1998) (in press)). However, recently obtained evidence
indicates that the alleles thought to be mutations are
actually part of the polygenic predisposition and are
required for sexual reproduction in marginal habitats.
What has not been previously appreciated, but which is
LO shown herein, is that it is the union of divergent
ecotypes (interracial or interspecific) through
secondary contacts that permits apomixis to arise (see
also Linnean).
In view of the foregoing, it will be appreciated
that providing methods for producing apomictic plants
would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide
methods for creating apomictic plants from sexual plants
?0 without using mutagenic procedures or plants that are
already apomictic.
Additional objects and advantages of the invention
are set forth in the detailed description or will be
appreciated by the practice of the invention.
To address the foregoing objects, and in accordance
with the invention as described herein, the present
invention provides methods for producing apomictic
plants from two or more sexual plants of the same or
related species.
One step of the method involves obtaining two
sexual lines whose female reproductive phenotypes differ
such that under the same environmental conditions (day
length, light intensities, temperature regimes, etc.) an
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11
appropriate degree of asynchrony in female developmental
schedules between the two lines occurs. Appropriate
degrees of asynchrony include but are not limited to
situations in which megasporogenesis in one line is
initiated at about the same tinne embryo sac formation is
initiated in the other line relative to the development
of nongametophytic ovule and ovary tissues (nucellus,
integuments, pericarp, etc) and other phenological
factors such as photoperiod-regulated floral induction
LO times. The accelerated line (.Line undergoing embryo sac
development) would have already accomplished floral
induction and megasporogenesi;~.
Another step of the method involves making
amphiploids of the appropriately-divergent sexual lines
(using standard chromosome doubling techniques), if they
are not already polyploid, and hybridizing the two
sexual amphiploid lines to induce apomixis.
Hybridization may precede amphiploidization. In such
cases, the amphiploidization may involve standard
?0 mitotic spindle inhibitors, such as colchicine, or rely
on the formation of Bm hybrids (fertilization of an
unreduced egg) to produce apomictic triploids. Other
processes are included in the examples described below.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIGS. lA-M show schematic representations of the
development of representative types of sexual (mixis)
and apomictic embryo sacs (see B.M. Johri et al.,
Comparative Embryology of Angiosperms, Vol. 1 and 2
(Springer-Verlag, 1992); Asker & Jerling). Polygonum-
type development is the norm and is expressed
exclusively in >99 0 of all angiosperm species. The
horizontal rows) of numbers associated with each
developmental type denote stage-specific gene
expression: (1) premeiotic int:erphase and early meiotic
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12
prophase (crossing-over and envelopment of MMCs in
callose), (2) meiotic divisions, (3) megaspore
maturation (digestion of callose from and initial
vacuolization of the surviving megaspore), (4) embryo
sac development, (5) double fertilization and early
endosperm formation, and (6) embryogenesis (usually
initiated after early endosperm formation). Parallel
rows of numbers indicate hypothesized asynchronous
expression of duplicate genes, the members of which
LO originate from different genomes in polyploids or
segments of different ancestral genomes in
paleopolyploids. Gaps in the numbers represent
mutations (mostly null alleles) and deletions. Note
that gaps are most prevalent among the paleopolyploid
L5 polysporic types and least prevalent among the polyploid
apomictic types. Some null-allele formation probably
enhances seed set in apomicts. The arbitrary
elimination of gene duplications in developmentally-
asynchronous polyploids and paleopolyploids appears to
?0 represent a major evolutionary process in which new
reproductive types (apomixis, polyspory, and
polyembryony) evolve (Linnean).
FIGS. 2A-B show examples of the geographic
distributions of two plant genera expected to provide
~5 adequate divergence in photoperiod responses and
reproductive schedules for producing apomictic plants:
(A) diploid sexual Antennaria aromatica (small triangles
in Montana), A. corymbosa (two circles by the Idaho
Montana border, one circle in Wyoming near the Montana
30 border, one circle in northern Colorado), A. marginata
(squares in New Mexico and Arizona), A, media/pulchella
(stars in California), A. microphylla (one circle in
Alberta, one circle near the border of Idaho, Montana,
and Wyoming, one circle in north central Wyoming, and
35 one circle in southern Colorado), A. racemosa (one
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13
circle in British Columbia, two circles near the Idaho
Montana border, and one circle in Montana near the
Wyoming border), A, rosulata (stars in Utah, Colorado,
Arizona, and New Mexico), A. umbrinella (large triangles
in Idaho, Montana, and Wyoming), A. friesiana (small
stars in Alaska), A, densifolia A. E. Porsild. (circles
in the Yukon), A. monocephala DC. (large stars in
Alaska, the Yukon, and the Northwest Territories), and
polyploid apomictic A. rosea (small squares); (B)
diploid sexual Tripsacum bracum Gray (large square in
Mexico), T. dactyloides (atars in the US), T.
dactyloides ~var hispidum (Hitchc. ) De Wet and Harlan
(large star in Mexico), T. latifolium Hitchc. (small
square in Mexico), T. laxum Nash (large circles in
Mexico), T. maizar Hernandez and Randolph (triangle in
Mexico), T. manusoroides de Wet and Harlan (small stars
in Mexico near the Gulf of Mex.ico), T. pilosum 5cribner
and Merrill (small circle's in Mexico), and T.
zopilotense Hernandez and Randolph (small stars in
~0 Mexico but not near the Gulf: of Mexico). Clones of
diploid sexual T. cundinamarce de Wet and Timothy, T.
australe var. australe Cutler and Anderson, and T.
dactyloides var. meridionale de Wet and Timothy are
obtainable from Columbia (near 15 N lot. 85 W long. )
and are not on the map.
FIG. 3 shows the developmental stage (premeiotic or
preapomeiotic, meiotic or apomeiotic, multinucleate
embryo sac) of pistils by pistil length for diploid
(sexual) and tetraploid (dip:losporous) T. zopilotense
and T. dactyloides. Pistil lengths (mm) were <0.50,
0.50-0.75, 0.75-1.00, 1.00-1.25, 1.25-1.50, 1.50-1.75,
1.75-2.00, 2.00-2.25, 2.25-2.5c), >2.5 for categories 1 -
10, respectively. Note that (a) the diploid T.
zopilotense proceeds through t:he three stages of female
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14
development more rapidly than the diploid T.
dactyloides, (b) the tetraploids begin apomeiosis sooner
than the diploids begin meiosis, and they proceed
through the three stages much more rapidly than the
diploids (meiosis is skipped in the tetraploids), and
(c) the diplosporous T. dactyloides proceeds through the
three stages more rapidly than the diplosporous T.
zopilotense (summarized from 0. Leblanc & Y. Savidan,
Timing of Megasporogenesis in. Tripsacum Species
LO (Poaceae) as Related to the Control of Apomixis and
Sexuality, 8 Polish Botanical Studies 75-81 (1994)
(incorporated herein by reference)).
FIG. 4 shows the distribution of 190 pistils from
sexual Elymus scabrus and 690 pistils from apomictic
Elymus rectisetus by stage of megasporogenesis (meiotic
or apomeiotic MMCs and mononucleate megaspores) or
embryo sac development (multinucleate embryo sacs) and
corresponding stage of pollen development (1 - PMC
meiosis II and tetrads; 2 = free microspores; 3 = pollen
?0 grains with incipient germ pore; 4 - vacuolate
uninucleate pollen prior to formation of Ubisch
granules; 5 - early Ubisch granule formation to early
endothecial wall thickening; 6 - endothecial wall
thickening; 7 = uninucleate pollen without subdivision;
35 8 = pollen with dark-stained single nucleus; 9 = pollen
with faint-stained single nucleus; 10 - binucleate
pollen). Bars within stages of female development and
above pollen stage designations represent cumulative
percentages of the total number of pistils sampled up to
30 the given pollen stage (cumulative across previous
pollen stages). Few MMCs were found among sexual
pistils after stage three. Thus, bars in this category
are nearly equal thereafter. In contrast, bars in the
sexual mononucleate megaspore category continue to
35 increase in length from stage three through 10, which
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indicates that most pistils in these stages had advanced
beyond the MMC stage. At stage 10, two of three sexual
pistils contained multinucleate embryo sacs. Among
apomictic pistils, binucleate embryo sacs were first
5 observed at pollen stage three with a large percentage
of multinucleate embryo sacs (>2) occurring by pollen
stage six (modified from C.F. Crane & J.G. Carman,
Mechanisms of Apomixis in Elymus rectisetus from Eastern
Australia and New Zealand, 74 Amer. J. Bot. 477-96
10 (1987) (incorporated herein by reference)).
DETAILED DESCRIPTION
Before the present methods for producing apomictic
plants are disclosed and described, it is to be
understood that this invention is not limited to the
15 particular configurations, process steps, and materials
disclosed herein as such configurations, process steps,
and materials may vary somewhat. It is also to be
understood that the terminology employed herein is used
for the purpose of describing particular embodiments
only and is not intended to be limiting since the scope
of the present invention will be limited only by the
appended claims and equivalents thereof.
It must be noted that, as used in this
specification and the appended claims, the singular
forms "a," "an," and "the" include plural referents
unless the context clearly dictates otherwise.
In describing and claiming the present invention,
the following terminology will be used in accordance
with the definitions set out below.
Historically, the term apomixis, when applied to
flowering plants (angiosperm.s), has included various
forms of vegetative propagation. The term apomixis is
limited herein, however, to those processes routinely
resulting in asexual reproduction without conjunction of
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16
gametes of opposites sexes (parthenogenesis) from
unreduced eggs. These processes are frequently
described as forms of gametophytic apomixis and involve
anomalies that disrupt normal ovule development. In
contrast, bispory and tetraspory (collectively referred
to as polyspory herein) also disrupt ovule development,
but normal, reduced eggs that require fertilization are
formed (FIGS. 1J-M). Cytological similarities between
apomixis and polyspory have recently been identified
LO (U. S. Serial No. 60/037,211; see also Linnean). These
similarities suggest commonalities in the origins of
apomixis and-polyspory.
Megasporogenesis (female meiosis) in angiosperms
occurs in megaspore mother cells (MMCs), and the female
.5 gametophyte (embryo sac) normally develops from one of
four meiotic products (megaspores). Most angiospermous
embryo sacs are eight-nucleate (FIG. 1A) and contain an
egg (fertilized to form the embryo), two synergid cells,
a central cell composed of two polar nuclei (fertilized
?0 to form the endosperm), and three antipodal cells
(Polygonum-type embryo sac). Each nucleus is originally
haploid and is derived from the nucleus of the surviving
megaspore through three sequential endomitotic divisions
(FIG. lA). Apomixis interrupts this process, resulting
25 in diplospory (FIGS. 1C-F) or apospory (FIGS. 1G-H).
In the form of apomixis known as diplospory, an
unreduced embryo sac forms from a MMC in which meiosis
is disturbed and replaced by precocious embryo sac
formation (FIGS. 1C-F). The completion of embryo sac
30 formation is also precocious, and the unreduced egg
divides parthenogenetically to form a proembryo, often
prio r to fertilization (e.g., Linnean). The form of
apomixis known as apospory is similar to diplospory
except the unreduced embryo sac forms from a somatic
35 cell of the ovule wall near the MMC (FIGS. 1G-H).
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During apospory, either the meiotic MMC or the young
sexual embryo sac aborts and i.s replaced by one or more
aposporic embryo sacs (Asker & Jerling), which also tend
to develop precociously (Linnean; Asker & Jerling).
Hence, both of these anomalies result from the premature
expression of genes required for embryo sac formation,
and most if not all apomicts are facultatively sexual,
which means sexuality has been retained during their
evolution and is occasionally expressed (Linnean; Asker
LO & Jerling) .
Tetraspory, in which meiotic nuclear divisions
(karyokineses) occur but cell divisions (cytokineses) do
not (FIGS. 1I-K; M.T.M. Willemse & J.L. van Went, The
Female Gametophyte, 'tar, B.M. Johri, Embryology of
'5 Angiosperms 159-96 (Springer-Verlag, 1984), incorporated
herein by reference; B.M. ~ohri et al, Comparative
Embryology of Angiosperms, Vol. 1 and 2 (Springer-
Verlag, 1992) (incorporated herein by reference)), has
also been attributed to a "precocious gametophytization"
?0 of the MMC (E. Battaglia, ThE~ Evolution of the Female
Gametophyte of Angiosperms: ~~n Interpretative Key, 47
Annali di Botanica 7-144 (1989), incorporated herein by
reference)). Both tetrasporic and diplosporic MMCs
undergo vacuolization, which normally occurs in the
35 surviving sexual megaspore during the onset of embryo
sac formation. Such vacuolization also occurs in
aposporous initials (Linnean). In both diplospory and
tetraspory, embryo sac formation continues without
interruption. In normal species, megasporogenesis and
30 embryo sac development are temporally separated during
which time callose (a f3, 1-3 gl.ucan) in the walls of the
surviving megaspore is catabo:Lized (Linnean).
The MMC walls of dip:~osporic and tetrasporic
species lack callose, which normally envelops MMCs of
35 monosporic and bisporic species during early prophase
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18
(FIGS. lA-B, L-M) and is deposited in the cross walls
during megasporogenesis (Linnean). These deposits are
catabolized following meiosis, which permits rapid
megaspore expansion at the onset of embryo sac
formation. The absence of callose in diplosporic and
tetrasporic species permits the precocious expansion of
MMCs (FIGS. 1C-F, I-K) and is further evidence of MMC
gametophytization (Linnean; E. Battaglia, 47 Annali di
Botanica 7-144 (1989); M.D. Peel et al, Megasporocyte
_0 Callose in Apomictic Buffelgrass, Kentucky Bluegrass,
Pennisetum squamulatum Fresen, Tripsacvm L. and Weeping
Lovegrass, 37 Crop Science 724-32 (1997) (incorporated
herein by reference)). Forms of polyspory, like forms
of apomixis, are derived anomalies of widespread
_5 polyphyletic origin (Linnean).
There are many forms of polyembryony, which is the
formation of more than one embryo per ovule (synergid
and antipodal embryony, cleavage polyembryony,
adventitious embryony) (Linnean). Like apomixis and
?0 polyspory, the various forms of polyembryony are
polyphyletically derived and involve temporally and
spatially-misplaced developmental programs.
Phylogenetic and cytological studies shed light on
the origins of apomixis. Cytological comparisons
?5 indicate that some apomictic types resemble unusual
sexual types more than other apomictic types, e.g.
Adoxa-type tetraspory (sexual) is identical to
Antennaria-type diplospory except the nuclear divisions
leading to a tetranucleate embryo sac are meiotic in
30 tetraspory but mitotic in Antennaria-type diplospory
(FIGS. lE & lI). Likewise, Ixeris-type diplospory (FIG.
1D) is identical to sexual bispory (FIGS. 1L & 1M)
except meiosis I fails in the former. Both Ixeris-type
diplospory and bispory (and apospory and tetraspory)
35 occur in the genus Erigeron. Phylogenetic data have
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19
been analyzed and it has been found that apomixis and
polyspory often occur together at the generic and
familial levels (highly significant associations).
Likewise, preleptotene chromo~;ome condensations and the
formation of nonfunctional megasporocytes that are
subsequently replaced by functional megasporocytes occur
in species closely related to apomictic or polysporic
species, which also suggests similar mechanisms of
evolution.
LO High chromosome base n~imbers (x z 10) usually
indicate paleopolyploidy, which means polyploidy
followed by diploidization with or without ascending or
descending aneuploidy. Multiple base numbers per genus
also reflect paleopolyploidy. It has been found that
chromosome base numbers for 80'-~ of all genera identified
as containing apomictic, pol:~sporic, or polyembryonic
species. Statistical analyses indicated that polysporic
and polyembryonic species are generally paleopolyploid,
while apomicts, which are generally polyploid (Asker &
20- Jerling), often cantain primary genomes. Furthermore,
genera with polysporic but not apomictic species had
more x values per genus (2.7 1: 0.4 SE) than genera with
apomictic but not polysporic apecies (1.7 0.1). This
means apomicts tend to have balanced sets of duplicate
genes (primary genomes) and po:Lysporic and polyembryonic
species usually have imbalanced sets of genes
(paleopolyploid genomes, i.e. partially duplicated or
triplicated due to aneuploidy). Hence, in the present
invention it is shown that in polyploids composed of
3~0 reproductively divergent ecotypes (a) interactions among
balanced genomes (contain:ing complete sets of
reproductive genes) are required for certain female
developmental sequences, e.g. megasporogenesis, etc., to
be completely replaced by developmental sequences that
normally occur later in development, such as occurs in
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apomixis, (b) interactions among unbalanced
"paleopolyploid" genomes containing incompletely-
duplicated or triplicated sets of reproductive genes are
required for only portions of certain female
5 developmental sequences to be asynchronously replaced or
duplicated by other portions of developmental sequences,
such as occurs during polyspory, polyembryony,
preleptotene condensations, and MMC replacements, and
(c) apomixis, with its long-term reproductive stability,
10 may, when influenced by paleopolyploid processes, be an
evolutionary springboard (rather than a dead end) in the
evolution of normal and developmentally-novel
paleopolyploid sexual species and genera.
Heterokaryon studies with yeast shed light on the
I5 types of developmental mechanisms that may cause
apomixis, polyspory, and polyembryony. Entire cell
cycle stages are skipped when yeast cells in G1 are
fused with cells in S-phase, i.e. Gl chromosomes
replicate precociously. The rate of initiation of
20 replication depends on the S:Gl nucleus ratio in the
heterokaryon. In mitotic yeast cells fused with
interphase cells, the interphase nuclei (all stages)
prematurely enter mitosis (B. Lewin, Genes V, (Oxford
University Press, 1994) (incorporated herein by
reference)).
Apomixis may occur in an analogous manner. If
embryo sac development signals from one genome are
superimposed on megasporogenesis signals from another
genome, meiosis may be skipped (diplospory) or embryo
sac development may be ectopic (apospory). Accordingly,
apomictic-like tendencies occur in polyploids only if
major differences in timing of megasporogenesis and
embryo sac development (relative to development of
nongametophytic ovule and ovary tissues) exist among the
ancestral ecotypes or species (Table 1). Such natural
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21
variation might only be frE:quently found in highly
cosmopolitan species, i.e. species with broad
latitudinal and ecological distributions.
Table 1 depicts a model of how asynchronously-
expressed duplicate genes cau;>e diplospory and apospory
in polyploids containing two genomes divergent in the
temporal expression of female developmental schedules
(floral induction, megaspore j=ormation, and gametophyte
LO development). Italicized developmental phases encoded
by genome I are skipped because of precociously-
expressed check-point genes from genome II.
Table 1
Developmentally-critical
stages*
Genome 1 3 q
2
1.5 Geriome I ArchesporeMeiosis
Embr
o sac
D
bl
y ou
(unmodified) e
fertilization/
early embryony
Genome I Embryo Double Fertilization
(modified) sac fertilization/of central
cell
early embryonyonly
Genome II Meiosis Embryo Double Fertilization
sac fertilization/of central
cell
early embryonyonly
?0 * Ovary development is initiated by a compromise between
developmental signals from genome I, which evolved in
short days and long seasons (lower latitudes), and
genome II, which evolved in long days and short seasons
(higher latitudes) . Thus, the initiation and pace of
?5 ovary development assumes an "intermediate phenotype" in
a manner similar to other intermediate phenotypes
observed in amphiploids derived from morphologically-
distinct parents (leaf lengths and widths, plant height,
etc . ) .
30 1. At the beginning of stage l, genome II produces
precocious signals for mE~iosis, which fail because
the archespore mother cell has not yet formed, i.e.
it develops at an intermediate rate dictated by the
intermediate phenotype.
35 2. At the beginning of stagE~ 2, end-of-meiosis check-
point signals from genomE~ II terminate meiosis and
synchronize genome I with genome II in a manner
similar to that observed in asynchronous yeast
heterokaryons (reviewed herein). If meiosis is
40 successfully terminated, one of several forms of
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22
diplospory (FIG. 1) occurs, i.e. an embryo sac
forms directly from the megasporocyte (Antennaria-
type diplospory) or young female meiocyte
(Taraxacum or Ixeris-types of diplospory). If
meiosis is unsuccessfully terminated, apospory
(FIG. 1) may occur, i.e. one or more embryo sacs
may form from adjacent nucellar cells. This occurs
primarily in species containing multiple or ill-
defined archegonial cells. In both apospory and
diplospory, a genetically unreduced embryo sac
develops. Development of the nongametophytic
tissues of the ovule and ovary continues to occur
according to the intermediate-phenotype (delayed)
schedule. In contrast, gametophyte (embryo sac)
development continues to occur precociously because
the embryo sac development genes of genome I (in
the embryo sac only) are synchronized with those of
genome II.
3. Signals from the two synchronized genomes induce
egg formation and parthenogenesis, both of which
occur precociously in most apomicts relative to the
development of nongametophytic tissues of the ovule
and ovary.
4. Pollination occurs according to the intermediate
phenotype schedule, but the egg is no longer
receptive and in many cases has already divided.
The central cell, if not autonomous, is fertilized,
and the endosperm and parthenogenetic embryo
develop.
This duplicate-gene asynchrony hypothesis explains
at a rudimentary level many genomic peculiarities of
species exhibiting reproductive anomalies as well as
many inconsistencies in the apomixis literature. For
example, apomixis, polyspory, and polyembryony are rare
but tend to occur together in cosmopolitan families
because sufficient ecotypic variation in reproductive
start-times, etc., is rare in most families but is high
in cosmopolitan families. Sexual reproduction of the
monosporic Polygonum-type occurs facultatively in
apomictic and polysporic species (Linnean) because,
barring deletions or mutations, each parental genome
contains genes required for normal reproduction, and
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23
growing conditions may occasionally favor the expression
of one genome over the other causing sexual development
to occur, as occurs in Dicanth:ium, Themeda, and numerous
other apomicts. Facultativeness is influenced by (a)
differential silencing of genorles, which could be caused
by differences in genetic background, or (b)
environmental factors that reduce the degree of
asynchrony by accelerating or decelerating gene
expression from one genome (photoperiod or temperature
_0 response, etc.) relative to that of another, thus
allowing sexual development to occur. Polyspory and
polyembryony result from the competitive expression of
grossly imbalanced genomes (incomplete sets of
reproductive genes) in which some checkpoint systems are
_5 missing. In contrast, competitive expression among
genomes is terminated by checkpoint genes in apomicts,
which generally contain balanced sets of reproductive
genes, thus allowing a smooth transition to apomixis
(Table 1). At least one of the two parental genomes of
:0 an apomict must have sufficient DNA to extend the
duration of reproductive development (meiotic durations,
etc) such that sufficient asynchrony can be expressed.
Hence, apomixis is seldom found in annuals, which have
little DNA and rapid meioses. Likewise, polyhaploidy
?'5 may obliterate asynchrony causing a reversion to
sexuality. Apomixis is mu~~h more prevalent among
outcrossing species than inbreE:ding species because they
are more prone to form interecotypic or interspecific
polyploids when secondary contacts occur, e.g. during
3>0 the frequent climatic shifts associated with the eight
major glaciations and numerous minor glaciations of the
Pleistocene (L.A. Frakes et al., Climate Modes of the
Phanerozoic (Cambridge University Press, 1992)
(incorporated herein by reference)). Likewise, more
35 apomicts are allopolyploid than autopolyploid because
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24
polyploidization, which is generally essential to the
asynchronous expression of different genomes with
respect to female development (Linnean), by BIII hybrid
formation is expected to occur more frequently in
interspecific hybrids than interracial hybrids.
Likewise, the chances of BIII hybrid formation occurring
in mostly sterile interecotypic or interspecific F1
hybrids that are annual is low compared to those that
are perennial, which flower every year for many years.
This factor further limits the chances of annuals
becoming apomictic and further explains the low
frequency of naturally-occurring apomictic annuals.
Finally, ambiguous outcomes regarding the sexuality of
progeny are expected when an apomict is crossed with a
L5 sexual or with another apomict, regardless of the
closeness or wideness of the cross. The mode of
reproduction expressed in the progeny will depend on how
the added or removed genome(s) affect asynchrony, and
this cannot be predicted without some a priori knowledge
~0 of the female developmental schedules encoded by the
involved genomes (Linnean). The ability of the
duplicate-gene asynchrony hypothesis to adequately
explain these many phenomena, which have been considered
major inconsistences in the apomixis literature, is
25 strong evidence for its validity.
The distribution patterns of many apomicts indicate
a Pleistocene origin (G. L. Stebbins, Chromosomal
Evolution in Higher Plants (Addison-Wesley, 1971)
(incorporated herein by reference); Asker & Jerling),
30 i.e. the geographic distributions and centers of
diversity of many apomicts are centered near the margins
of the Pleistocene glaciations but their ranges often
encompass the ecological ranges of the putative sexual
progenitors, which lie north and south of the glacial
35 margins. Eight major glaciations, which covered up to
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200 of the earth's surface, occurred during the
Pleistocene. These were separated by warm interglacial
periods lasting for several. thousand to a hundred
thousand years each. Likewise, most of the major
5 glacial events consisted of glacial advances interrupted
by minor interglacial periods .lasting for a few thousand
years (L.A. Frakes et al., Climate Modes of the
Phanerozoic (Cambridge Univer:~ity Press, 1992). Hence,
during the Pleistocene, the northern latitudes of North
10 America and Eurasia were repeatedly deglaciated and
revegetated by cosmopolitan t~ixa capable of adapting to
cool climates, long days, and short growing seasons.
A precocious meiosis and embryo sac development in
young ovules is an adaptation to short summers in high
15 latitudes. Glacial advances, which followed the
numerous interglacial peri~~ds, cooled the lower
latitudes permitting higher latitude flora to invade
lower latitude flora. This provided numerous
opportunities for ecotypes with a putatively-precocious
20 female development (from higher latitudes, i.e.
temperate to arctic climates) to form polyploids with
ecotypes (or different species) with a putatively-
delayed female development (from lower latitudes, i.e.
tropic to temperate climates). Such polyploids exhibit
25 asynchronous female development, i.e. apomixis.
Another class of adaptations to high and low
latitudes includes flowerin<~ responses to specific
changes in photoperiod during the changing of seasons.
Many plants have been categorized according to their
responses to photoperiod, e.g. "long day plants" are
adapted to higher latitudes anc~ flower in the spring and
early summer when days are long, "short day plants" are
often found in lower latitudes (tropics) and flower
during the tropical "winter" wizen days are short, "dual-
day-length plants" require either short or long days to
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26
induce reproductive bud formation followed by long or
short days, respectively, to cause the formed buds to
mature into flowers, "intermediate-day plants" will not
flower if days are too long or too short, and "day-
s neutral plants" show little adaptation to day length and
flower induction occurs under a broad range of day
lengths. Several other specialized categories exist
(Salisbury & Ross, Plant Physiology (1992) (incorporated
herein by reference)).
Salisbury and Ross selected 85 species, from among
approximately 300 species of plants studied to date for
flowering responses to different photoperiods. These 85
species distinctly represent the specific photoperiod
response categories listed above, and 67 different
genera are represented by these 85 species. It is noted
herein (for the first time) that 330 of these genera (22
of 67) contain species with female reproductive
anomalies (gametophytic apomixis, polyspory, or
polyembryony; compare Salisbury & Ross Table 23-1 with
the appendix in Linnean). This is a 9-fold increase in
the number of genera expected if reproductive anomalies
occur independently of adaptations to distinct
latitudes, i.e. only 3.80 of genera are known to express
reproductive anomalies. Thus, if reproductive anomalies
occur independently of distinct adaptations to
photoperiod, then only 3.80 (not 330) of the genera
identified as containing species with distinct
photoperiod adaptations should have also expressed
reproductive anomalies. This 33o is broken down as
follows: 12o contain gametophytic apomicts (compared
with to of all angiospermous genera, i.e. a 12-fold
increase), 13o contain polysporic species (compared with
1.60 of all angiospermous genera, i.e. an 8-fold
increase), and 7.5o contain polyembryonic species
(compared with 1.7% of all angiospermous genera, i.e.
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27
a 4.4-fold increase). '.Chis highly significant
association between genera. that contain plants
expressing reproductive anomalies and genera that
contain plants expressing distinct adaptations to
photoperiod (different latitudes) is taken as additional
evidence for the duplicate gene asynchrony hypothesis.
It is believed that amphiploids containing the genomes
of multiple ecotypes divergent: in these characteristics
(distinctly-different photoperiod responses and
LO distinctly-different times and rates of female
development) are apomictic because of asynchronous
female development, which causes the formation of
unreduced embryo sacs followed by parthenogenesis (Table
1) .
The discoveries made in the present invention, as
described above, largely invalidate important
assumptions currently being relied on by scientists
attempting to transfer apomixis to sexual species or to
induce its expression de novoloy causing mutations. The
underlying assumptions for t:he introgression approach
(wide hybridization with an apomictic relative) are that
apomixis is controlled by one or a few "apomixis genes"
and that these genes) can function appropriately in
other species. The duplicate-gene asynchrony hypothesis
states that apamixis is caused by asynchronous
(overlapping) expression of whole sets of genes that are
duplicated across interracial (interecotypic) or
interspecific genomes and that control entire female
developmental sequences in ovules (MMC differentiation,
meiosis, megagametophyte development, embryogenesis).
Thus, only interactions between gene sets from
specifically-divergent genomes will cause an appropriate
degree of asynchrony and induce apomixis. Such
specificity varies from specie=s to species, i.e. a gene
cassette that induces apomix:is in one species may not
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28
function appropriately to induce apomixis in another
species. Furthermore, it may be impossible to induce
apomixis in a particular species whose photoperiod
responses and female developmental sequences are average
with regard to timing (possibly the vast majority of
species) without actually replacing (by plant breeding
or genetic engineering) its current female reproductive
gene cassettes, which code for temporally-average
photoperiod responses and sequences of female
.0 development, with two divergent gene cassettes that
provide the appropriate degree of asynchrony. It may be
argued that one or a few master genes control the timing
of these sequences, and that only the master genes need
to be transferred, i.e. the master genes of one genome
~5 may control the gene cassettes of another genome.
However, as noted by Wilson, Breeding for Morphological
and Physiological Traits, in K.J. Frey (ed), Plant
Breeding II (Iowa State University Press, 1981)
(incorporated herein by reference), photoperiod
?0 responses are quantitative traits. Furthermore,
phylogenetic data indicate that apomixis is lost when
cassettes of genes are fragmented by paleopolyploid
processes. Thus, while the transfer of apomixis from a
wild species to a crop species through wide
?5 hybridization and backcrossing may be possible, many
previously unforeseen and poorly understood
complications are to be expected, including the
requirement of transferring many genes possibly from
more than one chromosome.
30 The main assumption of the mutation breeding
approach is that apomixis is a single gene or near
single gene trait. In the absence of intergenomic
asynchrony, only mutations that mimic portions of the
many asynchronous interactions responsible for apomixis
35 (all cellular responses from unreduced embryo sac
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29
formation through parthenogenesis) can be expected. It
is difficult to conceive of an accumulation of mutations
that induce an apomixis-like reproductive behavior
without deleteriously affecting other cellular,
reproductive, or whole plant processes. The asynchrony
hypothesis predicts that the mutation approach will meet
with major complications.
The main assumption of the sexual plant
hybridization approach (A. l~rnst, Bastardierung als
i0 Ursache der Apogamie im Pflamzenreich, Fischer, Jena
(1918)) is that apomixis originates as a fertility
restoration mechanism following wide hybridization and
amphiploidization. Thousands of wide hybrids and
amphiploids have been made by man, but only rarely has
_5 apomixis been observed among the progeny (Asker &
Jerling; Linnean). The asynchrony hypothesis explains
this observation in that the large degree of asynchrony
required to induce apomixis is only rarely observed in
nature. An example of such raise events in nature is the
?0 evolution and radiation of new apomicts in the Rosaceae,
Asteraceae, Poaceae, and other families that occurred
during the Pleistocene glaciations when grossly
different ecotypes of the same species and of closely-
related species were repeatedly brought together by
35 climatic shifts where they underwent hybridization and
subsequently formed interracial or interspecific
apomictic polyploids. Thus, apomixis arises
fortuitously only in a very low percentage of man made
wide hybrids or amphiploids. In contrast, the
30 asynchrony hypothesis predicts that amphiploids
developed from appropriately-selected or bred parental
ecotypes or lines (breeding and selection based on
cytologically-observable temporal differences in
photoperiod responses and timing of female development)
35 will be apomictic.
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The invention described herein relies on a specific
combination of technologies that explicitly identify, a
priori, germplasm that when combined through
hybridization and amphiploidization cause gametophytic
5 apomixis to be expressed. The a priori identification
of specific combinations of sexual germplasm that confer
apomixis when combined as polyploids (by conferring
asynchrony in photoperiod responses and other forms of
female reproductive timing) differentiates the present
LO invention from the prior art.
The present invention does not depend on the
existence of apomixis genes that will be appropriately
expressed in different taxa (genomes). In fact, the
invention relies on the concept that such genes do not
15 exist. The present invention does not rely on the
existence of alleles of genes that when mutated will
induce apomixis. Again, the invention relies on the
concept that such alleles will probably express
deleterious pleiotropic effects when mutated and will
?0 not produce desirable apomictic forms. The present
invention does not rely on fortuitous or previously-
produced wide hybrids that may or may not express
apomixis. In contrast, the invention identifies a
priori parentage or gene cassettes that when hybridized
or combined yield apomixis. The underlying concepts and
practices of the claims listed herein are fundamentally
different from all other methods of producing apomicts
previously conceived or currently in practice throughout
the world.
30 Others have proposed making apomictic plants by
molecular marker assisted introgression of genes for
apomixis from wild apomictic relatives to crop species.
The data reviewed herein imply that such markers will be
of limited use in that the genes they locate will confer
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31
apomixis only when numerous other genes are also
transferred, which could gre<~tly reduce the agronomic
desirability of the crop. These apomixis transfer
programs have as long range objectives the identifying
and patenting of a universal "apomixis gene" or a
universal "apomixis gene cassette". As taught herein,
the genes) from one genome, which are responsible for
conferring apomixis in a polyploid species containing
two genomes with different temporal schedules of female
development, will only be useful for conferring apomixis
in other species or ecotypes if the recipient species
contains a genome that is appropriately divergent from
the donor genome with regard to the encoding of female
developmental schedules. A gene cassette from a single
genome probably does not exist for conferring apomixis
universally.
The present invention is directed to processes for
producing gametophytic apomicts (plants expressing
apospory or diplospory) from plants that typically
2a undergo normal sexual reproduction.
The present invention is specifically directed to
the production of gametophytic apomicts by combining
through hybridization and amprLiploidization two or more
sexual plants of the same oz' closely related species
that differ in the time and/or duration in which
megasporogenesis, megagametogenesis (embryo sac
formation), pollination, fertilization, and embryony
occur relative to (a) the environmental conditions in
which the plants are grown (photoperiod responses, etc.)
and (b) gross developmental events in the plants such as
ovule initiation, integument growth and development, and
pistil length and width.
The present invention is also directed to the
amounts of asynchrony in fema7_e developmental schedules
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32
required among the two or more sexual species used to
produce apomictic amphiploids.
The present invention is also directed to the
selection pressures and breeding schemes used to create
the desired divergence in female developmental schedules
(reproductive asynchrony) among sexual lines not
originally expressing the required female developmental
schedules.
It is convenient to separate the process of the
present invention into four categories: (a) selection or
production of sexual germplasm appropriate for use in
producing apomictic plants from sexual plants, (b)
hybridization processes, (c) amphiploidization
processes, and (d) procedures for verifying expression
of apomixis.
Selecting and Breeding Sexual Germplasm for Producing
Apomicts
A feature of the present invention is the
accelerated simulation of processes responsible for the
evolution of apomixis from sexual plants in nature. The
natural process requires tens of thousands of years,
i.e. it may require glaciers to advance, which causes
plant populations separated for thousands of years (and
adapted to different climates and photoperiods) to be
reunited. In the present invention, the duration of the
entire process is reduced from tens of thousands of
years to only a few years.
A preferred method of selecting germplasm for
producing apomictic plants from sexual plants involves
the identification of plants of the same species or
closely related species that contain ecotypes
photoperiodically adapted to broadly-divergent latitudes
(long day plants, short day plants, day neutral plants,
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33
etc., Salisbury & Ross, Plant Physiology (1992). Many
such "cosmopolitan" taxa exist in the Poaceae,
Asteraceae, and Rosaceae (Linne~an; see examples, infra).
Groups of germplasm are selected such that they
represent extremes in (a) latitude in which the ecotypes
were derived, (b) flowering response to different
photoperiods, and (c) timing of megasporogenesis,
megagametogenesis, and embryony relative to the
development of nongametopytic ovule and ovary tissues.
LO Plant breeding is required as part of the preferred
method of obtaining germplasm for producing apomictic
plants from sexual plants when appropriate degrees of
divergence in female developmental schedules does not
exist among currently available varieties or lines. The
L5 preferred method involves (a) identifying several or
more varieties or ecotypes adapted to higher latitudes
and several or more varieties c>r ecotypes of the same or
closely related species that are adapted to lower
latitudes, (b) crossing the varieties within each of the
?0 two latitudinal adaptiveness categories, (c) selecting
germplasm from the Fls based on appropriate degrees of
divergence in flowering response to photoperiodic
treatments as well as timing of megasporogenesis,
megagametogenesis, and embryony, and (d) continued
?5 selection for each of these l.rafts using conventional
breeding regimes (e. g., mass selection, single seed
descent). These methods ar~~ well known to persons
skilled in the art, e.g., Poehl.man, Breeding Field Crops
(Van Nostrand Reinhold, 1987) (incorporated herein by
30 reference).
Hvbridizati n_n_ Proce~~P~
The hybridization and amphiploidization processes
are facilitated when the selected species are dioecious
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34
or self incompatible, have a short juvenile phase, and
are amenable to efficient procedures for cytogenetic and
embryological analyses of root tips and ovules,
respectively. The use of male sterile lines or
emasculation procedures are required if the plants are
not dioecious or self incompatible. Hybrids are
produced between sexual varieties or lines that display
appropriate degrees of divergence in photoperiod
responses and female developmental schedules.
Intraspecific hybrids are made using standard techniques
as taught in plant breeding texts, e.g. Poehlman,
Breeding Field Crops (1987). The successful production
of interspecific or intergeneric hybrids may require
hormone treatments to the florets and embryo rescue
procedures as taught in recent references involving wide
hybridization, e.g. Z.W. Liu et al., Hybrids and
Backcross Progenies between Wheat (Triticum aestivum L.)
and Apomictic Australian Wheatgrass [Elymus rectisetus
(Nees in Lehm.) A. Love & Connor]: Karyotypic and
Genomic Analyses, 89 Theor. Appl. Genet. 599-605 (1994)
(incorporated herein by reference). Hybrids are
verified by their intermediate phenotype.
~mphiploidization Pro s
The chromosome numbers of hybrids are doubled using
standard colchicine techniques, e.g. J. Torabinejad et
al., Morphology and Genome Analyses of Interspecific
Hybrids of Elymus scabrus, 29 Genome 150-55 (1987)
(incorporated herein by reference). Alternatively,
recently developed tissue culture techniques may be
used, e.g. O. Leblanc et al., Chromosome Doubling in
Tripsacum: the Production of Artificial, Sexual
Tetraploid Plants, 114 Plant Breed. 226-30 (/995)
(incorporated herein by reference); Salon & Earle,
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Determination of Mode of Rssproduction of Synthetic
Tetraploids of Eastern Gamag:rass, Agron. Abs. Pg 114
(I994) (incorporated herein by reference); Cohen & Yao,
In Vitro Chromosome Doubling of Nine Zantedeschia
5 Cultivars, 47 Plant Cell, Tiss. Org. Cult. 43-49 (1996)
(incorporated herein by reference); and Chalak & Legave,
Oryzalin Combined with Adventitious Regeneration for an
Efficient Chromosome Doubling of Trihaploid Kiwifruit,
16 Plant Cell Rep. 97-100 (199F>) (incorporated herein by
_0 reference). Partially amphiploid 2n + n BIII hybrids are
often produced in low frequencies (0.5% to 30) when
interspecific Fls are backcrossed, e.g. Z.W. Liu et al.,
89 Theor. Appl. Genet. 599-605 (1994), and this
frequency may be much higher if tendencies for apoz~ixis
.5 (unreduced egg formation) exist: in the hybrids as taught
in 0. Leblanc et al., Reproductive Behavior in Maize-
Tripsacum Polyhaploid Plants;: Implications for the
Transfer of Apomixis into Maize, 87 J. Hered. 108-111
(1996) (incorporated herein by reference). Thus, a
?0 preferred method for doubling chromosomes of
intraspecifac and interspecific: hybrids is to use one or
more of the colchicine (or othEer known spindle inhibitor
chemical) treatment methods lasted above. Likewise, a
preferred method for doubling chromosomes of
?5 interspecific hybrids involves backcrossing to one of
the sexual parents and counting chromosomes in root tips
to determine partial amphiploidy (usually triploidy).
This is followed by backcrossing to the other parent to
obtain a full amphiploid, o~_ to the same parent to
30 obtain a partial amphiploid (three genomes from one
parent and one genome from the other).
Amphiploidization may precede or follow hybridization.
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36
Procedures for V rifyinq the Expression of Apomixis
The expression of apomixis in synthetic amphiploids
is verified by analyses of megasporogenesis and embryo
sac development as taught by J.G. Carman & S.L. Hatch,
Aposporous Apomixis in Schizachyrium. Poaceae:
Andropogoneae, 22 Crop Sci. 1252-55 (1982) (incorporated
herein by reference), for aposporous apomixis and C.F.
Crane & J.G. Carman, 74 Amer. J. Bot. 477-96 (1987)
(incorporated herein by reference), for diplosporous
LO apomixis. Progeny testing is also useful, as taught in
Asker & Jerling.
Some of the features of the present invention may
be better appreciated by reference to specific examples.
It should be understood that the following examples are
illustrative in nature rather than restrictive, and they
are meant to demonstrate the basic teachings and
concepts of the present invention rather than to limit
the invention. It is expected that one of ordinary
skill in the art will be able to use the information
~0 contained in the examples and elsewhere herein to apply
the present invention to situations not specifically
described herein.
Example 1
Selecting and Collecting Germplasm - Dicots
It is a feature of the present invention to provide
procedures for selecting and collecting the most
appropriate lines from within a species or group of
closely related species for the purpose of producing
apomictic plants. These procedures are believed to
mimic and greatly accelerate natural processes that
initiate the evolution of apomictic plants from sexual
plants, i.e. those natural processes that cause
secondary contacts to occur among taxa adapted to
greatly divergent climates and photoperiods.
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37
In this example, there i.s illustrated a preferred
procedure for use with plants from the subclass
Dicotyledonae. In Example 2 there is illustrated a
. preferred procedure for use with plants from the
subclass Monocoty.ledonae. The dicotyledonous example
involves sexual species from the genus Antennaria (x =
14). It is expected that one of ordinary skill in the
art could successfully apply these procedures to many
species, including various dic~otyledonous crops, such as
strawberry, Raphanobrassica, potato, cherry, apple, and
sugar beet.
The presently preferrect procedure of selecting
appropriate lines of a given species or closely related
group of species begins with (a) identifying geographic
distributions from the literature and/or from field
studies, and (b) collecting all information concerning
the floral biology (photoperi.od responses, embryology,
etc.) and any specific adaptations of the various
ecotypes especially those at the latitudinal extremes.
For example, the dicotyledonous genus Antennaria
contains 33 sexual species (:mostly diploids with some
tetraploids) and five large highly polymorphic polyploid
agamic (apomictic) complexes. They are dioecious,
herbaceous, perennial, and usually stoloniferous.
Apomixis in Antennaria occurs naturally in one of six
Glades, the Catepes, which contains sexual diploids (2x)
and sexual and apomictic pol~~ploids ranging from 4x to
12x. All members of this group are stoloniferous and
sexually dimorphic (Bayer, A Phylogenetic Reconstruction
of Antennaria Gaertner (Asteraceae: Inuleae), 68 Canad.
J. Bot. 1389-97 (1990) (incorporated herein by
reference); Bayer, Evolution of Polyploid Agamic
Complexes with Examples from Antennaria (Asteraceae),
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38
132 Opera Botanica 53-65 (1996) (incorporated herein by
reference)).
Extensive ecological, morphological, and genetic
studies indicate that the five polyploid agamic
complexes of the Catepes, A. alpina (L.) Gaertn., A.
howellii E. L. Greene, A. parlinii Fern., A. parvifolia
Nutt., and A. rosea, have evolved from among the sexual
species. For example, the center of diversity for the
A. rosea agamic complex is the Rocky Mountains of
LO western North America (near the glacial margins), and
its range is from New Mexico and southern California,
north to Alaska and the Northwest Territories, and east
through Alberta, Saskatchewan, the northern Great Lakes
and with disjunct populations in Atlantic Canada. The
L5 sexual species believed to have been involved in the
initial evolution of A. rosea and its subsequent
polymorphic expansion include A. aromatica Evert, A.
corymbosa E. Nelson, A. pulchella E. Greene, A.
marginata E. Greene, A. ncicrophylla Rydb., A. racemosa
30 Hook., A. rosulata Rydb. and A. umbrinella Rydb. (Bayer,
132 Opera Botanica 53-65 (1996).
The sexual relatives of apomictic A. rosea meet the
geographic and reproductive criteria listed herein for
producing apomictic plants from sexual plants. They
25 have a very broad latitudinal distribution and
differences in flowering times, have been noted, e.g.
some apomictic forms flower three weeks later than
nearby sexual forms (G.L. Stebbins, 94 Bot. Gaz. 322-45
(1932)). Finally, species of Antennaria are easily
30 grown in cultivation, readily hybridized
interspecifically (due to lack of internal reproductive
isolating mechanisms), and can produce two seed
generations per year when vernalized.
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39
Ramets of Antennaria are collected from sites (FIG.
2A) that provide plant matf~rials representative of
extremes and midpoints in latitudinal, ecological, and
morphological diversity. Collections are made during
the flowering season (June, for lower latitudes; July,
for higher latitudes) when species can be confirmed and
both staminate and pistillate plants needed for crossing
can be identified. The preparation and execution of
collection trips involve the study of herbarium voucher
specimens and the attainment of appropriate collection
permits. Roots of the clones are washed clean and
packed in moist sphagnum. Shipments of plants are made
by courier every few days during the collection trips to
assure survival. The cutting's are potted in standard
potting mix and misted during establishment. It will be
appreciated that collection and establishment procedures
are expected to vary somewhat with each dicotyledonous
species.
Example 2
?0 Selecting and Collec-t;na rmr~lasm - Monoco
In this example, there is described an illustrative
procedure for selecting and collecting germplasm from
plants of the subclass i~Ionocotyledonae. This
monocotyledonous example involves sexual species from
the genus Tripsacum (x = 18). It is expected that one
of ordinary skill in the art could successfully apply
these procedures to many species, including various
monocotyledonous crops, such as sorghum, wheat, barley,
rice, and maize.
This example involves th.e monocotyledonous genus
Tripsacum, which is endemic to the new world and is
found from 42° N to 24° S latitude (de Wet et al,
Systematics of Tripsacum dactyloides (Gramineae), 69
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Amer. J. Bot. 1251-57 (1982) (incorporated herein by
reference), and from 0 to 2600 m above sea level
(Berthaud et al, Tripsacum: Diversity and Conservation,
in Taba, Maize Genetic Resources (CIMMYT, 1995)
5 (incorporated herein by reference)). They are
monoecious perennials, and the male and female flowers
are segregated from each other along the spike. As is
typical with many agamic complexes, variation for key
characters overlaps among the 16 species, which makes it
~0 difficult to classify certain individuals. The T.
dactyloides agamic complex is the most diverse in the
genus. Its range encompasses that of the entire genus,
and both sexual diploids and apomictic polyploids are
found throughout (de Wet et al., 69 Ame n J. Bot. 1251-
~5 57 (1982); Berthaud et al., Tripsacum: Diversity and
Conservation, ~n Taba, Maize Genetic Resources (CIMMYT,
1995) ) .
Tripsacum diploids are sexual, and all naturally-
occurring polyploids studied embryologically are
?0 apomictic (Burson et al, Apomixis and Sexuality in
Eastern Gamagrass, 30 Crop Sci. 86-89 (1990)
(incorporated herein by reference); 0. Leblanc et al,
Megasporogenesis and Megagametogenesis in Several
Tripsacum Species (Poaceae), 82 Amer. J. Bot. 57-63
~5 (1995) (incorporated herein by reference)). This
differs from Antennaria, in which sexual autotetraploids
occur in nature. Recently, sexual Tripsacum amphiploids
have been produced by colchicine doubling of diploids
adapted to temperate North American climates (Salon &
30 Earle, Determination of Mode of Reproduction of
Synthetic Tetraploids of Eastern Gamagrass, Agron Abs
114 (1994); Salon & Pardee, Registration of SG4X-1
Germplasm of Eastern Gamagrass, 36 Crop Sci. 1426 (1996)
(incorporated herein by reference)), and by crossing
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41
diploids adapted to tropical Mesoamerican climates
followed by colchicine doub_Ling (O. Leblanc et al.,
Chromosome Doubling in Trip,sacurn: the Production of
Artificial, Sexual Tetraploid Plants, 114 Plant Breed
226-30 (1995) (incorporated herein by reference)).
Sexual Tripsacum diploids (and artificial sexual
amphiploids) meet the criteria listed herein for
producing apomictic plants from sexual plants. They
have a broad geographic distribution with sexual
~0 ecotypes distributed throughout (FIG. 2B). The ecotypes
are easily grown in cultivation and are readily
hybridized. Their ovules are readily examined by
interference contrast and fluorescence microscopy, and
ecotypic variation in female developmental schedules
:.5 exists (FIG. 3). Finally, tropical ecotypes remain
green year round and flower in the late fall or early
winter under short day conditions (10 to 12 h days). In
contrast, temperate ecotypes require vernalization
followed by long days (14 t:o 16 h). Tropical and
?0 temperate ecotypes will not flower again until specific
induction stimuli are repeated.
Ramets of Tripsacum are collected from those sites
that will provide plant materials representative of
extremes and midpoints in latitudinal, ecological, and
?5 morphological diversity, e.g. FIG. 2B. Collections are
made during the flowering sE:ason (June and July for
higher latitudes; September and October for lower
latitudes) when species can be confirmed or from living
nurseries in the United States and Mexico (CIMMYT). The
30 preparation and execution procedures for collecting
Tripsacum germplasm are the same as those presented for
Antennaria (Example 1). It will be appreciated that
collection and establishment procedures are expected to
vary somewhat with each monocotyledonous species.
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42
Example 3
Ouantifyinq Effects of Different Photop~riods on
Flowering
It is a feature of the present invention to provide
procedures for quantifying the effects of different
photoperiods on floral development in sexual lines
selected as being the most appropriate for producing
apomictic plants (Examples 1 and 2). It will be
appreciated that many procedures for quantifying such
effects have been published in the recent literature,
and one skilled in the art may find that procedures
other than those described herein are better suited for
certain species.
A presently preferred method for quantifying
photothermal responses of Antennaria species, which are
native to temperate climates (intermediate latitudes)
through alpine climates (high latitudes), has been
modified from the classic studies on Oxyria digyna
(Mooney and Billings, Comparative Physiological Ecology
of Arctic and Alpine Populations of Oxyria digyna, 31
Eco. Monog. 1-29 (1961) (incorporated herein by
reference)). A set of ecotypes (12 clones of each) is
chosen from among the ecotypes identified as being the
most appropriate for producing apomictic plants (Example
1), i.e. they represent latitudinal extremes and
midpoints. At six weeks after flowering, four clones of
each ecotype are placed in a vernalization growth
chamber set for 4°C (constant) and a 6 h photoperiod (dim
light). Four weeks later, four additional clones of
each ecotype are added to the vernalization chamber, and
this is followed by a third set at eight weeks.
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43
All clones are removed. from the vernalization
chamber at 12 weeks, which provides three different
vernalization exposure group:> (4, 8, and 12 weeks).
The four clones of each ecotype of each exposure group
are randomly assigned to two flower-inducing
photoperiods (12 and 16 h) each set for a 22/17 °C
day/night temperature regime. Time intervals from the
end of vernalization to btzd formation, archespore
formation, megasporogenesis, megagametogenesis, and
_0 'flowering (dependent variables) are determined for all
clones. Each dependent variable is subjected to
analysis of variance and analyzed as a factorial
experiment (3X2Xnumber of ecotypes) with replication
(repeated with a duplicate set of ecotypes) . Cluster
_5 analysis (Sneath & Sokal, Tfumerical Taxonomy: The
Principles and Practice of Numerical Classification
(1973) (incorporated herein by reference)) is used to
group ecotypes exhibiting similar attributes of female
development (using phenoloc~ical, cytological, and
'0 photothermal data), and the results are used to predict
either asynchronous (apomixis) or synchronous (normal)
female development (Table 1) when artificial amphiploids
are produced between reproductively divergent ecotypes
or reproductively similar ecot=ypes, respectively.
'S Example 4
Ouantpfv,'_na Effects of Diyferent Pho~~eriods on
Flowering
The presently preferred method for quantifying
photothermal responses of Tripsacum species, which are
30 native to tropical climates (low latitudes) through
temperate climates (intermediate latitudes), differs
from that used for Antennaria (Example 3). Tropical
ecotypes often remain green year round and flower in the
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44
late fall or early winter under short day conditions (10
to 12 h days). Temperate ecotypes often require
vernalization followed by long days (14 to 16 h).
Tropical and temperate ecotypes will not flower again
S until specific induction stimuli are repeated. The
presently preferred method takes advantage of these
distinctions. A set of ecotypes (16 clones of each) are
chosen to represent latitudinal extremes, midpoints, and
other significant forms of divergence in female
development. Flowering is induced in each ecotype after
which noninducive conditions (13 h photoperiod, 30/25 °C
day/night temperatures) are maintained for a minimum of
six weeks after flowering has ceased. Four clones of
each ecotype are then placed in a vernalization growth
chamber set for 6 °C (constant) and a 13 h photoperiod
(dim light). Four weeks later, four additional clones
of each ecotype are added to the chamber. This is
followed by a third set at eight weeks. The clones in
the vernalization chamber are removed at 12 weeks, which
provides four different vernalization exposure groups
(0, 4, 8, and 12 weeks).
After vernalization, the four clones from each
ecotype of each exposure group are randomly assigned to
two flower-inducing treatment combinations (two clones
in each) defined by two photoperiods (10 and 16 h), each
set for a 30/25 °C day/night temperature regime. Time
intervals from the end of vernalization to floral bud
formation, culm bolting, archespore formation,
megasporogenesis, megagametogenesis, and flowering
(dependent variables) are determined for all clones.
Each dependent variable is subjected to analysis of
variance and analyzed as a factorial experiment
(4X2Xnumber of ecotypes) with replication (repeated with
a duplicate set of ecotypes). Cluster analysis is used
to group ecotypes exhibiting similar phenological,
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cytological, and photothermal characteristics, and the
results are used as in the Ant.=nnaria example to predict
apomictic-like or normal devs~lopment in artificially-
produced interecotypic amphip:Loids (Table 1).
5 Example 5
Ouantifyina DiveraencP in Female Developmen al hP~3,o P~
It is a feature of the present invention to provide
procedures for quantifying divergence among ecotypes in
female developmental schedules. A presently preferred
10 method, which is used with both dicotyledonous (e. g.
Antennaria) and monocotyledonous (e. g., Tripsacum)
plants, is to measure time intervals between floral bud
formation, archespore form<~tion, megasporogenesis,
megagametogenesis, flowering, fertilization, and early
15 embryo development (2 to 7.6 cell stage) using a
combination of noninvasive measurements and destructive
sampling. This information is obtained after the
ecotypes chosen in Examples 1 and 2, i.e. those that
represent latitudinal and other ecological extremes,
20 have been grown in uniform conditions. Data gathered in
Examples 3-5 are obtained simultaneously using the same
sets of plants.
Cytological analyses of the female meiotic
prophase, dyad, tetrad, and degenerating megaspore
25 stages and the l, 2, 4, and 8 nucleate embryo sac stages
are conducted, and the following data are obtained for
each ovule analyzed: meiotic or embryo sac development
stage, pistil length and width, inner and outer
integument lengths, and meioc:yte or embryo sac length
30 and width. Pistils for cytological analysis are killed,
fixed, cleared, observed, and measured as in C.F. Crane
& J.G. Carman (74 Amer. J. E4ot. 477-96 (1987)), J.G.
Carman et al., Comparative Histology of Cell Walls
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46
During Meiotic and Apomeiotic Megasporogenesis in Two
Hexaploid Australian Elymus Species, 31 Crop Sci. 1527-
32 (1991) (incorporated herein by reference); M.D. Peel
et al., 37 Crop Sci. 724-32 (1997) (incorporated herein
by reference); and M.D. Peel et al., Meiotic Anomalies
in Hybrids Between Wheat and Apomictic Elymus rectisetus
(Nees in Lehm.) A. Love & Connor, 37 Crop Sci. 717-23,
(1997) (incorporated herein by reference)).
Developmental stage data are graphed against (a) pistil
LO and integument lengths and widths (raw data) and (b) the
lengths and widths of these structures represented as
percentages of their mature lengths and widths (measured
at stigma exsertion). The likeness of ecotypes with
respect to female developmental schedules is tested by
L5 analysis of variance, and diagrams patterned after FIGS.
3 and 4 and Table 2 are produced and used with the
cluster analyses of Examples 3 and 4 to predict
apomictic-like or normal development in artificially-
produced interecotypic amphiploids (Table 1).
?0
CA 02280145 1999-08-04
WO 74 PCT/L1S98/02034
98/333
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Example 6
Obtaining Greater Divergence in Female Developmental
Schedules
It will be appreciated that sufficient divergence
in (a) flowering responses to different photoperiods and
(b) female developmental schedules will not be expressed
among extant ecotypes of many cosmopolitan species even
though sufficient genetic variability to establish such
divergence by breeding may exist within their primary
0 gene pools, i.e. within each cosmopolitan species as a
whole. It is a feature of the present invention to
provide breeding guidelines for increasing such
divergence. As noted by D. Wilson, Breeding for
Morphological and Physiological Traits, in K.J. Frey
.5 (ed), Plant Breeding II (Iowa State University Press,
1981) (incorporated herein by reference), many
morphological and physiological traits, including
flowering response to day length, are quantitatively
inherited, which means they are influenced by many
:0 genes. Thus, much progress towards increasing the day
length in which plants respond by flowering can be
expected by intercrossing lines already showing some
tendencies for this trait and selecting from among the
progeny those lines that show greater tendencies. Much
'5 progress can be expected by repeating this process over
several generations. In a similar manner, significant
decreases in the day length in which plants respond by
flowering can be expected by intercrossing lines already
showing this tendency and following a similar regime of
30 repeated selection and breeding. The traits for which
it is presently preferred that divergence be maximized
by such breeding schemes include (a) flowering responses
to different photoperiods, i.e. producing long and short
day ecotypes, and (b) accelerated and delayed
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initiations of archespore formation, meiosis, embryo sac
development, etc, relative to the development of
nongametophytic ovule and ovar~l tissues.
It will be appreciated that sufficient divergence
in floral development will generally not be expressed
among extant ecotypes of non-cosmopolitan species even
though sufficient genetic variability to establish such
divergence by breeding may exi:>t within their secondary
and tertiary gene pools, i.e. within the same genus,
_0 tribe, or family. It is contemplated that wide
hybridization and even genetic. engineering may in the
future be used to incorporate into targeted species
genes for
(a) appropriate flowering responses and (b) appropriate
.5 divergence in female developmental schedules.
Example 7
Makinq Apomictic Plants from ~>exual Lines Divergent in
Floral Development
The techniques in Example. 1 through 6 are used as
?0 guidelines to obtain three ox' more lines of the same
species (or closely related group of species) distinctly
adapted to long days (14 to 20 h) and generally an early
archespore development/early meiosis/early gametophyte
development relative to the development of
25 nongametophytic ovule and ovary tissues (nucellus,
integuments, pericarp, etc). The same techniques are
used as guidelines to obtain three or more lines of the
same species (or group of species) distinctly adapted to
short days (10 to 12 h) and generally a late archespore
30 development/late meiosis/late gametophyte development
relative to the development of nongametophytic ovule and
ovary tissues. The several lines of each category
(long-day plants and short-day plants, etc) are selected
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such that they form a continuum with regard to the day
length in which flowering responses are induced, e.g.
somewhat long, long, and very long and somewhat short,
short, and very short. The lines are selected such that
5 the initiation of embryo sac formation (degenerating
megaspore stage) in one set of lines (usually the long-
~day-adapted lines) occurs at about the same time as
female meiotic prophase through metaphase is occurring
in the other set of lines relative to the development of
10 the nongametophytic tissues of the ovule and ovary.
Amphiploids are then produced using the standard
procedures described above (colchicine induction or
through repeated production of B._II hybrids) or other
appropriate procedures. Standard hybridization
15 procedures are used for producing hybrids among
Tripsacum species. For Antennaria, pistillate plants
are isolated by placing pollination bags (made from
laboratory tissues, e.g. KIMWIPES) over the entire
capitulescence. Pollination is accomplished by rubbing
20 receptive pistillate inflorescences together with
staminate heads at anthesis. Unpollinated control
capitulescences are used to verify absence of apomixis
of each parent clone. This is especially important with
tetraploid clones in which either amphimictic or
25 apomictic reproduction occurs. The pollination bags
hold the fruits as they mature, and no embryo rescue is
required.
At least three of the nine possible combinations of
parents (one from each adaptation group) are made into
30 amphiploids initially: the somewhat early line with the
somewhat late line, the early line with the late line,
and the very early line with the very late line. These
are checked for the expression of apomixis as described
above. Additional amphiploids from the nine
35 possibilities are made if apomixis is not expressed. It
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will be appreciated that the genetic background in which
the lines are derived may influence the expression of
apomixis. Thus, the seleci:ion or production of
additional lines incorporating different genetic
5' backgrounds may occasionally be necessary.
Example 8
Producing Apomictic Monocotyledonous and Dicotyledonous
Plants
The techniques set forth in Examples 1 through 7
0 are used to obtain apomict:ic plants from sexual
dicotyledonous or sexual monocotyledonous plants.
The highly efficient production of apomictic plants
from sexual plants of many angiospermous genera is
obtained by following the practices taught herein.
Apomixis has been occasionally observed in man-made
hybrids and amphiploids involving both monocotyledonous
and dicotyledonous plants (Lirinean; Asker & Jerling).
The reasons offered for these infrequent occurrences are
:0 speculative and fail to teach the duplicate gene
asynchrony concept. Furthermore, they are not supported
by phylogenetic, genetic, physiological, or
developmental studies. The common explanation given for
these reports is that specific apomixis genes) are
'5 present in the parents but are suppressed by diploidy or
an inappropriate genetic background (Asker & Jerling).
This is reasonable for examples involving agamic
complexes. For example, G.L. Stebbins, Cytology of
Antennaria. I. Normal Species, 94 Bot. Gaz. 134-51
30 (1932), documented aposporous embryo sac formation in a
hybrid between sexual Antennaria neglecta and sexual
Antennaria plantaginifolia. Likewise, Nordberg,
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Embryological Studies in the Sanguisorba minor Complex
(Rosaceae), 120 Botaniska Notiser 109-19 (1967)
(incorporated herein by reference). produced all
combinations of hybrids between two sexual tetraploid
and one sexual octaploid Sanguisorba spp, and all of the
resulting tetraploid and hexaploid F,s produced
aposporous embryo sacs in which the unreduced eggs
formed embryos either parthenogenetically or after
fertilization. Furthermore, A. Jankun & M. Kovanda,
_0 Apomixis at the Diploid Level in Sorbus eximia
(Embryological Studies in Sorbus 3), 60 Preslia, Praha,
193-213 (1988) (incorporated herein by reference),
documented fully functional, high frequency apospory and
diplospory in both diploid and tetraploid Sorbus eximia,
L5 which is a geographically-restricted hybridogenous
species derived from Sorbus aria and Sorbus torminalis,
both of which are sexual diploids. These three examples
involve agamic complexes in which apomixis genes) could
have remained unexpressed in the sexual diploid
?0 ~ progenitors. In contrast, other examples exist that
involve taxa unrelated to agamic complexes.
Low frequency apomixis (parthenogenesis from
unreduced eggs) was reported in three trispecific
hybrids in the Triticeae (A. Mujeeb-Kazi, Apomictic
25 Progeny Derived from Intergeneric Hordeum-Triticum
Hybrids, 72 J. Hered. 284-85 (1981) (incorporated herein
by reference); A. Mujeeb-Kazi, Apomixis in Trigeneric
Hybrids of Triticum aestivum/Leymus
racemosusllThinopyrum elongatum, 61 Cytologia 15-18
30 (1996) (incorporated herein by reference); R. von
Bothmer et al" Complex Interspecific Hybridization in
Barley (Hordeum vulgare L.) and the Possible Occurrence
of Apomixis, 76 Theor. Appl. Genet. 681-90 (1988)
(incorporated herein by reference), and none involved
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Elymus rectisetus, the only apomict in the Triticeae, or
even other Elymus sp.
A much higher frequency of apomixis was observed in
hybrids between sexual amphiploids of Raphanus sativus
and Brassica oleraceae. From 3E~ to 70% of ovules in six
of 10 hybrids produced contained from 1.6 to 2.9
aposporic embryo sacs (S. Elle~rstrom & L. Zagorcheva,
Sterility and Apomictic Em~~ryo Sac Formation in
Raphanobrassica, 87 Hereditas 107 (1977) (incorporated
0 herein by reference) , and a maternal descendant of an
aposporic Raphanobrassica was documented (S. Ellerstrom,
Apomictic Progeny from Raphanobi:-assica, 99 Hereditas 315
(1983) (incorporated herein by reference). Apospory is
not expressed elsewhere in the parental species, genera,
.5 family, or entire Brassicales (Linnean), which strongly
suggests that apospory did not in this case surface as
a result of a gradual accumulation of developmentally
suppressed apomixis genes. To this effect, S.
Ellerstrom & L. Zagorcheva, 87 Hereditas 107 (1977),
:0 stated:
"In our opinion it see::ns therefore, more
justified to conclude that the formation of
aposporic embryo-sacs in Raphanobrassica is
caused by physiological disturbances, as a
?5 result of defective cooperation between the
two parent genomes in the hybrid, rather than
to assume the presence of specific genes
governing the formation o~= such embryo-sacs."
Sven Ellerstrom died shortly after this research was
30 conducted, and follow-up studies were not performed.
Neither S. Ellerstrom & L. Zago rcheva, 87 Hereditas 107
(1977) (incorporated herein by reference), nor S.
Ellerstrom, 99 Hereditas 315 (1983) (incorporated herein
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by reference), speculated as to the nature or cause of
the intergenomic physiological disturbances responsible
for apospory in Raphanobrassica beyond the wide
hybridization explanation offered by Ernst. Nor did
they identify how such disturbances might explain the
evidence for relatively simple Mendelian inheritance in
many apomicts and the fact that some apomicts appear to
have arisen from autopolyploidy. These deficiencies
were resolved in a study of the phylogeny and genomic
p composition of reproductive anomalies in angiosperms
(Linnean) wherein asynchronous expression of duplicate-
genes was identified. This concept explains essent~.ally
all major inconsistencies in the apomixis literature and
is supported by numerous phylogenetic, genetic, genomic,
5 and physiological studies (Linnean; M.D. Peel et al., 37
Crop Sci. 724-32 (1997); M.D. Peel et al., Crop Sci 37,
717-23 (1997)).
It will be appreciated that the present invention
may be embodied in other specific forms without
.0 departing from its spirit or essential characteristics,
which reside in the discovery that apomixis is caused by
asynchronous expression of duplicate genes for female
developmental pathways. The described steps and
materials are to be considered in all respects only as
>5 illustrative and not restrictive, and the scope of the
invention is indicated by the appended claims rather
than be the foregoing description. All changes which
come within the meaning and range of equivalency of the
claims are to be embraced within their scope.
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