Understanding Apomixis: Recent Advances and
Remaining Conundrums
Ross A. Bicknella and Anna M. Koltunowb,1
a Crop and Food Research, Private Bag 4704, Christchurch, New Zealand
b Commonwealth Scientific and Industrial Research Organization, Plant Industry, Adelaide, Glen Osmond,
South Australia 5064, Australia
INTRODUCTION
It has been 10 years since the last review on apomixis, or asexual
seed formation, in this journal (Koltunow, 1993). In that article,
emphasis was given to the commonalties known among
apomictic processes relative to the events of sexual reproduction.
The inheritance of apomixis had been established in some
species, and molecular mapping studies had been initiated. The
molecular relationships between apomictic and sexual reproduction,
however, were completely unknown. With research
progress in both sexual and apomictic systems in the intervening
years, subsequent reviews on apomixis in the literature have
considered the economic advantages of providing apomixis to
developing and developed agricultural economies (Hanna, 1995;
Savidan, 2000a) and strategies to gain an understanding of
apomixis by comparison with sexual systems (Koltunow et al.,
1995a; Grimanelli et al., 2001a). The potential to ``synthesize
apomixis'' in agricultural crops in which it is currently absent
by modifying steps in sexual reproduction and the possible
ecological consequences of the release of ``synthesized apomicts''
in nature also have been discussed (van Dijk and van
Damme, 2000; Grossniklaus, 2001; Spillane et al., 2001).
Recently, comparative developmental features of apomixis have
been considered in light of the now considerable knowledge
accumulated about ovule and female gametophyte development,
and seed formation in sexual plants (Koltunow and
Grossniklaus, 2003). In this review, we focus on the initiation
and progression of apomixis in plants that naturally express the
trait. Since 1993, there has been a growing understanding of the
complexity that underlies apomixis; some contentious issues
have been resolved and others raised. There also have been
significant advances in terms of new model systems and
approaches being used to study apomixis. We structure the
wider discussion around the knowledge of apomixis we have
accumulated from our study of Hieracium species, or hawkweeds,
a model system we established, and consider additional
factors that should be taken into account to induce apomixis in
crops. The continued comparative analyses of apomictic and
sexual reproduction at the fundamental level in appropriate
model systems remains essential for the development of successful
strategies for the greater application and manipulation
of apomixis in agriculture.
WHAT IS APOMIXIS?
Apomixis in flowering plants is defined as the asexual formation
of a seed from the maternal tissues of the ovule, avoiding
the processes of meiosis and fertilization, leading to embryo
development. The initial discovery of apomixis in higher plants is
attributed to the observation that a solitary female plant of
Alchornea ilicifolia (syn. Caelebogyne ilicifolia) from Australia
continued to form seeds when planted at Kew Gardens in
England (Smith, 1841). Winkler (1908) introduced the term
apomixis to mean ``substitution of sexual reproduction by an
asexual multiplication process without nucleus and cell fusion.''
Therefore, some authors have chosen to use apomixis to describe
all forms of asexual reproduction in plants, but this wider
interpretation is no longer generally accepted. The current usage
of apomixis is synonymous with the term ``agamospermous''
(Richards, 1997). Because seeds are found only among angiosperm
and gymnosperm taxa, this definition of apomixis limits its
use to those groups. In lower plants, phenomena similar to
apomixis are known, but discussion remains about the use of
this term in cases in which the reproductive structures involved
are different yet are considered analogous (Asker and Jerling,
1992).
PREVALENCE OF APOMIXIS
Although it is sometimes referred to as a botanical curiosity,
apomixis is far from rare, being relatively prevalent among
angiosperms, with a pattern of distribution that suggests that it
has evolved many times. It has been described in >400 flowering
plant taxa, including representatives of >40 families (Carman,
1997), and it is well represented among both monocotyledonous
and eudicotyledonous plants; curiously, though, it appears to
be absent among the gymnosperms. These estimates are
almost certainly very conservative. Unequivocal confirmation
of apomixis requires the simultaneous examination of both
genetic and cytological evidence (Nogler, 1984a). Embryological
examination of plant taxa for apomixis has not been exhaustive,
and supporting genetic evidence is uncommon even when
1 To whom correspondence should be addressed. E-mail: anna.
koltunow@csiro.au.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.017921.
The Plant Cell, Vol. 16, S228­S245, Supplement 2004, www.plantcell.org  2004 American Society of Plant Biologists
apomixis has been declared for a given plant. It seems likely that
as our understanding of this phenomenon grows and methods to
determine its presence improve, many more angiosperm taxa
will be found to include apomictic representatives and some
suspected cases will be revised. A recent study by Plitman (2002)
supports this prediction.
Several authors have noted a marked bias in the distribution of
apomixis among angiosperms (Asker and Jerling, 1992; Mogie,
1992; Carman, 1997; Richards, 1997). Of the plants known to
use gametophytic apomixis (Figure 1), 75% of confirmed examples
belong to three families, the Asteraceae, Rosaceae, and
Poaceae, which collectively constitute only 10% of flowering
plant species. Conversely, although apomixis is known among
the Orchidaceae, the largest flowering plant family, it appears to
be comparatively uncommon among these plants. Some authors
have postulated that the current patterns of distribution may
reflect the predisposition of certain plant groups to the unique
developmental and genetic changes that characterize apomixis
(Grimanelli et al., 2001b). This hypothesis appears intuitively
attractive, but like many issues associated with apomixis, it
remains conjecture until it is tested experimentally. Some of this
bias also might relate to the ease of embryological examination in
some plant groups or to data accumulated from embryological
investigations associated with activities in crop improvement.
There are other noted associations between apomixis and
various plant life history traits that provide insight into the nature
and possible ecological role of this phenomenon. Apomixis
frequently is associated with the expression of mechanisms that
limit self-fertilization (autogamy). Many apomictic plants belong
to genera in which sexual members predominantly exhibit
physiological self-incompatibility, dioecy, or heterostyly (Asker
and Jerling, 1992). In some cases, it is clear that the apomicts
themselves have retained such mechanisms. Dioecy is known
in a number of apomicts, such as Antennaria (O'Connell and
Eckert, 1999), Cortaderia (Philipson, 1978), and Coprosma
(Heenan et al., 2002). Similarly, self-incompatibility, a known
characteristic of the sexual biotypes of Hieracium subgenus
Pilosella (Gadella, 1984, 1991; Krahulcova` et al., 1999), was
demonstrated recently in the apomictic species H. aurantiacum
and H. piloselloides (Bicknell et al., 2003).
Apomictic species also are almost invariably perennials, and
they often use a vegetative mechanism of asexual reproduction,
such as stolon or rhizome growth. Thus, in the field, through
a combination of apomixis and vegetative division, apomicts can
form large clonal stands, and these may persist through long
periods of time. Apomixis also frequently leads to the formation
and maintenance of numerous morphologically distinct, yet
interfertile, varieties growing true to type from seed. The taxonomy
of such agamic complexes can be a difficult and contentious
task (Dickinson, 1998; Horandl, 1998). Examples of
genera in which the apomictic mode of reproduction is strongly
combined with morphological polymorphism include Alchemilla,
Hieracium, Poa, Potentilla, Ranunculus, Rubus, and Taraxacum
(Czapik, 1994). Apomicts are found more commonly in habitats
that are frequently disturbed and/or either where the growing
season is short, such as arctic and alpine sites, or where other
barriers operate to inhibit the successful crossing of compatible
individuals, such as among widely dispersed individuals within
a tropical rain forest (Asker and Jerling, 1992). There also are
clear indications that glaciation events defined the distribution
patterns of many agamic complexes (Asker and Jerling, 1992).
Gametophytic apomixis is known among herbaceous and tree
species, but it is considerably more common among the former.
This may simply be a reflection of the predominance of the trait in
the Poaceae and the Asteraceae, both of which are composed
largely of herbaceous species. Similarly, gametophytic apomixis
(Figure 1) is common among plants that have dehiscent fruits,
as seen in the Poaceae, Asteraceae, and Ranunculaceae, but
again, this may be more coincidence than causally linked.
MECHANISMS OF APOMIXIS
All known mechanisms of apomixis share three developmental
components: the generation of a cell capable of forming an
embryo without prior meiosis (apomeiosis); the spontaneous,
fertilization-independent development of the embryo (parthenogenesis);
and the capacity to either produce endosperm
Figure 1. Initiation and Progression of Apomictic Mechanisms Relative
to Events in the Sexual Life Cycle of Angiosperms.
The normally dominant vegetative phase of the life cycle is curtailed
in this figure to emphasize the events of gametophyte formation,
particularly the events in the ovule leading to sexually derived seeds
(pathway colored in yellow). Diplospory (purple) and apospory (red) are
termed gametophytic mechanisms because they initiate from a cell in
the position of the MMC or from other ovule cells, respectively, that
bypasses the events of meiosis and divides to mitotically to form an
unreduced embryo sac. Embryogenesis occurs autonomously from
a cell(s) in these unreduced embryo sacs. Endosperm formation may
require fertilization, or in a minority of apomicts it may be fertilization
independent. Adventitious embryony (green) is termed sporophytic
apomixis because embryos form directly from nucellar or integument
cells adjacent to a reduced embryo sac. The maturation and survival of
adventitious embryos is dependent on the endosperm derived from
double fertilization in the reduced embryo sac.
Apomixis in Plants S229
autonomously or to use an endosperm derived from fertilization
(Koltunow, 1993, Carman, 1997). That said, although broad similarities
can be seen at the level of the key events of apomixis, it is
probably true that there are as many mechanisms of apomixis
as there are plant taxa that express the trait. Some clear commonalities
have been identified, however, and these have been
used to categorize apomictic mechanisms into broad groupings.
Figure 1 portrays these mechanisms in relation to the temporal
sequence of events in the reproductive cycle of sexual plants.
Two main subgroups of mechanisms are apparent. In sporophytic
apomixis, or adventitious embryony, embryos arise spontaneously
from ovule cells late in the temporal sequence of ovule
maturation (Figure 1). Gametophytic apomixis operates through
the mediation of an unreduced embryo sac. Endosperm development
in these plants may be either spontaneous (autonomous)
or fertilization induced (pseudogamous) (Koltunow, 1993).
Gametophytic mechanisms are further subdivided based on
the cell type that gives rise to the unreduced embryo sac. In
diplosporous types, the megaspore mother cell (MMC) or a cell
with apomictic potential occupying its position is the progenitor
cell for the unreduced embryo sac. That cell may enter meiosis
but this aborts, and development proceeds by mitotic division to
achieve embryo sac formation (meiotic diplospory). Alternatively,
that cell might undergo direct mitosis to form an unreduced
embryo sac (mitotic diplospory). In aposporous apomicts, one or
more somatic cells of the ovule, called aposporous initials, give
rise to an unreduced embryo sac. Aposporous initials can
differentiate at various times during ovule development. Meiotically
reduced and aposporous embryo sacs might coexist in
the ovule, or the aposporous embryo sac might continue
development while the reduced sexual embryo sac degenerates
(Figures 1 and 2). The further subdivision of gametophytic
mechanisms (not shown in Figure 1) has been based on
characteristics related to the involvement, or avoidance, of the
different phases of meiosis, the number of mitotic divisions, and
the eventual form of the embryo sac (Crane, 2001).
The existence of apomixis in 40 plant families and the diversity
of apomictic processes suggests that the routes that led to the
evolution of apomixis may be as diverse as the known cytological
mechanisms. Morphological comparisons of structures formed
during sexual and apomictic reproduction provide hints that the
two reproductive pathways may share common elements, but
there also are numerous abnormalities. In the case of diplosporous
and aposporous embryo sac formation, a meiotic tetrad is
not observed. Positional differentiation of aposporous initials
(and adventitious embryos) occurs most frequently adjacent to
cells undergoing sexual reproduction, but they do not necessarily
arise from the nucellus, because epidermal and integumentary
origins have been described (Koltunow and Grossniklaus, 2003).
Irregularities also occur in the development of diplosporous and
aposporous embryo sacs, with structural abnormalities more
prevalent in apospory. Common abnormalities include a lack of
polarization of nuclei during early mitotic divisions, odd or
excessive numbers of nuclei varying in size, monopolar embryo
sacs, embryo sacs with inverted polarity or an abnormal situation
of the egg apparatus, and increased numbers of apparent polar
nuclei. In some cases, the final embryo sac structure is quite
stably different and used as a diagnostic tool, the best example
being the four-nucleus Panicum-type embryo sac (Koltunow,
1993; Czapik, 1994).
Most apomicts produce viable pollen. Even in diplosporous
apomicts, defects in the meiotic events of female gametophyte
development are not automatically extended to male gametophyte
formation or function. The presence of viable pollen
provides the possibility of fertilization of unreduced eggs. In
apomicts such as Taraxacum (Cooper and Brink, 1949; Richards,
1997), Poa, Parthenium, Tripsacum (Asker and Jerling, 1992),
and Hieracium piloselloides (Koltunow et al., 1998), embryo formation
can be precocious, initiating before anthesis or even
before the opening of the flower, limiting the possibility of unreduced
egg cell fertilization. In many sexual species, the egg cell
has an incomplete cell wall, and synthesis of a complete cell wall
occurs only in the zygote after fertilization. In the aposporous
apomict Pennisetum ciliare, however, a complete cell wall forms
around the unreduced egg cell before the arrival of the pollen
tube containing the two sperm cells (Vielle et al., 1995). P. ciliare
is a pseudogamous species, requiring fertilization to initiate
endosperm development. The early development of a complete
cell wall around the unreduced egg of this species appears to
serve as a means to avoid fusion of the second sperm cell with
the unreduced egg cell at the time of fertilization-induced
endosperm formation. Naumova and Vielle-Calzada (2001)
further noted that polysomes, endoplasmic reticulum, Golgi
bodies, and mitochondrial cristae were more abundant and more
developed in unreduced egg cells found in mature P. ciliare
aposporous embryo sacs than in the meiotically derived egg
cells of this species. These observations are consistent with
precocious egg cell maturation and suggestive of a loss or truncation
of the quiescent phase of egg cell development, a common
feature of sexually reproducing plants.
In apomicts, embryo and endosperm pattern formation may or
may not be conserved relative to that observed in related sexual
plants, although the latter has been least studied (Koltunow,
1993; Czapik, 1994). Apomicts also appear to tolerate imbalances
in parental gene dosage in their endosperm and still form
viable seeds (Grimanelli et al., 1997; Koltunow and Grossniklaus,
2003). Understanding how this occurs and its impact on seed
quality is particularly relevant for the installation of apomixis in
cereals in which imbalances in parental gene dosage in endosperm
are not tolerated.
THE POTENTIAL VALUE OF APOMIXIS IN AGRICULTURE
Apomixis is an attractive trait for the enhancement of crop
species because it mediates the formation of large genetically
uniform populations and perpetuates hybrid vigor through
successive seed generations. Many agronomic advantages of
apomixis can be envisioned: the rapid generation and multiplication
of superior forms through seed from novel, currently
underused germplasms; the reduction in cost and time of
breeding; the avoidance of complications associated with sexual
reproduction, such as pollinators and cross-compatibility; and
the avoidance of viral transfer in plants that are typically
propagated vegetatively, such as potatoes (Hanna, 1995;
Jefferson and Bicknell, 1995; Koltunow et al., 1995a, Savidan,
S230 The Plant Cell
2000a, 2000b). The value of these opportunities will vary between
crops and between production systems. For farmers in the
developed world, the greatest benefit is expected to be the
economic production of new, advanced, high-yielding varieties
for use in mechanized agricultural systems. Conversely, for
farmers in the developing world, the greatest benefits are expected
to relate to the breeding of robust, high-yielding varieties
for specific environments, improvements in the security of the
food supply, and greater autonomy over variety ownership
(Bicknell and Bicknell, 1999; Toenniessen, 2001).
However, apomixis is very poorly represented among crop
species. The main exceptions to this appear to be tropical and
subtropical fruit trees, such as mango, mangosteen, and Citrus,
and tropical forage grasses, such as Panicum, Brachiaria,
Dichanthium, and Pennisetum. It is possible that the low representation
of apomixis among crops arose unintentionally from
a protracted human history of selecting superior plants for future
cultivation. Selection for change over a parental type would work
against a mechanism such as apomixis that acts to maintain
uniformity. The presence of the trait among tropical fruit and
Figure 2. Early Events of Embryo Sac Formation in Ovules of Sexual and Apomictic Hieracium Plants.
The early events of reduced embryo sac formation in sexual plants and in apomicts are colored yellow, and aposporous embryo sac formation is colored
red. The numbers represent morphological stages of capitulum development as defined by Koltunow et al. (1998), and mature embryo sac structures
are not shown for stages P4 and D3. In stage A3.4, multiple embryo sacs form with few nuclei, and these coalesce to form a single embryo sac
(Koltunow et al., 1998). The presence of callose in the walls of MMCs in ovules at stages 2 or 3 and in meiotic tetrads in ovules at stages 3 or 3/4 is shown
by the fluorescence of trapped aniline blue dye after exposure to UV light (Tucker et al., 2001). Meiotic tetrads in stages A3.4 and D2 are rare because of
the presence of multiple aposporous initials and embryo sacs that physically distort and/or crush the structure. Aposporous initial cells do not contain
callose in their cell walls (Tucker et al., 2001). Stage D2 also forms isolated embryos external to an embryo sac (Koltunow et al., 2000). aes, aposporous
embryo sac; ai, aposporous initial; em, embryo; es embryo sac; et, endothelium; fm, functional megaspore; mmc, megaspore mother cell.
Apomixis in Plants S231
grass crops may be a reflection of this effect, because focused
efforts to improve these crops are comparatively recent events.
There also are few apomictic species of significant relatedness
available for use in introgression programs, which may explain at
least some of the difficulties experienced when attempts have
been made to introduce apomixis into crops through hybridization.
For example, major programs aimed at introducing apomixis
into maize (Sokolov et al., 1998; Savidan, 2000a, 2001) from the
wild relative Tripsacum dactyloides have been under way now for
decades, yet they have proven unsuccessful in terms of generating
apomictic plants with agronomically acceptable levels of
seed set. Difficulties also have been encountered in efforts to
produce apomictic lines of hybrid millet (Morgan et al., 1998;
Savidan, 2001). Even if successful, it seems likely that introgression
lines would provide limited flexibility in terms of
practical capacity to manipulate apomixis in agricultural breeding
systems. Current breeding efforts with apomictic crop species,
such as the forage grasses Brachiaria and Panicum, are
frustrated by the need to use complex breeding strategies to
accommodate the inaccessibility of the female gamete to generate
hybrid progeny (Valle and Miles, 2001). We believe,
therefore, that the best solution would be the introduction of
apomixis into crops in an inducible format, permitting its use
during seed increase but allowing for its silencing during hybridization.
To achieve this, information will be required concerning
the genes that control the trait, their interrelationship with
sexual processes, and the impact the trait might have on seed
yield, viability, and quality for a given plant.
EXPERIMENTAL APPROACHES AND MODEL SYSTEMS
IN APOMIXIS RESEARCH
Current research on apomixis generally is divided into two
complementary approaches: the evaluation of the trait in natural
apomictic systems, and the ``synthesis'' of the trait through the
directed modification of reproductive events in a sexual species.
Of the native monocot apomictic systems under study, most
work focuses on aposporous plus pseudogamous species in the
genera Panicum (Savidan, 1980; Chen et al., 1999), Pennisetum
and Cenchrus (Roche et al., 1999), Brachiaria (Pessino et al.,
1997, 1998), Paspalum (Martinez et al., 2001), and Poa (Naumova
et al., 1999; Albertini et al., 2001a, 2001b) and the diplosporous
plus pseudogamous genus Tripsacum (Grimanelli et al., 1998a,
1998b; Sokolov et al., 1998). The eudicot genera under study
include the daisy genera Taraxacum and Erigeron (diplosporous
plus autonomous) (Noyes and Rieseberg, 2000; van Dijk et al.,
2003) and Hieracium (aposporous plus autonomous) (Koltunow
et al., 1998, 2000). Hypericum perforatum (aposporous plus
pseudogamous), a member of the St. John's wort family, has
been well studied (Matzk et al., 2001, 2003), and Boechera
holboellii (syn. Arabis holboellii; diplosporous plus pseudogamous)
(Naumova et al., 2001; Sharbel and Mitchell-Olds, 2001),
a relative of Arabidopsis, also is receiving attention for its
potential to exploit the vast genetic and molecular resources
developed in Arabidopsis. We suspect that these efforts may
become more focused in coming years as the relative advantages
of one or a small number of these systems leads to their
predominant use (Bicknell, 2001). Among the sexual species
under study for the synthesis of apomixis, there is little doubt that
Arabidopsis, rice, and maize will remain the systems of choice
because of the availability of genetic and molecular tools and, for
the latter crops, the strong economic and social drivers seeking
the installation of the trait (Khush, 1994; Savidan, 2000a).
HIERACIUM SUBGENUS PILOSELLA, A MODEL SYSTEM
FOR APOSPOROUS PLUS AUTONOMOUS
GAMETOPHYTIC APOMIXIS
During the last 10 years, Hieracium subgenus Pilosella has
been developed into a model system for the genetic and molecular
study of gametophytic apomixis (Bicknell, 1994a, 2001;
Koltunow et al., 1998; Koltunow, 2000). These plants are highly
suited for molecular studies because they are small, herbaceous
perennials with a rapid generation time (3 to 6 months) and
a long-day photoperiodic response that allows them to be
flowered on demand at any time of the year (Yeung, 1989). Some
species include both sexual and apomictic biotypes, and hybrids
formed between most of the species have proven to be fertile.
This allows the inheritance of apomixis to be studied using
intraspecific hybridization, in contrast to many other apomictic
plant systems. Methods for the micropropagation (Bicknell,
1994b), anther culture (Bicknell and Borst, 1996), genetic transformation
(Bicknell and Borst, 1994), and progeny class estimation
(Bicknell et al., 2003) of Hieracium have been developed to
further facilitate its use in the molecular study of apomixis. The
plants also are very amenable to vegetative propagation, which
is of particular value when genetic or molecular manipulations
affect flower form and/or seed set. Vegetative propagation also is
the preferred mode of stock maintenance, because a fraction of
the progeny is not true to type (see below).
In all of the wild-type apomicts of Hieracium studied to date,
both the embryo and endosperm arise spontaneously inside an
unreduced aposporous embryo sac (Figure 2). The initiation of
apospory is stochastic in these plants, but in each form, the
majority of aposporous initial cells tend to differentiate at
a particular time relative to the concurrent sexual process in
the ovule. This makes the plants valuable for examining factors
that influence the timing of aposporous initial formation. In most
of the apomictic Hieracium plants characterized to date, the
sexual process usually ceases soon after apospory initiates
(Koltunow et al., 1998, 2000; Bicknell et al., 2003). This provides
an opportunity to examine signaling between apomictic and
sexual pathways and to examine factors that mediate the
survival of one mode of embryo sac formation over the other
(Figure 2). Whether one or many aposporous embryo sacs
initiate, almost always a single one survives; nevertheless, in
some species, multiple embryos can form in an individual
aposporous embryo sac. Thus, the mechanisms that limit
aposporous embryo sac and embryo frequency can be
examined.
Endosperm development was examined recently in the autonomous
apomict H. piloselloides (tall hawkwed) and compared
with that in a sexual biotype, H. pilosella (mouse ear hawkweed).
In the apomict, autonomous endosperm development initiates
primarily, but not exclusively, from fused polar nuclei. In contrast
to the fertilization-dependent endosperm development in the
S232 The Plant Cell
sexual plant, migrating clumps of nuclei form during early
endosperm development in the apomict, but as nuclei numbers
increase, this is rectified so that the cellularization and maturation
events are cytologically indistinguishable in the two types (M.
Tucker and A.M. Koltunow, unpublished results). Apomictic
Hieracium plants exhibit developmental abnormalities reminiscent
of epigenetic phenomena. For example, there are disturbances
in the pattern of embryo formation and phyllotaxis
during early seedling growth in apomictic Hieracium that are
reset eventually to a normal pattern with subsequent plant
growth (Koltunow et al., 1998, 2000).
Sexual reproduction, or components of it, are not excluded
completely in apomictic Hieracium. In some ovules, either
apospory does not initiate or it fails and the meiotic process
proceeds to completion. In addition, fertilization may or may not
occur in these plants irrespective of the reduced or unreduced
nature of the egg. Thus, Hieracium is facultative for apomixis,
because a fraction of the seedlings are nonmaternal in genotype.
DIFFERENT PROGENY TYPES FOUND IN
FACULTATIVE APOMICTS
The coexistence of apomixis and sexuality is certainly not unique
to Hieracium. For all of the mechanisms of apomixis known, and
for almost all of the genotypes studied in depth, it has been
reported that some degree of sexuality remains possible in
apomictic plants; thus, most are regarded as facultative. True
obligate apomicts, those able to form seeds by apomixis alone,
are very uncommon (Asker and Jerling, 1992). Facultative,
gametophytic apomicts are capable of hybridization using either
a reduced or an unreduced egg cell. The previously described
mechanisms of early egg cell wall completion and precocious
embryogenesis of unreduced eggs are by no means fully
penetrant. This can lead to the formation of two different types
of hybrid progeny. Reduced hybrids result from the fusion of
a reduced egg cell with a reduced sperm cell, whereas unreduced
hybrids typically result from the mediation of an unreduced
egg cell. In the nomenclatural system of Rutishauser
(1948), these are referred to as BII and BIII hybrids, respectively.
Harlan and De Wet (1975) termed them n1n and 2n1n hybrids,
respectively, whereas Asker (1977) used the terms R- and
U-hybrids. Unfortunately, all of these terms are still used in
the literature, but it does appear that the system of Harlan and De
Wet (1975) is now the most popular choice, and we will continue
with its use in this article. A further progeny class of note in these
plants are n10 seedlings, or ``polyhaploids.'' These plants result
from the parthenogenetic development of a reduced egg cell,
leading to the formation of a plant with a ploidy state less than
that of the parent. Although male meiosis and male gamete
function are typically normal in gametophytic apomicts, the
formation and use of unreduced pollen also has been observed
(Matzk et al., 2001). Both the formation of unreduced gametes
and the parthenogenetic development of unfertilized egg cells
are widely recorded phenomena in sexual species (Grossniklaus,
2001); however, they are typically much more frequent events
among apomicts. In the case of H. aurantiacum and H. piloselloides,
the rates of parthenogenesis (which includes 2n10 and
n10 progeny) were 97.6 and 98.0%, respectively (Bicknell et al.,
2003) (Figure 3) compared with reported examples of 0.11% for
maize and 0.018% for Datura (Kimber and Riley, 1963).
An important practical outcome of the diversity of progeny
types produced by apomictic Hieracium, and for that matter by
most other apomicts, is that the progeny of these plants cannot
be considered to be uniformly of clonal, maternal origin. In
particular, because different progeny classes result from the
different actions of segregation (meiosis) and recombination
(fertilization), different inheritance ratios are expected in each
progeny class, and this fact must be considered in any analysis of
trait transmission. Even in sporophytic apomicts such as Citrus,
in which spontaneous embryogenesis adjacent to a reduced
embryo sac is the defining manifestation of this form of apomixis
(Koltunow et al., 1995b), some authors have noted that unreduced
embryo sacs do also form (Naumova, 1992). Plants with
unexpectedly high ploidy levels are frequently observed in
breeding populations of Citrus, and it seems likely that such
individuals arise following the fertilization of an unreduced egg
cell, in a manner similar to that seen in gametophytic apomicts.
Given the importance of understanding the relative levels of
different progeny types among the seedlings produced by
Figure 3. Progeny Types Formed in Crossed Hieracium Species
Facultative for Aposporous Plus Autonomous Apomixis.
Most of the progeny are maternal (2n10), because there is no genomic
contribution from a male gametophyte. Nonmaternal seedlings form
a smaller subset that can be subdivided into a number of types.
Seedlings also are derived from the use of a reduced egg that undergoes
autonomous embryogenesis (n10); alternatively, the egg can be fertilized
with a sperm cell from self (s) or nonself (ns) pollen to give rise to
hybrids that arise from sexual reproduction (n1ns or n1nns, respectively).
Unreduced egg cells also can be fertilized with sperm cells from self or
nonself pollen to give rise to hybrid progeny (2n1ns or 2n1nns, respectively)
that have increased ploidy relative to the maternal parent. D3
ovules appear to be strictly self-incompatible, whereas in A3.4, progeny
arising from self-pollination are evident (data are from Bicknell et al.,
2003).
Apomixis in Plants S233
a facultative apomict, there has been a strong need for robust
methodologies to determine these levels and to isolate representative
subpopulations for separate analysis. For many years,
the nonmaternal (``aberranť' or ``off-type'') progeny of apomicts
were identified through morphological scoring, chromosome
counting, or progeny cross methods, which were too inaccurate
and too expensive to warrant their use on any scale. Recently,
methods based on flow cytometry and transgenesis have been
published that address these limitations. The method of Matzk
et al. (2000), which applies a flow cytometric test to individual
seeds, is proving to be particularly versatile in that it permits the
rapid screening of previously uncharacterized material for signature
characteristics of apomixis (Matzk et al., 2001, 2003). In
Hieracium, a method based on the inheritance of positive and
negative selectable transgenic markers has been described
(Bicknell et al., 2003). This method, which is based on the survival
of different progeny types when placed on selective media,
provides the added versatility of being nondestructive and less
expensive to apply. It also provides a mechanism for the direct
isolation of aberrant progeny individuals. Its use, however, is
limited to plants that can be transformed genetically, and it is less
suited for survey studies that assess and compare a large
number of genotypes.
The facultative expression of apomixis, therefore, is an experimental
complexity in the study of these plants. However, it
has proven to be a valuable tool for exploring the unique biology
of apomixis, particularly in the analysis of interactions between
apomixis and sexuality. For example, polyhaploidy (n10) was
used to isolate diploid apomictic plants of the species
H. aurantiacum and H. piloselloides (Bicknell, 1997; Koltunow
et al., 2000; Bicknell et al., 2003), demonstrating that apomixis
can occur in both diploid and polyploid members of this species.
Similarly, the quantification of hybrid (2n1n and n1n) progeny in
both H. aurantiacum and H. piloselloides indicated that a selfincompatibility
mechanism was acting to influence progeny
formation in these plants despite the autonomous nature of
apomixis in Hieracium (Bicknell et al., 2003) (Figure 3).
Another area of study has been the origin of multiple embryos,
a common occurrence in the seeds of many apomicts. The formation
of multiple viable embryos, or polyembryony, is evident in
up to 10% of H. aurantiacum seeds and 17% of H. piloselloides
seeds (Koltunow et al., 2000; Bicknell et al., 2003). The origin of
the additional embryos remains a mystery, because in the
majority of ovules the sexual process terminates and a single
aposporous embryo sac is present at the initiation of embryogenesis.
We previously hypothesized that the additional embryos
might arise from trapped aposporous initials or perhaps
endosperm cells that had altered in fate (Koltunow et al., 1998).
From a study quantifying progeny types (Bicknell et al., 2003), it
was found that meiotically derived seedlings were seven times
more likely to arise alongside viable, asexually derived embryo(s)
in a polyembryonic seed than from a single embryo seed. The
reason why a partnering asexual embryo is present so often
remains unclear. One possibility is the formation of adventitious
embryos either from reprogrammed aposporous initials or from
other somatic cells. This is not completely speculative, given that
an experimentally derived diploid form of H. piloselloides readily
forms adventitious embryos (Koltunow et al., 2000) (Figure 2).
Alternatively, embryogenic development may proceed from both
coexisting aposporous and reduced sexual embryo sacs, an
event that might have proven too difficult to detect cytologically
because of its rarity.
The interplay between sexuality and apomixis in facultative
plants provides an opportunity to consider the evolutionary and
ecological implications of these different modes of reproduction
(Krahulcova` et al., 2000; Houliston and Chapman, 2001). For
example, Bicknell et al. (2003) noted that in H. aurantiacum and
H. piloselloides, the rates of seedlings derived from meiotic egg
usage (n1n and n10) were very similar, 2.60 and 2.85%,
respectively (Figure 3), yet the modes of aposporous embryo sac
development are very different in these plants (Koltunow et al.,
1998). This result indicates that the rate of meiotic egg use giving
rise to viable progeny in these plants is unlikely to be a chance
event; rather, it is a characteristic optimzed under selection to
reflect the relative costs and opportunities of sexual and asexual
reproduction in these plants. This proposal is supported by
evidence for the operation of a self-incompatibility mechanism in
these plants (Bicknell et al., 2003). Field evidence further indicates
that sexuality is an important driver in the maintenance of
diversity in these plants. Chapman et al. (2000) noted that the
patterns of genetic diversity seen among Pilosella officinarum
(syn. Hieracium pilosella) clones at five dispersed sites in New
Zealand suggested the frequent action of hybridization, despite
the relatively recent introduction of this weed into that country
and the predominantly apomictic nature of the clones studied
(Houliston and Chapman, 2001). Similar findings have been
reported for the related daisy genus Taraxacum (van der Hulst
et al., 2000).
CALLOSE DEPOSITION: EARLY CLUES TO APOMIXIS
CELL TYPE IDENTITY?
One means of assessing the relationships between sexual and
apomictic pathways is to use developmental markers that
identify specific cells or structures in sexual systems and to
examine their expression in apomictic plants. One of the earliest
used was the biochemical detection of callose, a b-1,3-linked
glucan that accumulates in a distinctive manner during female
gametophyte development in sexual plants. The walls of the
MMC (or megasporocyte), the tetrad of megaspores (Figure 2),
and degenerating megaspores are marked by the temporary
accumulation of callose. It is lost from the walls of the selected
megaspore during its expansion and is absent once the mitotic
events of embryo sac development initiate.
Differences in callose deposition during gametophyte development
in sexual and apomictic plants have been noted, and
in the case of Tripsacum, this has been used as a screening tool
to identify apomicts (Leblanc and Mazzucato, 2001). In both
diplosporous Tripsacum and Elymus, which exhibit diplospory
(Carman et al., 1991), callose is absent from the walls of the cell
that initiates diplospory, which is otherwise identified by position,
size, and appearance as the MMC. Given that residual sexuality
was low in the apomicts examined, it was not possible to directly
compare meiotic and diplosporous processes in the same material;
however, callose was present in the MMC of sexual
S234 The Plant Cell
accessions. In aposporous species in which the initiation of both
sexual and apomictic processes can be observed simultaneously,
callose was completely absent from aposporous initial
cells in Poa pratensis, Pennisetum (Peel et al., 1997), Brachiaria
(Dusi and Willemse, 1999), and Hieracium (Tucker et al., 2001)
but evident in the MMC (Figure 2). Studies in aposporous Poa
and Pennisetum also have noted differences in callose distribution
in the adjacent MMC. This was not obvious in aposporous
Hieracium, but an early appearance of initials correlated with
the persistence of callose (Tucker et al., 2001). Furthermore,
changes in b-1,3-glucanase expression postulated by Peel et al.
(1997) to coincide with aposporous initial formation were not
evident in Hieracium with respect to mRNA distribution in the
plants examined, although changes in protein activity cannot be
discounted (Tucker et al., 2001). Assuming that apomixis is an
aberrant form of sexual reproduction in all of the species
mentioned above, then the lack of callose in cells that initiate
diplospory and apospory implies that they do not share identity
with functional MMCs. Peel et al. (1997) have proposed that the
MMC of diplosporous plants undergoes ``precocious gametophytization,''
which could be interpreted as a change in identity,
but other markers are required to clarify this possibility.
MOLECULAR RELATIONSHIPS BETWEEN SEXUAL AND
APOMICTIC PATHWAYS
Considerable information has now been accumulated concerning
the regulation of ovule and female gametophyte development
in sexual plants, and this provides molecular tools for the
comparative analysis of sexual and apomictic reproduction
(Koltunow and Grossniklaus, 2003). Tucker et al. (2003) provided
strong evidence that sexual and apomictic reproduction in Hieracium
are related developmental pathways that share common
regulatory programs (Eckardt, 2003). They examined the expression
patterns of several reproductive marker genes from Arabidopsis
in sexual and apomictic Hieracium. These markers were
fusionconstructsofb-glucuronidase(GUS)andavarietyofArabidopsis
genes as promoter:GUS or chimeric protein fusions. The
markers included SPOROCYTELESS (SPL/NOZZLE), which is
required for male and female sporogenesis in Arabidopsis
(Schiefthaler et al., 1999; Yang et al., 1999), SOMATIC EMBRYO
RECEPTOR KINASE1 (SERK1), which is thought to play a role in
embryogenesis (Hecht et al., 2001), and three independent FIS
class genes, mutations in any one of which result in fertilizationindependent
endosperm development (Luo et al., 2000). An
astonishing conservation of expression pattern was observed in
apomictic and sexual Hieracium plants using these chimeric
genes that for the most part also reflected patterns observed in
Arabidopsis (Tucker et al., 2003). AtSPL:GUS was expressed in
MMCs but not in aposporous initials, providing the first molecular
evidence that these cells develop from somatic cells that do not
share identity with MMCs. SERK1:GUS expression was conserved
in pattern in sexual and apomictic plants, and the three
FIS chimeric genes were expressed coordinately during the early
events of seed development in Hieracium.
The main differences in spatial and temporal expression pattern
occurred early in ovule development and, in marked contrast
to Arabidopsis AtFIS2:GUS, was expressed at the completion of
meiosis in both sexual and apomictic Hieracium (Figure 4). The
spatial pattern of expression, however, differed in sexual and
apomictic plants. In sexual Hieracium, expression of AtFIS2:GUS
was observed in the three megaspores destined to degenerate,
was absent from the single cell layer enveloping them (nucellar
epidermis) destined for degeneration, and was absent from the
selected megaspore until the first nuclear division of embryo sac
formation. By contrast, in two apomictic Hieracium species with
differing modes of aposporous embryo sac formation, AtFIS2:
GUS expression was observed in all four megaspores and
the enveloping nucellar epidermis, all of which were destined
for degeneration. Expression of AtFIS2:GUS was absent from
aposporous initials until their first nuclear division, similar to the
expression profile observed for the functional megaspore in
sexual plants (Figure 4). We speculate that the spatial differences
in AtFIS2:GUS expression in sexual and apomictic plants might
reflect gene expression shifts associated with aposporous initial
cell commitment to the events of embryo sac initiation and the
concomitant displacement of the sexual pathway. Therefore, FIS
genes might play a different role in Hieracium relative to that in
Arabidopsis that could be related to the capacity for apomixis
and autonomous seed development (Eckardt, 2003). The isolation
and characterization of endogenous Hieracium FIS genes
is under way (Koltunow and Tucker, 2003).
The findings of Tucker et al. (2003) indicate that sexual and
apomictic pathways in Hieracium share common gene expression
profiles and thus common molecular regulatory features,
indicating that they are not distinct pathways (Figure 4). The form
of apomixis in Hieracium (apospory coupled with autonomous
embryo and endosperm development) appears to differ from
sexual reproduction in a very limited number of ways. Specifically,
it is comparable to sexual development except for two
specific switch points: meiosis and fertilization. For apomixis to
occur in Hieracium, the developmental program of one or at most
a few cells is altered, and this appears to be sufficient to enable
them to bypass meiosis yet mimic the normal program seen in
the descendants of a selected megaspore. Later development
bypasses fertilization but still moves through the normal embryo
and endosperm developmental programs (Figure 5). These data
suggest that apomixis is a deregulation of the sexual reproductive
program in space and time, leading to cell fate changes
and the omission of steps critical to sexual progression. It is
conceivable that different types of apomixis, as seen in other
species, might arise depending on when and where this deregulation
occurs (Koltunow and Grossniklaus, 2003). Figure 1
also portrays this concept in the sense that it depicts the
three broad classes of apomictic mechanisms as operating
within the framework of the sexual reproductive cycle. Differential
and subtractive hybridization have been attempted to isolate
genes relevant to apomictic reproduction (Vielle-Calzada et al.,
1996; Guerin et al., 2000; Pessino et al., 2001). Although not
exhaustive, these have failed to find genes specific to structures
arising from apomictic development when the expression of
these genes has been examined (Guerin et al., 2000). Although
negative in context, this observation further supports the proposal
that sexual and apomictic developmental pathways differ
primarily at the level of regulation of common elements.
Apomixis in Plants S235
Figure 4. AtFIS2:GUS Expression during Early Ovule Development in Hieracium.
Ovules from sexual Hieracium P4 ([A] to [D]), apomictic Hieracium D3 ([E] to [H]), and apomictic Hieracium A3.4 ([I] to [L]) were stained with GUS and
viewed whole mount using dark-field microscopy ([A], [E], and [I]) or Nomarski differential interference contrast microscopy. The numbers at the top
right indicate the ovary stage (Koltunow et al., 1998). Bars  50 mM in (A), (E), (I), and (L), and bars  25 mM in (B) to (D), (F) to (H), (J), and (K).
(A) P4 ovule in the chalazal (ch) to micropylar (mp) orientation showing GUS stain in pink.
(B) Enlarging selected spore (ss) and blue GUS-stained degenerating megaspores (dms) surrounded by the nucellar epidermis. Indicated structures are
outlined with a black line.
(C) Enlarging functional megaspore (fm) with a large nucleus (n) chalazal to degenerated megaspores. Indicated structures are outlined with a black line.
(D) Ovule containing an early embryo sac (es) containing dividing embryo sac nuclei (esn) and surrounded by the endothelium (et).
(E) D3 ovule showing the corresponding stage of apomictic development to (A).
(F) Enlarging aposporous initial (ai) at the corresponding stage of apomictic development to (B). The aposporous initial, outlined with a broken line, is
forming in a slightly different plane to the other structures, which are outlined with an unbroken line.
(G) Enlarging aposporous initial cell above two smaller initials.
(H) D3 ovule containing a dividing aposporous embryo sac (aes) with embryo sac nuclei (esn) at the corresponding stage of apomictic development
to (D).
(I) A3.4 ovule showing the corresponding stage of apomictic development to (A) and (E).
(J) Enlarging aposporous initials at the corresponding stage of apomictic development to (B) and (F). Multiple aposporous initial cells are indicated with
broken lines, and some form in slightly different planes compared with the sexual structures; these are outlined with an unbroken line.
(K) Enlarging aposporous initial cells.
(L) Aposporous embryo sac structures at the corresponding stage of apomictic development to (D) and (H).
Figure reprinted from Tucker et al. (2003) with permission.
S236 The Plant Cell
SIGNALS FROM OTHER OVULE TISSUES?
In sexual species such as Arabidopsis, the development of both
the embryo sac and the embryo is influenced by signals from
surrounding sporophytic ovule tissues (Ray et al., 1996; Gasser
et al., 1998; Schneitz, 1999). Some preliminary lines of evidence
suggest that the sequence of apomictic events in Hieracium is
relatively flexible, influenced by information from surrounding
ovule tissues. Ovule identity and form were altered significantly in
flowers that develop from ectopic meristems in H. piloselloides
plants expressing the rolB oncogene from Agrobacterium
rhizogenes. This gene is known to alter plant growth morphogenesis
and cellular sensitivity to auxin. In structurally deformed
ovules characterized by elongated and distorted funiculi and
aberrant integuments, the sexual process ceased earlier, before
meiosis, and a higher frequency of aposporous initials was
observed. Surprisingly, however, embryos and endosperm continued
to develop (Koltunow et al., 2001). A diploid apomict, D2
(n10 progeny from D3; Figure 3), was found to exhibit similar but
less severe defects in funiculus and integument formation than
those observed in ovules from rolB plants. The developmental
events of apomixis in D2 ovules phenocopied those found in rolB
ovules in terms of the increased frequency of apomictic initiation
and continuation of the process in malformed ovules (Koltunow
et al., 2000). However, the D2 plant also is capable of forming
isolated embryos that are found external to an embryo sac
structure, mechanistically resembling the process of adventitious
embryony (Koltunow et al., 2000) (Figure 2). These embryos
might arise from aposporous initial cells diverted to a different
fate as a result of alterations in ovule structure (Figure 5).
Alternatively, signaling changes resulting from ovule malformation
may stimulate a parthenogenetic program in a set of somatic
cells in a manner possibly reminiscent of stress-induced somatic
embryogenesis (Mordhorst et al., 1997). All of these possibilities,
including the role of auxin in apomictic events in Hieracium,
require further investigation.
THE GENETIC BASIS OF APOMIXIS
The first known study of inheritance in an apomictic plant was
unknowingly conducted by Gregor Mendel (1869) on Hieracium,
ironically selected to assist in corroborating his laws of inheritance
(reviewed by Correns, 1905; Nogler, 1994). It is not known
how many crosses Mendel conducted on Hieracium because
most of the data have been lost, but extrapolations from the
information that is available indicates that he performed many
thousands of crosses over a period of >10 years. By direct
contrast to his observations in pea, the Hieracium F1 hybrids
showed extensive segregation, whereas the F2 ``hybrids'' did not
segregate and uniform progeny were obtained consistently. In
correspondence with Na¨ geli, a Hieracium specialist (July 1870),
Mendel noted the ``almost opposed behavior'' in the two systems
``both [of which represented] the emanation of a higher universal
law.'' By the turn of the 20th century, apomixis was a known
phenomenon in plants, and Ostenfeld (1904, 1906, 1910) returned
to the study of inheritance in Hieracium. He conducted
several cross combinations, including repetitions of Mendeľs
work, and, along with Rosenberg (1906, 1907), correctly noted
the expression of apomixis in this genus. It had taken almost
40 years to explain Mendeľs data.
The inheritance of gametophytic apomixis has since been
reported to be associated with the transfer of either a single locus
or a small number of loci in most of the systems studied to date.
In the aposporous grasses Pennisetum (Sherwood et al., 1994),
Panicum (Savidan, 1983), and Brachiaria (Valle et al., 1994),
apomixis is reported to be simply inherited, with the trait
conferred by the transfer of a single dominant factor. Simple
dominant inheritance also has been reported for apospory in the
dicotyledonous genera Ranunculus (Nogler, 1984b) and Hieracium
(Bicknell et al., 2000). Among the diplosporous apomicts,
independent inheritance of diplospory and parthenogenesis
have been observed in the dandelion Taraxacum (van Dijk et al.,
1999) and in Erigeron (Noyes, 2000; Noyes and Rieseberg, 2000),
Figure 5. A Model for Apomixis in Hieracium (Modified from Tucker et al., 2003).
Apomixis in Plants S237
whereas Voigt and Burson (1983) reported the simple dominant
inheritance of diplospory in Eragrostis curvula, the weeping
lovegrass. Similarly, the inheritance of diplospory in Eastern
gamagrass (Tripsacum dactyloides) is reported to be simple and
dominant (Leblanc et al., 1995). There is evidence of segregation
ratio distortion in some of these systems, often because the
dominant factor(s) associated with apomixis also appears to
confer gamete lethality, restricting its transfer to some gamete
genotypes (Nogler, 1984b; Grimanelli et al., 1998a; Roche et al.,
2001a; Jessup et al., 2002).
From these and other older studies, it is widely generalized that
there is either one locus, or only a small number of loci, involved
in the inheritance of apomixis in native systems. There are,
however, some important caveats associated with the interpretation
of these data. In almost all of the cases mentioned, the
inheritance of apomixis was recorded after crosses between
closely related sexual and apomictic species. Very often in these
studies, the expression of apomixis restricts the use of the
apomict to the pollen parent. Reciprocal crosses are seldom
reported, and the influence of maternal effects remains largely
untested. Additionally, particular care must be exercised in
scoring progeny during these studies. Apomixis is a complex
trait. Ideally, it should be measured strictly through the production
of genetically identical seedlings, coupled with an
embryological study to confirm the reproductive mode of each
progeny plant and an assessment of ploidy in all individuals
(reviewed by Nogler, 1984a). This is often so time consuming that
correlated characteristics are used as a measure of apomixis
(for review, see Bicknell, 2001; Leblanc and Mazzucato, 2001).
Examples include the development of tetranucleate embryo sacs
in Panicum (Savidan, 1980) and the formation of globular
embryos after emasculation in Hieracium (Bicknell et al., 2000).
In all such cases, there are opportunities for inaccuracies in the
estimation of intact, functional apomixis. For example, in
Panicum, an overestimate may result if <100% of the embryo
sacs observed form functional unreduced embryos, whereas in
Hieracium, the embryos formed are clearly the result of
parthenogenesis but it is not clear if they are derived from
unreduced eggs. Indeed, from a reexamination of the data
collected during earlier studies of Hieracium, including our own
(Bicknell et al., 2000), together with a new analysis of inheritance
in this system, we now believe that the number of controlling
loci has been underestimated. Rather than a single locus being
involved, it appears that as many as three unlinked dominant
factors may need to be inherited to ensure the transfer of
autonomous aposporous apomixis as an intact trait in Hieracium
(our unpublished data). Recent studies also suggest that three
unlinked loci are required for the inheritance of autonomous
diplosporous apomixis in Taraxacum officinale (van Dijk et al.,
2003).
Another difficulty with the interpretation of some of the published
inheritance data for apomixis is that it often treats
apomixis as a qualitative trait, one that is either inherited or not.
As mentioned above, apomixis is expressed facultatively in most
plants, and the level of viable asexual seed formation can vary
considerably between individuals. A common observation from
inheritance studies is that the level of apomixis expressed by
many of the F1 progeny is well below that of the apomictic parent
(Nogler, 1984b; Koltunow et al., 2000). This effect can be quite
profound. Most of the apomictic F1 progeny observed in an
inheritance study conducted by the authors on Hieracium expressed
the trait at <5% of the total seed set, whereas apomixis
was rated at 98% in the apomictic parent (Bicknell et al., 2000;
Koltunow et al., 2000; R. Bicknell, unpublished results). Attempts
to introgress apomixis from wild relatives to crops through serial
backcrossing also provide an indication of this phenomenon.
Tripsacum dactyloides is an apomictic relative of maize. Savidan
and co-workers have been involved in an effort to develop
apomictic maize for many years through the introgression of
Tripsacum DNA into Zea mays. Their efforts are particularly
impressive for the significant amount of effort involved and for the
intelligent use of advanced technologies for the rapid screening
of large hybrid populations. Despite their dedication and the
apparently ``simple'' inheritance of the trait, however, although
significant progress has been made to bring their materials to the
BC4 generation, the integration of intact, functional apomixis into
a genotype with just the 20 chromosomes of maize remains an
intractable goal. Among the BC4 plants reported to date, no
fertile apomictic individuals have been confirmed with fewer than
16 complete Tripsacum chromosomes (Savidan, 2001). Similar
outcomes have been reported for the introgression of apomixis
into pearl millet (Pennisetum glaucum) from related apomicts
(also reviewed by Savidan, 2001).
These data and experiences have led some in the field of
apomixis to speculate that the trait may not be as simply inherited
or as simply controlled in natural systems as is often reported.
Rather, some believe that individuals within a typical agamic
complex, both sexual and apomictic, may share a predisposition
to express this trait (Grimanelli et al., 2001b; Sharbel and
Mitchell-Olds, 2001). The nature of this predisposition may be
a developmental characteristic, such as the possible presence of
a large, nutritive nucellus in Citrus (Koltunow et al., 1995b) or
a nutritive integument in Hieracium (Koltunow et al., 1998) and
Taraxacum (Cooper and Brink, 1949), or it may be genetic, such
as the apparent linkage grouping in Tripsacum necessary for
interspecific transfer (Savidan, 2001).
WHY ARE GAMETOPHYTIC APOMICTS POLYPLOIDS?
Gametophytic apomicts, irrespective of the mechanism they
use, are almost invariably polyploids, yet sexual members of the
same or closely related species are very commonly diploids
(Asker and Jerling, 1992). The reason(s) for this association remains
unclear. It is, however, potentially a critical issue, because
a frequently stated aim of current research is the installation of
apomixis into diploid crop species. Three main theories have
been forwarded. Some have proposed that the optimum expression
of apomixis may be achieved only in conjunction with
a polyploid genome (Quarin et al., 2001). Because rare diploid
gametophytic apomicts have been reported (Asker and Jerling,
1992; Bicknell, 1997; Kojima and Nagato, 1997; Naumova et al.,
1999; Koltunow et al., 2000), polyploidy does not appear to be
absolutely required for the expression of the apomixis. In these
examples, however, asexual seed formation often was poor,
so polyploidy may enhance the expression of apomixis in
many systems rather than ensuring its presence per se. Some
S238 The Plant Cell
indications of how this might operate come from yeast (Galitski
et al., 1999) and Arabidopsis (Lee and Chen, 2001), in which
alterations in ploidy status are known to affect methylation and
the expression of different alleles. Conversely, there are intriguing
examples of apomixis being expressed in previously
sexual plants after chromosome duplication (Nygren, 1948;
Quarin et al., 2001). However, there is some debate in these
cases about the possibility of innate predisposition, because the
plants used were sexual members of groups containing
apomicts (Quarin et al., 2001). Furthermore, the reverse has
been described by Asker (1967): a sexual plant was recovered
after the doubling of an apomictic biotype of Potentilla argentea.
Finally, polyploidy has been induced in a large number of plants,
and apomixis is reported very seldom in the products.
An apparent interspecific hybrid origin also is a common
feature among apomicts, and the combination of polyploidy and
hybridity is believed to have resulted in allopolyploidy in many
gametophytic apomicts (Ellerstrom and Zagorcheva, 1977;
Carman, 1997, 2001; Roche et al., 2001a). The action of tetrasomic
inheritance in many systems, however, also indicates the
presence of autopolyploidy, or possibly segmental allopolyploidy,
in these plants (Pessino et al., 1999). Carman (1997, 2001)
postulated that a combination of hybridity and polyploidy can
lead to the disjunction of key regulatory events during critical
stages of megasporogenesis, megagametogenesis, and fertilization.
This in turn may lead not only to apomixis but also to other
unusual developmental events, such as polyspory and polyembryony.
Through a comprehensive survey of the botanical
literature, together with his own experimentation, Carman presents
compelling evidence that there are associations between
apomixis and these other phenomena, that hybridity between
related species has been a key factor in the formation of many
apomictic complexes, and that different types of apomixis and
related phenomena are all expected outcomes of a theoretical
model based on the disjunction of a relatively small number of
key regulatory events. Whether this is universally true, and
whether it can be used to harness apomixis in crop species, are
questions remaining to be answered.
Roche et al. (2001b) provided a refinement on Carman's
hypothesis, suggesting that supernumerary chromatin may be
the principal driver in this process. A hybrid origin, segmental
allopolyploidy, and the activity of reproductive drivers all are
reported characteristics of supernumerary chromatin biology
(McVean, 1995). There is growing evidence for the presence of
supernumerary chromatin in several apomictic species, and it is
clearly involved in the inheritance of apomixis in the grasses
Pennisetum squamulatum and Cenchrus ciliaris (Roche et al.,
2001a, 2001b, and references therein).
Matzk et al. (2003) recently combined a flow cytometric seed
screen for reproductive mode with chromosome counts and
found that apomictic Hypericum and Ascyreia plants had a higher
DNA content per chromosome than related sexual species. The
same appears to be true for apomictic Hieracium (M. Tucker, F.
Matzk, and A.M. Koltunow, unpublished results). An increased
genetic load mediated by transposon replication was postulated
by Matzk et al. (2003) as a mechanism by which the increased
DNA content of apomictic Hypericum and Ascyreia might arise.
We have evidence that at least four classes of transposons
are present in Hieracium species, but the relative content of each
in sexual and apomictic genomes has not been established
(M. Tucker, T. Tsuchiya, R. Bicknell, and A.M. Koltunow, unpublished
results). Therefore, the enlarged genomes of apomicts
might be more the consequence of asexual seed formation than
its cause, and this may have contributed to the apparent involvement
of supernumerary chromatin. This observation and
conclusion, however, contradict the hypothesis that sexual
species should have larger genomes than related asexual
species (Wright and Finnegan, 2001), because sexual reproduction
is thought to favor the spread of mobile elements
between individuals of a population and asexuality is thought
to prevent interindividual transfer (Hickey, 1982; Matzk et al.,
2003).
It also has been proposed that the inheritance of apomixis may
be favored by the mediation of a diploid or polyploid gamete
(Nogler, 1984b, 1986). This would lead to the rapid establishment
of polyploid agamic complexes. Under this proposal, the
creation of apomixis may occur in a diploid plant, but any further
dispersion of the trait throughout a species would require the
mediation of an unreduced gamete. As described above, unreduced
gametes, particularly unreduced eggs, are observed
commonly in most gametophytic apomicts, providing an opportunity
for this mechanism to play a role in the evolution of agamic
complexes. Nogler (1984b) noted that in Ranunculus auricomus,
a dominant allele conferring apomixis could be transferred only
through a diploid gamete. Haploid gametes were produced by
these plants, but their products all were sexual. This observation
led Nogler to speculate that the dominant allele may play
a gamete-lethal role when present in homozygous form in the
pollen. Similar observations have been made for Tripsacum
(Grimanelli et al., 1998a) and Pennisetum (Roche et al., 2001b;
Jessup et al., 2002). There are cases, however, in which there
does not appear to be a gamete-lethal effect associated with the
transfer of apomixis. In Hieracium, haploid gametes do transfer
competence for parthenogenesis, a component of apomixis
(Bicknell et al., 2000). Intriguingly, in this case, diploid apomictic
progeny could not be recovered, but selection against this class
appeared to be acting at the level of the zygote, not the gamete.
As mentioned above, there is growing evidence that natural
apomicts frequently carry a high genetic load of deleterious
alleles. This is expected to result in a ratchet effect, encouraging
the formation of polyploids and reducing the viability of any
diploid derivatives they may produce. Apomictic polyploids often
are capable of producing diploid progeny, typically through the
operation of polyhaploidy, a natural mechanism analogous to
haploid parthenogenesis that often is observed in apomicts
(Asker and Jerling, 1992). Diploids have been recovered among
the progeny of several apomictic species. However, they are
invariably considerably weaker than their polyploid parent, often
to the point of being barely viable, which we believe to be
a reflection of the inherited genetic load.
Similarly, we recently isolated a small number of genomic
sequences of putative reproductive importance from both sexual
and apomictic accessions of Hieracium. Sequence comparisons
indicated that the genes from the apomict typically contained
a higher frequency of transposon insertions and gene rearrangements
(A.M. Koltunow and M. Tucker, unpublished results). We
Apomixis in Plants S239
hypothesize, therefore, that in Hieracium at least, once a polyploid
apomict is formed, mutation rapidly increases its genetic
load and limits the viability of any future diploid derivatives. That
could clearly result in the haploid gamete lethality observed in
Ranunculus and also may explain the diploid zygote lethality
seen in Hieracium.
Finally, where apomixis requires the simultaneous inheritance
of critical alleles at several unlinked loci, as seen in Erigeron
(Noyes and Rieseberg, 2000) and Taraxacum (van Dijk and BakxSchotman,
in press), unreduced gametes would ensure the
intact transfer of the trait, essentially by acting as a single wholegenome
linkage group. In many cases, this would provide a clear
selective advantage because, typically, the inheritance of only
part of a mechanism of apomixis, such as parthenogenesis
without apomeiosis, leads to the production of disadvantaged
progeny (n10 progeny in this example; Figure 3). The complete
inheritance of apomixis through the mediation of an unreduced
gamete would clearly avoid this issue, but it also would lead to an
increase in ploidy over the parental state after fertilization. As
mentioned above, unreduced gametes, particularly unreduced
eggs, are relatively common in these plants, and frequently they
are involved in the formation of hybrid progeny (Figure 3)
(Chapman and Bicknell, 2000; Bicknell et al., 2003). Therefore,
in a manner similar to the role of genetic load, this effect can be
expected to stimulate the formation of polyploid agamic complexes
and to repress any reverse process by biasing against
diploid apomict survival.
GENE IDENTIFICATION IN APOMICTS
The identification of the genes involved in apomixis appears to be
tractable, because in most of the native apomictic systems under
study, only a small number of loci have been determined to be
critical for the inheritance of the trait. In most cases, a map-based
approach has been taken to attempt the cloning of the sequences
involved. The first and most comprehensive mapping
efforts in apomicts have been reported from groups working in
grass species, most of which are relatives of important cereal
crops. The dominant factor associated with the inheritance of
apomixis in the aposporous plus pseudogamous grass species
Pennisetum squamulatum appears to be hemizygous, because
no equivalent region has been found in sexual biotypes (OziasAkins
et al., 1993, 1998; Roche et al., 2001a). Intriguingly, the
same region was found in the apomictic relative Cenchrus ciliaris,
also as a hemizygous region (Roche et al., 1999, 2001a). Efforts
to isolate this region have revealed its complex nature. Twelve
molecular markers were reported to cosegregate with the
apospory-specific genomic region without separation, an early
indication that this region may be associated with a localized
suppression of recombination. After the isolation of BACs corresponding
to these markers, it became apparent that this region
of suppressed recombination spanned at least 50 Mbp in
P. squamulatum (Roche et al., 2002) and may be considerably
larger. Using the corresponding BAC clones as probes, Goel et al.
(2003) demonstrated that the apospory-specific genomic region
localizes to a single short arm of a P. squamulatum chromosome.
A similar effort is under way with the maize relative Tripsacum
dactyloides, an apomict that uses diplospory plus pseudogamy.
Again, markers were reported in linkage with the region
associated with the inheritance of diplospory in this plant
(Grimanelli et al., 1998b; Blakey et al., 2001). Suppressed recombination
clearly is also a feature of this region, frustrating efforts
to clone the critical genes involved in apomixis. Estimates of the
size of this region are less accurate than with Pennisetum, but it
appears to correspond to a 40-cM region of the sexual
Tripsacum map (Grimanelli et al., 2001a). Paspalum, another
grass genus, also is being used in a map-based cloning strategy.
The apospory plus pseudogamy mechanism of apomixis in this
plant is inherited as a simple dominant factor (Martinez et al.,
2001). Grasses are known for their high synteny. Pupilli et al.
(2001) noted the coinheritance of five rice markers with apospory
in Paspalum and are attempting to clone the locus through their
use as probes. Again, nonrecombination and hemizygosity were
detected in association with the locus (Labombarda et al., 2002).
Intriguingly, although all of the grasses mentioned above show
considerable synteny with the rice genome, in each case the
region associated with the inheritance of apomixis aligns to
a different region of that genome (Grimanelli et al., 2001a).
Albertini et al. (2001a, 2001b) also reported the isolation of
markers linked to apospory in Kentucky bluegrass (Poa
pratensis), and Pessino et al. (1997) noted similar findings for
Brachiaria. Among the eudicotyledonous systems under study,
progress is being made toward the mapping of ``apomixis''
genes in Taraxacum (van Dijk et al., 2003; Vijverberg et al., 2004)
and Erigeron (Noyes and Rieseberg, 2000). There is some
evidence for nonrecombinant sector(s) associated with apomixis
genes in Erigeron and for gamete selection in Taraxacum,
indicating that similar difficulties with cloning also may arise in
these plants.
Critically considered, the map-based cloning strategy described
above is clearly a valid and promising line of inquiry, but
success is certainly not proving to be either easy or assured. The
commonly observed suppression of recombination about loci
associated with apomixis is particularly problematic, because it
markedly reduces the power of the approach. Fine mapping
usually is attempted in this method to reduce the distance that
must be spanned by a genomic contig, but suppressed crossover
frequencies result in a paucity of recombinants and little
opportunity to refine the basic mapping data. It also should be
remembered that these plants are unlike familiar sexual models
such as Arabidopsis and rice. They typically have large, polyploid
genomes, and almost certainly they have high representations of
highly repetitive sequences, particularly those of transposons. In
most cases, mapping is proceeding in a triploid or tetraploid
highly heterozygous background. Fortuitously, the alleles associated
with apomixis typically are simplex and dominant,
simplifying phenotypic analysis of segregating progeny. However,
once a dominant allele is mapped, contigs must be formed,
taking care to ensure that the aligned genomic fragments are
from the same homolog. At this time, the hemizygosity seen in
Pennisetum and Paspalum may be an advantage, but it is
probably also associated with the suppression of recombination
mentioned above. Distances of 50 to 100 Mbp appear to be
commonly involved in these mapping efforts. Matzk et al. (2003)
have suggested that large, hemizygous arrays of DNA linked to
apomixis may be attributable to the segregation of markers with
S240 The Plant Cell
a retrotransposon(s) accumulated after the establishment of
apomixis. The presence of highly repetitive sequences clustered
about the locus of interest will further frustrate efforts in walking
and cloning. Additionally, so little is known of the molecular basis
of apomixis that the essential elements may not even be
recognized even if they were sequenced. Given the regulatory
nature of the trait, cis-acting factors, including chromatinmodeling
factors, are likely to be as critical as structural
sequences (Koltunow and Grossniklaus, 2003). Finally, many of
these plants cannot be transformed genetically, so functionality
testing of putative control elements is not yet possible. Efforts to
address this limitation apparently are under way (Dresselhaus
et al., 2001).
MUTAGENESIS STRATEGIES AND RECOVERED MUTANTS
In light of the difficulties described above, alternative strategies
to identify genes involved in apomixis are being tried based on
targeted mutagenesis. Tripsacum dactyloides, a diplosporous
plus pseudogamous apomict, can be crossed to maize and
apomictic progeny isolated from the hybrids formed. This feature
has been used to introduce Mutator transposable elements from
maize into an apomictic genotype, the progeny of which will then
be scored for insertional mutagenesis (Grimanelli et al., 2001b).
A transposon tagging strategy, based on the Ac/Ds elements
of Zea mays, also was implemented in Hieracium through the
introduction of engineered elements (Bicknell et al., 2001; Weld
et al., 2002). Unfortunately, although the elements were shown to
be operational in this plant, the tagging system is proving to be
too slow and too complex to be practical, so it has been dis-
continued.
During the course of the Ac/Ds work in Hieracium, two mutants
were identified, and these have been partially described (Bicknell
et al., 2001). In both mutants, apomixis was lost, yet the plants
remained able to form seed sexually. In one line, the plant was no
longer able to form aposporous initials, whereas the other formed
initials but these did not progress to form unreduced embryo
sacs. Intriguingly, in the former case, the rate of sexuality
remained at the wild-type level of 2%, whereas in the latter case,
sexuality increased to essentially 100% of the viable reproductive
effort of the capitulum. Together with evidence from inheritance
(Bicknell et al., 2000) and recent comparative marker
gene expression studies (Tucker et al., 2003), we interpret this
result to indicate that one component of apomixis in this plant
entails the formation of aposporous initials and another leads to
the suppression of sexual reproduction. The two mutants crosscomplement,
permitting the recovery of apomixis, which indicates
that they carry lesions in different genes. Another independent
mutant has since been obtained that has lost the
capacity both to form initial cells and to progress in the sexual
process (A.M. Koltunow and S.D. Johnson, unpublished results).
All three mutants were obtained as somaclonal variants, each
after a transformation experiment. However, there is no evidence
for insertional mutagenesis in any of these cases. Unfortunately,
although these mutants are providing valuable insight into the
cellular and genetic processes underlying the expression of
apomixis in Hieracium, without clear molecular tags they do not
provide a simple route to the identification of the sequences
involved. A g-induced mutation screen aimed at identifying lossof-function
apomictic mutants is providing promising results
(Bicknell et al., 2001). More than 70 mutants have been isolated
to date in which apomixis is lost yet sexuality is retained to
varying degrees. Apomictic mutants reflecting all of the phenotypes
described above have been obtained, together with
others indicating that several factors associated with the expression
of apomixis have been affected. Amplified fragment
length polymorphism now is being used to identify chromosomal
regions commonly lost in these plants and to provide probes to
those regions.
THE PROSPECT OF INSTALLING APOMIXIS IN CROPS
During the 10 years since the last Plant Cell review on apomixis,
many of the critical issues identified at the time have been
addressed, together with a consolidation of the approaches
being taken and a refinement of the methods and systems being
used to study functional apomixis. We now believe that
researchers in this field are in a much stronger position to predict
the successful installation of this characteristic into diploid crop
species. Some issues, such as the role of polyploidy, remain
incompletely resolved, but they are now framed by clear hypotheses
and these are being tested. One of the most important
conclusions during this 10-year period has been the understanding
that apomixis results from changes in a small number of
key events typical of sexual reproduction. This clearly points the
way toward identifying and manipulating those elements in
a ``synthetic'' approach to engineering asexual seed formation.
As many of the other articles in this special issue demonstrate,
advances in our understanding of the regulation of sexual reproduction
have been profound in recent years and are providing
critical insights for this line of inquiry.
Significant effort still is required on a number of fronts before
we can hope to successfully engineer a controlled, commercially
viable form of apomixis in a wide range of target crops. We have
no knowledge of the cues and genes that enable cells in the ovule
to switch to an apomictic pathway. The interaction between
embryo and endosperm development in apomicts is poorly
understood, as is the capability of apomicts to tolerate parental
genome imbalances during endosperm development that result
in seed sterility in sexual plants. The role of epigenetic factors in
the control of apomixis is unknown, yet they are widely implicated
in the control of sexual reproduction. Similarly, molecular
signaling events have been implicated in the development of
both sexual and asexual structures within the plant ovule, yet the
nature and role of these events in apomixis are largely unstudied
to date. We believe that the continued investigation of both native
apomicts and sexual systems will provide the best opportunity to
elucidate the genetic and physiological factors that contribute
to the control of apomixis and that this ultimately will permit the
controlled formation of maternally derived, genetically identical
seeds in flowering plants.
In conclusion and upon reflection, it is intriguing that Mendel
selected two plant taxa to examine the nature of inheritance
between generations. The study of Pisum, his first choice,
established the particulate nature of inheritance, demonstrated
trait segregation, confirmed the role of gametes as the couriers
Apomixis in Plants S241
of genetic determinants, and demonstrated the combined consequences
of segregation and recombination. In choosing pea,
Mendel is rightfully credited with the establishment of the field of
genetics as it is known today. Mendeľs second choice was
Hieracium, a plant that proved unsuitable for its original intended
purpose of verifying the findings made in pea. It is probably
for this reason, and because the results Mendel obtained in
Hieracium were reported only in abbreviated form, that this part
of his work has remained largely unnoticed to this day. Therefore,
there is a certain irony in noting that Hieracium is becoming such
a useful tool in the study of a very different form of reproduction,
that of facultative apomixis, >130 years after Mendeľs first
crosses with this plant were made.
ACKNOWLEDGMENTS
We are indebted to Gian Nogler and Sven Asker for their pioneering
work in apomixis and for their encouragement during the development
of Hieracium as a model system. We also are indebted to our colleagues
working with other functional apomicts for freely contributing their
thoughts, and particularly Peter van Dijk for his comments on the
manuscript. We thank Matthew Tucker for assistance with figures and
past and present members of our laboratories, including visitors on
sabbatical, for their efforts toward the analysis of apomixis in Hieracium.
Research in A.M.K.'s laboratory on apomixis has been supported by the
Australian Centre for International Agricultural Research, and research in
R.A.B.'s laboratory has been supported by The New Zealand Foundation
for Research Science and Technology.
Received October 2, 2003; accepted January 14, 2004.
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