Cytogenetics and Plant Breeding
Cytogenet Genome Res 120:358–369 (2008)
Pathways to doubled haploidy: chromosome
doubling during androgenesis
J.M. Seguí-Simarro F. Nuez
Instituto para la Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad politécnica de
Valencia, Valencia (Spain)
Accepted in revised form for publication by M. Schmid, 14 December 2007.
Abstract. Production of doubled haploid (DH) plants
through androgenesis induction is a promising and convenient
alternative to conventional selfing techniques for the
generation of pure lines for breeding programs. This process
comprises two main steps: induction of androgenesis
and duplication of the haploid genome. Such duplication is
sometimes indirectly induced by the treatments used to
promote androgenic development. But usually, an additional
step of direct chromosome doubling must be included in
Haploid individuals possess only a gametic number of
chromosomes, which makes them extremely useful from a
theoretical and practical point of view, for example, in studies
of induced mutagenesis where recessive mutations can
be easily detected without the masking effects of dominance,
or to reveal deleterious genes present in diploids of
cross-pollinating species. However, haploids tend to be
smaller in habit, less vigorous, more sensitive to disease and
stress sources and, most importantly, sterile. Therefore, it is
usually desired for practical purposes to obtain doubled
haploids (DHs). DH technology has emerged as an exciting
and powerful tool of pure line production for crop improve-
This work was supported by grants GV05-023 from Generalitat Valenciana
and AGL2006-06678 from the Spanish Ministry of Education and Science
(MEC) to J.M.S.S.
Request reprints from José M. Seguí-Simarro, Instituto para la
Conservación y Mejora de la Agrodiversidad Valenciana (COMAV)
Universidad politécnica de Valencia Ciudad Politécnica de la
Innovación (CPI), Edificio 8E – Escalera 9, Camino de Vera s/n
ES–46022 Valencia (Spain)
telephone: +34 387 7000, ext. 88472; fax: +34 96 387 9422
Fax +41 61 306 12 34
© 2008 S. Karger AG, Basel
the protocol. Duplication of the haploid genome of androgenic
individuals has been thought to occur through three
mechanisms: endoreduplication, nuclear fusion and c-mitosis.
In this review we will revise and analyze the evidences
supporting each of the proposed mechanisms and their
relevance during androgenesis induction, embryo/callus
development and plant regeneration. Special attention will
be devoted to nuclear fusion, whose evidences are accumulating
in the last years. Copyright © 2008 S. Karger AG, Basel
ment. The constant discovery of new protocols to obtain
DHs in an increasing number of agronomically interesting
species has boosted the application of DHs in breeding programs.
Besides the practical facet of this technique, it is a
valuable method for genetic cartography of complex traits,
transgenesis and genomics among others (see Forster et al.,
2007 for a review on applications of DHs).
In order to obtain a DH, two main steps should be usually
considered: (1) the induction of haploid development
and (2) the induction of chromosome doubling of the haploid
individual. There are several experimental pathways to
haploidy, including wide hybridization, parthenogenesis,
gynogenesis and androgenesis (Palmer and Keller, 2005;
Forster et al., 2007). The choice of method for haploid production
largely depends on the specific response of a given
species to each method. But in species that respond to different
methods, the most used by far is androgenesis, due to
its higher simplicity and efficiency. Androgenesis is defined
as a developmental route, alternative to zygotic embryogenesis,
whereby a haploid individual is obtained from a malederived
haploid (reduced) nucleus, thus having the genetic
traits of the male donor plant. At present, there are several
recent reviews on the different aspects of androgenesis induction,
treatments and potentialities (Datta, 2005; Maras-
Accessible online at:
chin et al., 2005; Germana, 2006; Pauls et al., 2006; Shariatpanahi
et al., 2006; Forster et al., 2007). However, reviews
on chromosome doubling (the second step) during androgenesis
are comparatively scarcer despite its importance.
This will be the main topic of this review.
In most of the species studied, chromosome doubling occurs
‘spontaneously’ in a percentage of individuals. This
percentage may be largely influenced by the in vitro conditions
used to induce androgenesis. But there are always a
number of induced embryos which do not undergo doubling
and finally become a haploid plant. This number of
individuals may oscillate enormously among species. Within
a species, there are differences among genotypes as well.
For example, doubling rates ranging from 0 to 21.4% have
been reported for different maize genotypes (Barnabas et
al., 1999), and from 10 to 40% in Brassica napus (Henry,
1998). As an exception, the frequency of ‘spontaneous’
(nondirectly induced) chromosome doubling in some elite
cultivars may be high enough – up to 87% in certain barley
cultivars (Hoekstra et al., 1993) – to skip the doubling step
and use the directly obtained DHs, discarding those (few)
haploid individuals. The mechanism by which androgenic
microspores, embryos or calli double their genome is not
well understood. There are examples of the occurrence of
endomitosis, nuclear fusion or endoreduplication, but some
of them are controversial and it is not known what factors
influence each of these processes. Given the relevance of the
doubling step for DH production, it is important to gain
deeper knowledge on the mechanisms intervening on this
step. In this paper we will review the different cellular mechanisms
known to lead to chromosome doubling during androgenesis,
including those indirectly promoted by the
treatment of androgenesis induction and those directly applied
to specifically duplicate the genome.
‘Spontaneous’ or indirect chromosome doubling
The use of the term ‘spontaneous’ chromosome doubling
is widely spread throughout the DH literature to refer to a
doubling event under experimental conditions not just suited
to promote duplication. However, we as well as other authors
(Kasha, 2005; Kasha et al., 2006) feel that such a process
seems far from being spontaneous, as many different in
vitro or ex vitro factors may be influencing duplication. As
pointed out by Henry (1998) and Kasha (2005), the microspore
stage at the time of anther/microspore inoculation,
the stressing treatments or the culture conditions used to
induce androgenesis do affect the frequency of chromosome
doubling. Several factors, like duration of inductive
conditions, temperature (heat and cold), mannitol pretreatments
and colchicine and other antimitotic drugs, used to
induce androgenesis, are known to influence the frequency
of genome duplication (Zhao and Simmonds, 1995; Henry,
1998; Kasha et al., 2001; Zhou et al., 2002a, b; Kasha, 2005;
Shim et al., 2006). The use of plant hormones for in vitro
cultures has also been directly related to DNA duplication
events (Joubes and Chevalier, 2000; Magyar et al., 2005).
Even the type of explant used to induce androgenesis can
have an impact. A striking example can be found in the
work on Brassica rapa of Sato et al. (2005), who found that
microspore culture yields up to a three-fold higher rate of
duplication than anther culture. Similar results have been
obtained in B. napus (Lichter et al., 1988) and B. oleracea
(Wang et al., 1999). On the other hand, chromosome doubling
is an event that also occurs spontaneously in nature
and has been historically referred to as spontaneous or natural
chromosome doubling (Jensen, 1974). Therefore, in order
to avoid misinterpretations it would be advisable to refer
to the doubling indirectly induced by the androgenesis induction
treatments as a sort of secondary or indirect effect
of the culture conditions, thus being ‘indirect’ or ‘indirectly-induced
Across the literature, four major mechanisms for plant
chromosome doubling have been proposed (Jensen, 1974;
d’Amato, 1984, 1989; Kasha, 2005) alternatively to the normal
cell cycle ( Fig. 1 A): endoreduplication (DNA duplication
without mitosis; Fig. 1 B), nuclear fusion (merging of
coalescing nuclei into a larger nucleus, mixing both DNA
contents; Fig. 1 C), endomitosis (mitosis in the absence of
both mitotic spindle and nuclear envelope breakdown;
Fig. 1 D) and c-mitosis (colchicine-induced collapse of the
mitotic spindle and breakdown of the nuclear envelope;
Fig. 1 E). All of these mechanisms have been proposed to
mediate chromosome doubling in specific situations. By far,
endoreduplication is the most common way to ploidy increase
during the normal life cycle of plants. In flowering
plants, it is believed to occur in 90% of the cases of doubling
(d’Amato, 1984). Conversely, endomitosis and nuclear
fusions have been rarely documented in angiosperms
(d’Amato, 1984) with the exception of fusion of sperm nuclei
and female polar nuclei involved in zygote and endosperm
formation (West and Harada, 1993).
However, the situation is somewhat different with respect
to chromosome duplication during an experimentally
induced process such as in vitro androgenesis. On the one
hand, soon after the demonstration of the experimental induction
of androgenesis (Guha and Maheshwari, 1964), evidences
favoring the occurrence of endoreduplication and
nuclear fusion began to accumulate. On the other hand, the
fact that colchicine can promote c-mitosis was known for
long and applied for practical purposes (Eigsti and Dustin,
1955). One of these applications of colchicine is to induce
c-mitosis in androgenic haploids so that DHs can be obtained.
In the following sections, we will present and discuss
the evidences and hypotheses accounting for the role and
mode of action of these three mechanisms in chromosome
doubling for DH production through androgenesis.
Endoreduplication ( Fig. 1 B) is characterized by one or
more extra rounds of chromatid duplication, in addition to
the normally-occurring during the phase of DNA synthesis
(S-phase) of the cell cycle. A parallel inhibition of mitosis
Cytogenet Genome Res 120:358–369 (2008) 359
Fig. 1. Diagram of the different alternatives for chromosome doubling compared with a normal cell cycle. ( A ) Normal
cell cycle. ( B ) Endoreduplication. (B) Putative pathway to re-enter the cell cycle with diplochromosomes after endoreduplication.
( C ) Nuclear fusion after defective cytokinesis. ( D ) Endomitosis. ( E ) C-mitosis after mitotic blockage. See text
for further details.
(M-phase) is also necessary. In this manner, the cell uncouples
S and M phases and exits the cell cycle. During the normal
life cycle of a plant, it is widely accepted that once a cell
enters endoreduplication cycles, it irreversibly differentiates
and there is no way back to re-enter into a new cell cycle
(Joubes and Chevalier, 2000; Larkins et al., 2001). However,
under special circumstances, such as in vitro culture conditions,
endoreduplicated cells may have the potential to dedifferentiate
and re-enter the mitotic cycle (Rao and Suprasanna,
1996). If an endoreduplicated cell re-enters the cell
cycle ( Fig. 1 B), chromosomes undergoing one round of endoreduplication
(diplochromosomes) would arrive at mitotic
metaphase with four sister chromatids and during anaphase
segregation, two-chromatid chromosomes migrate to
the future daughter cells, which will enter the next G1 phase
with a doubled DNA content. In other words, endoredupli-
Cytogenet Genome Res 120:358–369 (2008)
cation increases the number of chromatids of a chromosome
without a change in the number of chromosomes. If
more than one duplication round takes place, polytene
chromosomes are formed. Diplochromosomes or polytenic
chromosomes are generally originated without the usual
rounds of chromatin condensation and decondensation
(Sugimoto-Shirasu and Roberts, 2003), which makes it very
difficult to gain insights about their structure. However,
there is considerable information on the molecular machinery
involved in the regulation of endoreduplication, as well
as in the switch from the normal cell cycle to endoreduplication.
A normal cell cycle and endoreduplication cycle
seem to be mutually exclusive, but they share much of the
molecular machinery needed to enter into both DNA-replicative
processes (Traas et al., 1998). Constitutive expression
of genes involved in DNA replication stimulates both types
of cycles (Inze and De Veylder, 2006) which, in addition, are
commonly promoted by cyclin-dependent kinases. Proteolysis
of cyclin B by the anaphase-promoting complex (APC)
is common for both processes to license another replication
round (see Joubes and Chevalier, 2000; Inze and De Veylder,
2006 for detailed reviews on molecular aspects of endoreduplication).
However, there are some results that challenge
the view of a common machinery for both processes. Besides
topoisomerase II (topo II), it seems that plants also
have an old form of type II topoisomerase (topoisomerase
VI) for DNA decatenation (reviewed in Corbett and Berger,
2003). From several works with Arabidopsis mutants carrying
defective subunits of topo VI, it was shown that topo VI
seems to mediate additional rounds of endoreduplication
beyond 8C, whereas topo II alone suffices to accomplish the
first two rounds (reviewed in Sugimoto-Shirasu and Roberts,
2003). Since there is no evidence for different structural
intermediates in DNA packaging that could explain a
different role for each topoisomerase in normal and endocycle
DNA replication, it was proposed that possibly both
topoisomerases are differentially regulated in Arabidopsis ,
each regulation being particularly important for each type
of DNA replication (Corbett and Berger, 2003). Nevertheless,
there is still a long way to understand the fine details of
this intriguing process in plants.
Endoreduplication is related to cell expansion, differentiation
and high metabolic rates (Traas et al., 1998), which
is frequently seen in highly specialized and metabolically
active cells such as basal cells of the embryo suspensor or
endosperm cells (Nagl, 1976; d’Amato, 1989), among others.
Endoreduplication has also been proposed as an ontogenic
mechanism of species or cell types with low DNA contents
to compensate their evolutive lack of the minimal amount
of gene copies necessary for their normal metabolism (Nagl,
1976). Natural endoreduplication has been shown to be regulated
by genetic, environmental and developmental cues
(Traas et al., 1998). Endogenous levels of plant hormones are
also supposed to be involved (Joubes and Chevalier, 2000),
but there is much less information on this respect. Exogenous
application of plant growth regulators to cultured cells
has also an effect on DNA duplication, as they have been
related to in vitro ploidy shifts (Karp, 1994). But it has traditionally
been related to endomitosis events rather than to
endoreduplication (Joubes and Chevalier, 2000). However,
evidence is increasing regarding a role of auxins in the cell
decision between progression through the cell cycle or exit
to endoreduplication. Valente et al. (1998) observed that
auxin application (but not combined with cytokinin) can
induce endoreduplication in tobacco cell cultures. More
recently, auxin was demonstrated to have a clear role as
a modulator of the cell cycle/endoreduplication switch,
through regulation of levels of E2FB transcription factor
(Magyar et al., 2005).
With respect to androgenesis, endoreduplication has
long been suggested as a mechanism to explain the occurrence
of higher DNA contents in induced microspores and
pollen grains. In fact, most of the reported examples of endoreduplication
point to an event occurring during andro-
genesis induction or just after induction. Raquin et al. (1982)
showed in wheat that a significant percentage of uninucleate
microspores – at G2-phase (2C DNA content) by the time of
culture – increase their DNA content after the induction
treatment to 4C but keep their uninucleate status. Indirect
evidence for endoreduplication has also been observed in
induced uninucleate microspores of wheat and petunia (Raquin
et al., 1982), corn (Pretova et al., 1993) and rapeseed
(Binarova et al., 1993) as well as in the vegetative and/or
generative nucleus in bicellular pollen from rapeseed (Binarova
et al., 1993) and Datura innoxia (Sunderland et al.,
1974). In addition, endoreduplication has been proposed as
a mechanism to explain some types of polyploidies observed
in certain species. To explain the occurrence of triploid proembryos
from bicellular pollen grains of Datura innoxia ,
Sunderland et al. (1974) proposed the occurrence of endoreduplication
in the generative nucleus followed by a joint
segregation of the ‘generative’ diplochromosomes together
with the ‘vegetative’ chromosomes through a common
spindle. Raquin et al. (1982) pointed to endoreduplication
as the only possible mechanism to explain their observed
DNA increase in G2 microspores of petunia from 2C to 3C
DNA content. Sunderland et al. (1974) also proposed a combination
of endoreduplication in the generative nucleus and
nuclear fusion with two vegetative-descending nuclei to
produce the observed 4C proembryos. It appeared that endoreduplication,
alone or combined, is the most likely
mechanism to explain ploidy shifts towards triploidy or
polyploidy at early stages of microspore androgenic development.
Going one step further, endoreduplication was
proposed nearly ten years ago as the main mechanism for
early chromosome doubling during induction of microspore
embryogenesis (Rao and Suprasanna, 1996; Henry,
But there are still some loose ends. It is possible that the
use of plant hormones in most of the protocols to induce
androgenesis could favor endoreduplication as described
for other in vitro systems such as those mentioned above.
However, the early stages of androgenesis induction and microspore
embryogenesis do not match many of the characteristics
of cells typically undergoing endoreduplication. In
nature, endoreduplication is a dead end in the differentiation
process of certain cells to acquire improved metabolic
competences or larger sizes. But instead of differentiation,
expansion or high metabolic activity early stages of microspore
embryogenesis are characterized by dedifferentiation,
proliferative growth and low protein levels (Harada et al.,
1988; Pechan et al., 1991; Maraschin et al., 2005; Seguí-Simarro
et al., 2005). In some instances, the occurrence of
endoreduplication during androgenesis was inferred just
from the presence of nuclei larger than usual (Pretova et al.,
1993) and the existence of DNA levels higher than 2C (Binarova
et al., 1993). This evidence could be attributed to
endoreduplication events, but they could well be alternatively
interpreted as indicative of nuclear fusions, since
there is nothing in these results that unambiguously rules
out this possibility. A similar reinterpretation of Sunderland’s
evidences for endoreduplication (Sunderland et al.,
Cytogenet Genome Res 120:358–369 (2008) 361
Fig. 2. Examples of absent or defective cell walls, binucleated cells
and fusing nuclei in different androgenic systems. ( A ) Binucleated cell
within a rapeseed androgenic multicellular microspore, still surrounded
by the exine coat (ex). ( B ) Magnification of the binucleated cell
shown in A . Note the absence of cell wall in the cytoplasm (ct) between
the two interphasic nuclei (n) with active nucleoli (nu). ( C , D )
Meiocyte-derived tomato young callus. ( C ) Callus region showing multiple
binucleated cells. ( D ) Electron microscopy of a binucleated cell
where the nuclear envelopes (ne) are in contact at several regions.
( E , F ) Barley binucleated embryogenic microspore, v: vacuole. ( F ) Detail
of the tightly apposed nuclear envelopes. ( G ) Barley multinucleated
embryogenic microspore stained with Sytox Green (for nuclei, green)
and calcofluor (for cell walls, blue) and observed under a confocal laser
scanning microscope. The series shows five consecutive confocal
planes through which two nuclei in a same cell (white arrowheads) and
a large, fused nucleus (yellow arrowheads) can be followed. Images E,
F and G courtesy of Dr. P. González-Melendi. Bars in ( A , B ): 10 m; ( C ):
20 m; ( D–F ): 500 nm; ( G ): 100 m.
Cytogenet Genome Res 120:358–369 (2008)
1974) has also been recently proposed (Kasha, 2005; Shim
et al., 2006). The fact that those putative endoreduplicating
nuclei observed in maize (Pretova et al., 1993) or rapeseed
(Binarova et al., 1993) never proceeded further in embryogenesis
is an additional argument against endoreduplication
Nuclear fusion has also been proposed as a mechanism
of chromosome duplication during androgenesis for more
than 30 years. Pioneering authors proposed two possible
ways of nuclear fusion: (1) fusion of mitotic nuclei and (2)
fusion of nuclei at interphase. Fusion of mitotic nuclei was
proposed by Sunderland et al. (1974) as an explanation for
genome duplication in induced Datura pollen grains by
means of a synchronous entry into mitosis of both vegetative
and generative nuclei. According to Sunderland et al.
(1974) and Sunderland and Evans (1980), chromosomes
from both nuclei intermix and then segregate together
through a common mitotic spindle. This hypothesis, although
attractive, has been questioned due to the lack of
evidence for such a common spindle and most importantly,
to the unlikelihood of perfectly synchronized mitoses in
pollen grains. In wheat, mitotic synchronization was observed
in less than 4% of androgenic microspores (Raquin
et al., 1982). In a posterior paper, Sunderland (1974) reported
a ‘high frequency’ of 16 of these compound mitoses in
one anther, but unfortunately the percentage from the total
of pollen grains was not given.
In parallel, fusion of interphasic nuclei ( Fig. 1 C) has been
better documented. It consists of a normally-occurring
karyokinesis and nuclear reassembly, followed by a disrupted
cytokinesis that allows daughter nuclei to coalesce within
the same cytoplasm and finally fuse into a single, larger
nucleus with twice the chromosome number of the original
nucleus. As far as we are aware, the first visual evidence of
two fusing interphasic nuclei during androgenesis was provided
in barley (Chen et al., 1984a, b), the androgenic system
where nuclear fusion has been more and better studied. But
it has been in the last decade that examples of occurrence of
this mechanism in androgenic systems such as maize (Testillano
et al., 2004), barley (Kasha et al., 2001; González-Melendi
et al., 2005; Shim et al., 2006), wheat (Hu and Kasha,
1999) and recently tomato (Seguí-Simarro and Nuez, 2007)
and rapeseed ( Fig. 2 A, B; unpublished results) are accumulating.
In maize, Testillano et al. (2004) reported the occurrence
of incomplete or absent cell walls and therefore polynucleated
cells during the early stages of microspore embryogenesis.
They observed fusing, peanut-like shaped nuclei in
both embryo- and endosperm-like domains, and proposed
this mechanism to explain the observed ploidy shift to 2C
in the embryo-like domain and to higher ploidies in the endosperm-like
domain. In tomato, the presence of binucleated
cells and fusing nuclei in induced meiocytes and meiocyte-derived
mixoploid (C + 2C) calli ( Fig. 2 C, D) giving
ise to haploid, mixoploid and DH tomato plants (Seguí-Simarro
and Nuez, 2005, 2007) has been shown. In this case,
nuclear fusion was preceded and mediated by absence of cell
plates or severe defects in phragmoplast assembly during
cytokinesis. Recently, time-course studies have unambiguously
demonstrated that nuclear fusion takes place as early
as after the first symmetric division of the barley microspore
(Shim et al., 2006). Parallel ultrastructural analysis
revealed again the presence of tightly apposed, flat nuclear
envelopes prior to fusion ( Fig. 2 E, F), typical peanut-shaped
fusing nuclei and again, incomplete cell walls ( Fig. 2 G;
González-Melendi et al., 2005).
Another mechanism for doubled haploidy through nuclear
fusion was proposed to take place in induced pollen
grains of barley (Sunderland and Evans, 1980; Chen et al.,
1984a, b) and Datura (Dunwell and Sunderland, 1976). This
mechanism involved the merging of the vegetative and generative
nucleus of induced bicellular pollen, the so-called
route C of Sunderland (Sunderland, 1974; Sunderland et al.,
1974; Sunderland and Evans, 1980). A compilation of evidence
for this phenomenon can be found in Kasha (2005).
In order to permit nuclear coalescence within the same cytoplasm,
it was proposed that the generative cell wall, after
detachment from the intine, enters a process of callose dissolution
and wall fragmentation, prior to the dispersal of the
fragments (Dunwell and Sunderland, 1976). Alternatively,
failure during the assembly of the generative wall has also
been hypothesized (Kasha, 2005).
In this context, it is noteworthy to mention that in some
occasions, disruption of cytokinesis may also lead to the occurrence
of diploid heterozygous, non-DH individuals. In
barley, the fusion between the nuclei of two microspores was
described (Chen et al., 1984b). It was proposed that this phenomenon
could be due to incomplete walling during postmeiotic
cytokinesis, allowing for the microspores to stay
physically connected. Recently, this hypothesis has been
confirmed in tomato meiocytes induced to proliferate into
a callus (Seguí-Simarro and Nuez, 2007). If post-meiotic cytokinesis
is blocked before callus induction, neighbor meiotic
products end up fusing their nuclei. This may originate
the regeneration of non-DH individuals.
It can be concluded from previous data that disruption of
normal cytokinesis must be a prerequisite for a subsequent
nuclear fusion. But what is the cause of the disruption? As
mentioned, the in vitro culture environment is an important
source of stress in itself, and initial culture conditions promote
cell proliferation but may also cause defects in normal
cytokinesis. For example, multi-polar spindle formation,
cytoplasmic bridges across incomplete or fragmented cell
plates, micronuclei or chromosome fragmentation have
been described as collateral consequences of in vitro culture
(d’Amato, 1989). In Datura anther cultures, abnormalities in
post-meiotic cytokinesis including dyads, triads and binucleated
meiotic products were attributed to stress conditions
(Collins et al., 1974). Heat shock, widely used in species such
as rapeseed (Custers et al., 1994), wheat (Touraev et al.,
1996b), tobacco (Touraev et al., 1996a), eggplant (Dumas de
Vaulx and Chambonnet, 1982) or pepper (Dumas de Vaulx
et al., 1981) is known to destabilize microtubules and actin
filaments (Hause et al., 1993; Simmonds and Keller, 1999;
Gervais et al., 2000), both essential constituents of the phragmoplast
scaffold for cell plate formation. In vitro induced
changes in pH towards alkalinization have been described
to have an effect on the actin cytoskeleton (Pauls et al., 2006).
Obviously, even the transient use of microtubule-destabilizing
drugs (colchicine or other microtubule-based antimitotics)
as inductive agents for androgenesis or chromosome
doubling will also have an impact not only in the mitotic
spindle but also in the phragmoplast which is initially nucleated
from remnants of the spindle (Seguí-Simarro et al.,
2004). In the literature there are multiple examples illustrating
how induced failures on microtubule or microfilament
assembly and/or positioning may lead to absence of cytokinesis
or incomplete cell plates such as those observed prior
to nuclear fusion (Risueño et al., 1968; Yasuhara et al., 1993;
Valster et al., 1997; Gimenez-Abian et al., 1998). Given the
enormous amount of data obtained in recent years from genetic,
structural, molecular and signal transduction analysis
of cytokinesis (Jürgens, 2005), there are nearly hundreds of
potential targets to be affected by the plethora of individual
physicochemical agents present in the different culture media.
As mentioned, some of them are known, but a profound
and extensive study would be needed to clarify the different
levels at which the cytokinetic machinery can be affected by
the induction treatments.
Looking for cellular and molecular basis of
It is clear that prevention or abortion of cytokinesis must
be a prerequisite to overcome cell wall formation and trigger
nuclear fusion. However, this fact alone may not suffice for
the completion of the fusion process. There are many examples
of naturally occurring (tapetum, nuclear endosperm,
meiocytes) or experimentally-induced multinucleate
cells (Risueño et al., 1968; Nishihama et al., 2001; Park
and Twell, 2001) where such a fusion never takes place.
Thus, there must be some unknown force that allows for
two independent nuclear envelopes to become one (Chen et
al., 1984b; González-Melendi et al., 2005; Seguí-Simarro
and Nuez, 2007). It has been speculated with a role for actin
filaments in driving a nuclear approach (Shim et al., 2006),
but unfortunately, at this time very little is known about the
cellular and molecular mechanisms that drive nuclear fusion
in an induced process such as androgenic in vitro development.
However, some interesting ideas can be extracted
from the three naturally-occurring nuclear fusion events
that take place during the angiosperm life cycle.
In angiosperms, the first fusion occurs during megagametophyte
development by fusion of the two haploid polar
nuclei into a diploid secondary nucleus. The other two
fusions take place during double fertilization, where one
sperm fuses with the egg cell to give rise to the diploid zygote
and the other sperm fuses with the secondary nucleus
to generate the triploid endosperm (West and Harada, 1993).
Cytogenet Genome Res 120:358–369 (2008) 363
According to the excellent electron microscopic studies of
Jensen (1964) and Schulz and Jensen (1973), nuclear fusions
in female gametophytes start with the fusion of the endoplasmic
reticulum (ER) networks continuous with the outer
membrane of each of the fusing nuclei. Then, both nuclei
are brought closer apparently by shortening of the connected
ER cisternae, and several nuclear bridges trap small
pockets of cytoplasm. When a common, continuous outer
membrane can be clearly seen, the inner membranes merge
as well, allowing for the contact between both nucleoplasms.
A similar mechanism has also been described in in vitro
induced processes of nuclear fusion such as karyogamy in
electrofused egg and sperm cell protoplasts of maize (Faure
et al., 1993) or nuclear fusion in somatic protoplasts of soybean
and pea, with the exception that the ER seemed not to
be involved in this particular case (Fowke et al., 1977). Examples
of fusing membranes during androgenic development
are still scarce, but it seems to occur similarly to the
above described processes. As mentioned, coalescence of
nuclear membranes ( Fig. 2 D–F) seems to be a prerequisite
for nuclear fusion in androgenic systems (Shim et al., 2006;
Se guí-Simarro and Nuez, 2007). Recently, González-Melendi
et al. (2005) have illustrated the fusion of barley nuclei
during microspore-derived embryogenesis at the confocal
and transmission electron microscope level. The authors
show examples of fusing profiles at different regions of the
remarkably flat and closely apposed outer membranes, similar
to those previously reported by Chen et al. (1984a). In
this study the authors showed fusion of both symmetrically
and asymmetrically divided nuclei (vegetative and generative).
Interestingly, the authors remarked the consistent and
abundant presence of ER surrounding the generative cell,
and referred to the work of Dunwell and Sunderland (1974)
who showed quite similar results in tobacco. At that time,
the authors could not explain the significance of such an
accumulation, but it might well be an early marker of an
upcoming fusion event.
Thus, it seems that sequential fusion of membrane systems
is a common mechanism for nuclear fusion in all plant
cell types. But the question still remains as to why nuclear
membranes fuse under certain circumstances. By T-DNA
mutant analysis, two genes potentially involved in polar and
egg nuclear fusion, GFA2 and NFD1 , have been recently
characterized (Christensen et al., 2002; Portereiko et al.,
2006). In these mutants, migration of polar or egg and sperm
nuclei seems not to be affected and they get together at a
very close distance, but fail to fuse. It is suggested that both
gfa2 and nfd1 mutants are defective exactly at the step of
outer membrane/ER fusion. Another interesting observation
is that both GFA2 and NFD1 genes code for mitochondrial
proteins, present in mitochondria of all plant cell
types. Portereiko et al. (2006) suggested a role for mitochondria
in nuclear fusion, due to the clear association between
defective mitochondria and non-fused nuclei. However, the
mechanisms through which mitochondria can influence
nuclear membrane fusion remain obscure.
A more attractive possibility also deduced from the work
of Portereiko et al. (2006) relates to a role for the nfd1 muta-
Cytogenet Genome Res 120:358–369 (2008)
tion in altering the lipid composition of nuclear membranes.
NFD1 has a homolog in yeasts ( MRPL49 ) involved in phosphatidylcholine
(PC) biosynthesis (Hancock et al., 2006).
PC is the main lipid constituent of the plant nuclear envelope
(Philipp et al., 1976) and is known to stabilize lipid
membranes so that in PC-rich membranes fusion is inhibited
(Duzgunes et al., 1981). Thus, changes in the functional
properties of the nuclear envelope may depend on changes
of its phospholipid composition. Since the lipid composition
of nuclear membranes can be remodeled by regulating
the genes responsible for phospholipid biosynthesis (Santos-Rosa
et al., 2005), Portereiko et al. (2006) proposed that
inhibition of karyogamy in nfd1 mutants may be due to the
alteration of PC ratios in nuclear membranes.
In view of these results, it is tempting to speculate that
nuclear fusion during the early stages of microspore-derived
embryos could also be related to an altered lipid metabolism.
It is known that stressful in vitro culture procedures
have a wide impact on genic and metabolic pathways.
For example, in tobacco the direct effect of different in vitro
culture conditions over the cellular lipid contents and ratios
and in particular over PC biosynthesis has been demonstrated
(Chervin et al., 1995). Abiotic stresses such as salt,
cold, heat or mannitol also influence PC biosynthesis (Horvath
and Vanhasselt, 1985; Kinney et al., 1987; Tasseva et al.,
2004). Therefore, promotion of nuclear fusion during androgenic
development could also be influenced by altered
phospholipid ratios, in this case due to the in vitro environment.
Another interesting pathway to explore is a highly similar
process, the nuclear fusion during mating in yeasts. In
mating yeasts, nuclei orient along the cell axis with the aid
of microtubules (MTs). Then, nuclear membranes fuse in a
sequential manner as described above. Such similarities
may make it worth to search the plant genome for homologs
of yeast Kar2p, Kar4p, Kar5p, Kar8p, Prm2, Prm3p and
some SNARE proteins, known to have a role in this membrane
fusion process (Lahav et al., 2007). At least, SNARE
proteins have been shown to participate in other processes
of homotypic membrane fusion, like vesicle-vesicle or vesicle-cell
plate during plant cytokinesis (Jürgens, 2005).
In summary, the amount of evidence of nuclear fusion is
increasing in the last few years, as is the spectrum of androgenic
systems where nuclear fusion is being reported. Some
authors expressed certain doubts about nuclear fusion as
the main mechanism for chromosome doubling during androgenesis
(Henry, 1998), and most of the reports demonstrating
nuclear fusion do not exclude the possibility of endoreduplication
(Testillano et al., 2004; Shim et al., 2006) or
even endomitosis (Chen et al., 1984b) as a complementary
way, possibly acting later in development. But it is a fact that
in some species, mainly cereals, evidence for nuclear fusion
is conclusive (González-Melendi et al., 2005; Shim et al.,
2006). It could be argued that nuclear fusion is favored in
cereals, possibly due to the use of agents that favor such a
mechanism, as proposed for the use of mannitol in barley
(Kasha, 2005). Anyway, examples of nuclear fusion in mannitol-free
media can be found in maize (Testillano et al.,
2004), but also in more distant solanaceous species as in
Datura pollen grains (Sunderland et al., 1974) or in calli and
meiocytes of tomato (Seguí-Simarro and Nuez, 2007). It is
expected that in the near future, the application of powerful
microscopic techniques such as electron and laser confocal
and time-course microscopy to other androgenic systems
will yield more examples of nuclear fusion.
Endomitosis was first described in tapetal cells of Leontodon
by Meyer (1925), who observed an ‘internal division’
of chromosomes inside the nucleus with no evidence of
spindle fibers. Endomitosis ( Fig. 1 D) involves a failure in
the assembly of the mitotic spindle together with the absence
of nuclear envelope breakdown during mitosis. Chromosomes
normally duplicate their chromatids during Sphase,
condensate at prophase and sister chromatids separate
during metaphase, but they do not migrate since there
is no spindle assembly and the nuclear envelope is not dismantled.
Therefore, a cell with twice the original number of
chromosomes is formed. Endomitosis has been documented
in several animal groups, but rarely in angiosperms
(d’Amato, 1984). Therefore, it is not surprising that during
microspore embryogenesis, examples of endomitosis are
very scarce (Chen et al., 1984b), which makes this route negligible
for the purpose of this review.
C- m i t o s i s
Historically, the name of c-mitosis ( Fig. 1 E) has been applied
to an artificially-induced form of chromosome doubling
produced by colchicine, whereby mitosis is blocked to
a different extent depending on the dose of colchicine. At
high doses, mitosis is stopped at metaphase (c-metaphase).
Arrested c-metaphase cells have been extensively used for
cytogenetic analysis. Lower dosages allow sister chromatids
to detach from each other. Centromeres stay longer together
due to the lower turnover rate of kinetochore microtubules,
but they eventually separate yielding doubled chromosomes.
Colchicine is a natural alkaloid extracted from several
species of the genus Colchicum , especially C. autumnale . Its
action is exerted by binding to tubulin dimers, which hampers
de novo polymerization of microtubules (MT) and promotes
depolymerization of the existing ones, since the tubulin
turnover at the (–) end is not compensated at the (+)
end of the MT. Therefore, formation and persistence of the
mitotic spindle, phragmoplast or any other MT-based cytoskeletal
structure is compromised. This is reflected in most
cases in a blockage of mitosis during the assembly of the
anaphase spindle. But it is also possible that colchicine affects
the onset of cytokinesis and blocks phragmoplast formation,
which may cause defective walling and facilitate
nuclear fusion. This effect on nuclear fusion could be dosage-dependent,
with high doses promoting c-mitosis and
low doses promoting nuclear fusion (Kasha et al., 2006).
Moreover, it has been recently speculated that colchicine
may have additional indirect roles in chromosome doubling
also by endoreduplication, possibly by affecting the levels of
undegradable cyclin B-like proteins (Caperta et al., 2006).
Colchicine effects and reversibility can be modulated.
Time and colchicine concentration seem to have a key role
(Caperta et al., 2006). The right combination of time and
concentration appears critical for the promotion of either
c-metaphase arrest or chromosome doubling followed by
cell cycle progression. With respect to time, short-term exposures
to colchicine seem to work better than prolonged
treatments in terms of doubling frequency and avoidance of
polyploidies or embryo abnormalities (Chen et al., 1994;
Zhou et al., 2002a, b). Genotype is another factor that influences
the final action of colchicine. Colchicine is in general
effective for obtaining doubled haploids in dicotyledonous
species, but its effect is inconsistent in grasses. For example,
from three different maize genotypes exposed to colchicine
under the same conditions, doubling frequencies of 100%,
80% and 15.7% were reported (Barnabas et al., 1999). This
indicates that despite more or less general rules, conditions
must be set up independently to reach acceptable yields in
Colchicine can be applied at different moments during
the process of DH production. The most common stages to
apply the treatment are (1) application to parts or the whole
haploid plant, once regenerated, (2) application to developing
microspore-derived haploid embryos and (3) application
during androgenesis induction as a component of the induction
medium. Perhaps the easiest way is to apply colchicine
to the plant, once it is regenerated. Haploid plants can be totally
sunk into colchicine solutions for varying times, never
longer than a few hours. However, this method yields poor
efficiency of doubling and may carry a significant amount
of plant abnormalities and/or death. An additional drawback
is the generation of a considerable volume of toxic waste
that must be properly disposed of. An alternative is to apply
colchicine to specific plant organs such as roots, axillary
buds or apical meristems. There are different ways to apply
colchicine to plant buds: applying soaked cotton plugs to the
bud, or directly applying colchicine diluted in water, lanoline,
DMSO, or any other viscous substance capable of vehiculate
colchicine and remain in place for some time. However,
efficiency of these methods is not universally accepted.
Lotfi et al. (2003) unsuccessfully tried to duplicate haploid
melon buds with colchicine in lanolin. Alternatively, they
excised and in vitro cloned shoot tips from the same haploid
plants, and dipped regenerant shoots into an aqueous solution
of colchicine. By these means, Lotfi et al. (2003) reported
the occurrence of diploids as well as mixoploids and few
still haploids, even from the same colchicine-exposed explant.
From an economic point of view, this way is costly,
since it carries the need of maintaining chimeric plants from
which only some branches are useful, or implies the excision
and in vitro cloning of DH branches in order to obtain full
DH plants. In summary, in planta methods are easy but seem
not to be an efficient wide-range strategy.
Cytogenet Genome Res 120:358–369 (2008) 365
Colchicine can also be applied in vitro to developing androgenic
embryos (Pintos et al., 2007) or calli (Wan et al.,
1989; Ouyang et al., 1994), but it implies low survival rates
and chimerism. However, what seems to be more efficient
is to directly apply colchicine as soon as possible during the
early stages of haploid embryogenic development. When applied
before embryo/callus formation, the generation of chimeric
individuals can be greatly avoided. Besides, early application
is much more economic due to the small amount
of reagent used and the reduced volume of waste generated.
In maize (Saisingtong et al., 1996) and wheat (Barnabas et
al., 1991), application of colchicine directly to the anther
culture media results in beneficial effects in embryo production
from the embryogenic callus and an increased rate
of DH generation, while keeping all other parameters unchanged
with respect to control cultures (Barnabas et al.,
1999). In rapeseed, Chen et al. (1994) performed a comparative
study on the application of colchicine to isolated microspores,
developing embryos and regenerated plants.
They showed a nearly two-fold increase in doubling at the
stage of developing embryos, three-fold in regenerated
plants and nearly five-fold in isolated microspores. Their
results clearly supported the use of colchicine as soon as the
microspores are suspended in culture media.
The use of colchicine during the induction phase in rapeseed
has also the potential to induce androgenesis, which
allows for androgenesis induction and chromosome doubling
in just one step. Since the cytoskeleton is clearly involved
in the reprogramming of the microspore towards
androgenesis (Touraev et al., 2001; Aionesei et al., 2005), it
is reasonable to think that a cytoskeleton-affecting drug like
colchicine may also have a role in androgenesis induction.
It was proposed that colchicine-based MT depolymerization
may release the microspore nuclei from their peripheral
location, allowing for a central displacement which in
turn permits a symmetric division (Zoriniants et al., 2005).
In parallel, the induced increase in the cytoplasmic pool of
free tubulin dimers may block the synthesis of new pollenspecific
tubulin and eventually inactivate the gametophytic
program. The first use of colchicine to induce androgenesis
successfully produced embryos from rapeseed microspores
(Zaki and Dickinson, 1991). From then on, application of
colchicine to rapeseed microspores has been further studied
and refined (Chen et al., 1994; Zhao et al., 1996), reaching
frequencies of nearly 90% of DHs (Zhou et al., 2002a, b).
Thus, a clear relationship between cytoskeleton-affecting
treatments for androgenesis induction and chromosome
doubling was established in rapeseed. Such a direct relationship
has prompted some laboratories to focus on a search
for factors to induce high rates of both microspore embryogenesis
and chromosome doubling. Another interesting example
of this is the use of mannitol in cereals. As mentioned
in the previous section, mannitol is known to promote chromosome
doubling by nuclear fusion. But in addition, it may
promote androgenesis induction by disrupting MTs and
acting as an osmotic and starvation stressing agent (Kasha,
2005). The herbicide trifluralin would be another example
of ‘dual’ effector in rapeseed (Zhao and Simmonds, 1995).
Cytogenet Genome Res 120:358–369 (2008)
In addition to colchicine, there are other drugs that have
been successfully applied for chromosome doubling. Herbicides
like trifluralin or amiprophos-methyl (APM) were
used in wheat to obtain four- and five-fold higher duplication
yields than in controls, respectively (Hansen and Andersen,
1998). In rapeseed, trifluralin showed an even higher
performance than colchicine while being also effective in
inducing androgenesis (Zhao and Simmonds, 1995). In cork
oak, a comparative study revealed a higher efficiency of oryzalin
and APM compared to colchicine, which in addition
was found to induce necrosis in embryos (Pintos et al.,
2007). A similar comparison in rapeseed revealed comparable
duplication efficiencies for APM, oryzalin and trifluralin
with respect to colchicine, but clearly less toxicity due
to the lower concentration needed (Hansen and Andersen,
1996). It can be concluded that the main advantage of these
alternative drugs is that they can promote effects similar to
colchicine but at micromolar concentrations, as opposed to
the millimolar orders required for colchicine. However,
they are not proven useful in a wide range of species, as opposed
to colchicine. Other physicochemical methods traditionally
used to induce genome doubling include X- or gamma-rays,
heat and cold treatment and nitrous oxide (N 2 O)
gas (reviewed in Jensen, 1974). N 2 O has been used to induce
chromosome doubling in a variety of species, mostly cereals.
However, N 2 O is considered to be less effective than colchicine
An important aspect of the use of doubling agents is their
potential effect on somaclonal variation and their contribution
to the total variation observed in DHs. In this regard,
there are several studies indicating that genetic instability
contributed by the methods of DH production is negligible
(Baenziger et al., 1991; Finnie et al., 1991). In maize, somaclonal
variation produced by somatic callus-derived plants
and androgenic plants was recently compared (Barret et al.,
2006). The analysis of ZmTPA pong-like sequences showed
variation in callus-derived plants but not in plants of androgenic
origin. However, the authors did not exclude the possibility
of variation, arguing that their DNA samples corresponded
to young plant stages where chromosome doubling
is not completed. In other words, they speculated with chromosome
doubling as a cause of somaclonal variation. Nevertheless,
more specific studies have also been previously
conducted in maize to test the potential effect of chromosome
doubling agents such as colchicine, pronamide or
APM on somaclonal variation (Wan and Widholm, 1995).
In this study, the authors concluded that the use of such antimitotics
does not significantly affect the rates of variation,
as determined by the measurement of both qualitative and
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principal mechanism during androgenesis. However, at
present much of this evidence could be reinterpreted as nuclear
fusions. A similar situation could happen with c-mitosis.
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