Cytogenetics and Plant Breeding
Cytogenet Genome Res 120:358–369 (2008)
DOI: 10.1159/000121085
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 conve-
nient alternative to conventional selfing techniques for the
generation of pure lines for breeding programs. This pro-
cess 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 addition-
al 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 stud-
ies of induced mutagenesis where recessive mutations can
be easily detected without the masking effects of domi-
nance, 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
e-mail: seguisim@btc.upv.es
Fax +41 61 306 12 34 © 2008 S. Karger AG, Basel
E-Mail karger@karger.ch 1424–8581/08/1204–0358$24.50/0
www.karger.com
the protocol. Duplication of the haploid genome of andro-
genic individuals has been thought to occur through three
mechanisms: endoreduplication, nuclear fusion and c-mi-
tosis. In this review we will revise and analyze the evidenc-
es 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 accumu-
lating 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 pro-
grams. 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 usu-
ally considered: (1) the induction of haploid development
and (2) the induction of chromosome doubling of the hap-
loid 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 pro-
duction largely depends on the specific response of a given
species to each method. But in species that respond to dif-
ferent 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 embryogen-
esis, whereby a haploid individual is obtained from a male-
derived 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 in-
duction, treatments and potentialities (Datta, 2005; Maras-
Accessible online at:
www.karger.com/cgr
chin et al., 2005; Germana, 2006; Pauls et al., 2006; Shariat-
panahi et al., 2006; Forster et al., 2007). However, reviews
on chromosome doubling (the second step) during andro-
genesis are comparatively scarcer despite its importance.
This will be the main topic of this review.
In most of the species studied, chromosome doubling oc-
curs ‘spontaneously’ in a percentage of individuals. This
percentage may be largely influenced by the in vitro condi-
tions used to induce androgenesis. But there are always a
number of induced embryos which do not undergo dou-
bling and finally become a haploid plant. This number of
individuals may oscillate enormously among species. With-
in 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 mech-
anisms known to lead to chromosome doubling during an-
drogenesis, including those indirectly promoted by the
treatment of androgenesis induction and those directly ap-
plied 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 suit-
ed to promote duplication. However, we as well as other au-
thors (Kasha, 2005; Kasha et al., 2006) feel that such a pro-
cess 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 micro-
spore stage at the time of anther/microspore inoculation,
the stressing treatments or the culture conditions used to
induce androgenesis do affect the frequency of chromo-
some doubling. Several factors, like duration of inductive
conditions, temperature (heat and cold), mannitol pretreat-
ments 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 dou-
bling is an event that also occurs spontaneously in nature
and has been historically referred to as spontaneous or nat-
ural chromosome doubling (Jensen, 1974). Therefore, in or-
der to avoid misinterpretations it would be advisable to refer
to the doubling indirectly induced by the androgenesis in-
duction treatments as a sort of secondary or indirect effect
of the culture conditions, thus being ‘indirect’ or ‘indirect-
ly-induced chromosome doubling’.
Across the literature, four major mechanisms for plant
chromosome doubling have been proposed (Jensen, 1974;
d’Amato, 1984, 1989; Kasha, 2005) alternatively to the nor-
mal cell cycle (Fig. 1A): endoreduplication (DNA duplica-
tion without mitosis; Fig. 1B), nuclear fusion (merging of
coalescing nuclei into a larger nucleus, mixing both DNA
contents; Fig. 1C), endomitosis (mitosis in the absence of
both mitotic spindle and nuclear envelope breakdown;
Fig. 1D) and c-mitosis (colchicine-induced collapse of the
mitotic spindle and breakdown of the nuclear envelope;
Fig. 1E). All of these mechanisms have been proposed to
mediate chromosome doubling in specific situations. By far,
endoreduplication is the most common way to ploidy in-
crease during the normal life cycle of plants. In flowering
plants, it is believed to occur in ⬃90% of the cases of dou-
bling (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 re-
spect to chromosome duplication during an experimentally
induced process such as in vitro androgenesis. On the one
hand, soon after the demonstration of the experimental in-
duction of androgenesis (Guha and Maheshwari, 1964), ev-
idences 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 ob-
tained. 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
Endoreduplication (Fig. 1B) 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. (Bⴕ) Putative pathway to re-enter the cell cycle with diplochromosomes after endoredu-
plication. (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 uncou-
ples S and M phases and exits the cell cycle. During the nor-
mal 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 condi-
tions, endoreduplicated cells may have the potential to de-
differentiate and re-enter the mitotic cycle (Rao and Supra-
sanna, 1996). If an endoreduplicated cell re-enters the cell
cycle (Fig. 1Bⴕ), chromosomes undergoing one round of en-
doreduplication (diplochromosomes) would arrive at mi-
totic metaphase with four sister chromatids and during ana-
phase 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-
cation increases the number of chromatids of a chromo-
some 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 machin-
ery involved in the regulation of endoreduplication, as well
as in the switch from the normal cell cycle to endoredupli-
cation. 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-repli-
cative processes (Traas et al., 1998). Constitutive expression
of genes involved in DNA replication stimulates both types

360 Cytogenet Genome Res 120:358–369 (2008)

of cycles (Inze and De Veylder, 2006) which, in addition, are
commonly promoted by cyclin-dependent kinases. Prote-
olysis 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 endoredu-
plication). However, there are some results that challenge
the view of a common machinery for both processes. Be-
sides 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 carry-
ing 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 Rob-
erts, 2003). Since there is no evidence for different struc-
tural intermediates in DNA packaging that could explain a
different role for each topoisomerase in normal and endo-
cycle 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). Neverthe-
less, there is still a long way to understand the fine details of
this intriguing process in plants.
Endoreduplication is related to cell expansion, differen-
tiation 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 reg-
ulated 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. Exoge-
nous 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 tra-
ditionally 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 occur-
rence of higher DNA contents in induced microspores and
pollen grains. In fact, most of the reported examples of en-
doreduplication 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 (Ra-
quin 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 (Bina-
rova 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 pro-
embryos from bicellular pollen grains of Datura innoxia,
Sunderland et al. (1974) proposed the occurrence of endo-
reduplication 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 com-
bination of endoreduplication in the generative nucleus and
nuclear fusion with two vegetative-descending nuclei to
produce the observed 4C proembryos. It appeared that en-
doreduplication, alone or combined, is the most likely
mechanism to explain ploidy shifts towards triploidy or
polyploidy at early stages of microspore androgenic devel-
opment. Going one step further, endoreduplication was
proposed nearly ten years ago as the main mechanism for
early chromosome doubling during induction of micro-
spore embryogenesis (Rao and Suprasanna, 1996; Henry,
1998).
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 mi-
crospore embryogenesis do not match many of the charac-
teristics of cells typically undergoing endoreduplication. In
nature, endoreduplication is a dead end in the differentia-
tion process of certain cells to acquire improved metabolic
competences or larger sizes. But instead of differentiation,
expansion or high metabolic activity early stages of micro-
spore embryogenesis are characterized by dedifferentiation,
proliferative growth and low protein levels (Harada et al.,
1988; Pechan et al., 1991; Maraschin et al., 2005; Seguí-Si-
marro 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 (Bi-
narova et al., 1993). This evidence could be attributed to
endoreduplication events, but they could well be alterna-
tively interpreted as indicative of nuclear fusions, since
there is nothing in these results that unambiguously rules
out this possibility. A similar reinterpretation of Sunder-
land’s evidences for endoreduplication (Sunderland et al.,
Cytogenet Genome Res 120:358–369 (2008) 361

A B
C D
E F
G nucleus with twice the chromosome number of the original
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 surround-
ed by the exine coat (ex). (B) Magnification of the binucleated cell
shown in A . Note the absence of cell wall in the cytoplasm (ct) be-
tween the two interphasic nuclei (n) with active nucleoli (nu). (C , D)
Meiocyte-derived tomato young callus. (C) Callus region showing mul-
tiple 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) De-
tail 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.
362 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 embryo-
genesis is an additional argument against endoreduplica-
tion during androgenesis.
Nuclear fusion
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 vegeta-
tive 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, al-
though 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 ob-
served in less than 4% of androgenic microspores (Raquin
et al., 1982). In a posterior paper, Sunderland (1974) report-
ed 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. 1C) has been
better documented. It consists of a normally-occurring
karyokinesis and nuclear reassembly, followed by a disrupt-
ed cytokinesis that allows daughter nuclei to coalesce with-
in the same cytoplasm and finally fuse into a single, larger
nucleus. As far as we are aware, the first visual evidence of
two fusing interphasic nuclei during androgenesis was pro-
vided 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 (Tes-
tillano et al., 2004), barley (Kasha et al., 2001; González-Me-
lendi et al., 2005; Shim et al., 2006), wheat (Hu and Kasha,
1999) and recently tomato (Seguí-Simarro and Nuez, 2007)
and rapeseed (Fig. 2A, B; unpublished results) are accumu-
lating.
In maize, Testillano et al. (2004) reported the occurrence
of incomplete or absent cell walls and therefore polynucle-
ated cells during the early stages of microspore embryogen-
esis. 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 en-
dosperm-like domain. In tomato, the presence of binucle-
ated cells and fusing nuclei in induced meiocytes and meio-
cyte-derived mixoploid (C + 2C) calli (Fig. 2C, D) giving
rise to haploid, mixoploid and DH tomato plants (Seguí-Si-
marro 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 unambigu-
ously demonstrated that nuclear fusion takes place as early
as after the first symmetric division of the barley micro-
spore (Shim et al., 2006). Parallel ultrastructural analysis
revealed again the presence of tightly apposed, flat nuclear
envelopes prior to fusion (Fig. 2E, F), typical peanut-shaped
fusing nuclei and again, incomplete cell walls (Fig. 2G;
González-Melendi et al., 2005).
Another mechanism for doubled haploidy through nu-
clear 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 gen-
erative 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 evi-
dence for this phenomenon can be found in Kasha (2005).
In order to permit nuclear coalescence within the same cy-
toplasm, it was proposed that the generative cell wall, after
detachment from the intine, enters a process of callose dis-
solution 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 oc-
currence 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 phe-
nomenon could be due to incomplete walling during post-
meiotic 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 cy-
tokinesis is blocked before callus induction, neighbor mei-
otic 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 pro-
mote 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 binu-
cleated 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 phrag-
moplast 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-destabiliz-
ing drugs (colchicine or other microtubule-based antimitot-
ics) 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 nucle-
ated from remnants of the spindle (Seguí-Simarro et al.,
2004). In the literature there are multiple examples illustrat-
ing how induced failures on microtubule or microfilament
assembly and/or positioning may lead to absence of cytoki-
nesis 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 ge-
netic, 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 me-
dia. 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
nuclear fusion
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 ex-
amples of naturally occurring (tapetum, nuclear endo-
sperm, meiocytes) or experimentally-induced multinucle-
ate 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 fu-
sion in an induced process such as androgenic in vitro de-
velopment. However, some interesting ideas can be extract-
ed from the three naturally-occurring nuclear fusion events
that take place during the angiosperm life cycle.
In angiosperms, the first fusion occurs during megaga-
metophyte 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 zy-
gote 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 endo-
plasmic 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 connect-
ed 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 soy-
bean and pea, with the exception that the ER seemed not to
be involved in this particular case (Fowke et al., 1977). Ex-
amples of fusing membranes during androgenic develop-
ment are still scarce, but it seems to occur similarly to the
above described processes. As mentioned, coalescence of
nuclear membranes (Fig. 2D–F) seems to be a prerequisite
for nuclear fusion in androgenic systems (Shim et al., 2006;
Seguí-Simarro and Nuez, 2007). Recently, González-Melen-
di 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, sim-
ilar 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 genera-
tive). 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 sys-
tems 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 observa-
tion is that both GFA2 and NFD1 genes code for mitochon-
drial proteins, present in mitochondria of all plant cell
types. Portereiko et al. (2006) suggested a role for mitochon-
dria 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-
364 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 phos-
phatidylcholine (PC) biosynthesis (Hancock et al., 2006).
PC is the main lipid constituent of the plant nuclear enve-
lope (Philipp et al., 1976) and is known to stabilize lipid
membranes so that in PC-rich membranes fusion is inhib-
ited (Duzgunes et al., 1981). Thus, changes in the function-
al properties of the nuclear envelope may depend on chang-
es of its phospholipid composition. Since the lipid composi-
tion of nuclear membranes can be remodeled by regulating
the genes responsible for phospholipid biosynthesis (San-
tos-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-de-
rived embryos could also be related to an altered lipid me-
tabolism. It is known that stressful in vitro culture proce-
dures 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 demon-
strated (Chervin et al., 1995). Abiotic stresses such as salt,
cold, heat or mannitol also influence PC biosynthesis (Hor-
vath and Vanhasselt, 1985; Kinney et al., 1987; Tasseva et al.,
2004). Therefore, promotion of nuclear fusion during an-
drogenic development could also be influenced by altered
phospholipid ratios, in this case due to the in vitro environ-
ment.
Another interesting pathway to explore is a highly simi-
lar 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 mem-
brane 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 vesi-
cle-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 andro-
genic systems where nuclear fusion is being reported. Some
authors expressed certain doubts about nuclear fusion as
the main mechanism for chromosome doubling during an-
drogenesis (Henry, 1998), and most of the reports demon-
strating nuclear fusion do not exclude the possibility of en-
doreduplication (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 man-
nitol-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
Endomitosis was first described in tapetal cells of Leon-
todon by Meyer (1925), who observed an ‘internal division’
of chromosomes inside the nucleus with no evidence of
spindle fibers. Endomitosis (Fig. 1D) involves a failure in
the assembly of the mitotic spindle together with the ab-
sence of nuclear envelope breakdown during mitosis. Chro-
mosomes normally duplicate their chromatids during S-
phase, condensate at prophase and sister chromatids sepa-
rate during metaphase, but they do not migrate since there
is no spindle assembly and the nuclear envelope is not dis-
mantled. Therefore, a cell with twice the original number of
chromosomes is formed. Endomitosis has been document-
ed 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 neg-
ligible for the purpose of this review.
C-mitosis
Historically, the name of c-mitosis (Fig. 1E) has been ap-
plied to an artificially-induced form of chromosome dou-
bling 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 togeth-
er due to the lower turnover rate of kinetochore microtu-
bules, but they eventually separate yielding doubled chro-
mosomes.
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 ham-
pers de novo polymerization of microtubules (MT) and pro-
motes depolymerization of the existing ones, since the tu-
bulin 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 cyto-
skeletal 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 af-
fects the onset of cytokinesis and blocks phragmoplast for-
mation, which may cause defective walling and facilitate
nuclear fusion. This effect on nuclear fusion could be dos-
age-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 ex-
posures 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 influ-
ences 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
different genotypes.
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 develop-
ing microspore-derived haploid embryos and (3) application
during androgenesis induction as a component of the induc-
tion medium. Perhaps the easiest way is to apply colchicine
to the plant, once it is regenerated. Haploid plants can be to-
tally 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 draw-
back 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, lano-
line, DMSO, or any other viscous substance capable of ve-
hiculate colchicine and remain in place for some time. How-
ever, 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 solu-
tion of colchicine. By these means, Lotfi et al. (2003) report-
ed the occurrence of diploids as well as mixoploids and few
still haploids, even from the same colchicine-exposed ex-
plant. 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 an-
drogenic 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 ap-
plied before embryo/callus formation, the generation of chi-
meric individuals can be greatly avoided. Besides, early ap-
plication 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 pro-
duction from the embryogenic callus and an increased rate
of DH generation, while keeping all other parameters un-
changed with respect to control cultures (Barnabas et al.,
1999). In rapeseed, Chen et al. (1994) performed a compar-
ative study on the application of colchicine to isolated mi-
crospores, 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 rape-
seed has also the potential to induce androgenesis, which
allows for androgenesis induction and chromosome dou-
bling in just one step. Since the cytoskeleton is clearly in-
volved 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 depolymeriza-
tion may release the microspore nuclei from their periph-
eral 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 pollen-
specific 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 relation-
ship has prompted some laboratories to focus on a search
for factors to induce high rates of both microspore embryo-
genesis and chromosome doubling. Another interesting ex-
ample of this is the use of mannitol in cereals. As mentioned
in the previous section, mannitol is known to promote chro-
mosome 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).
366 Cytogenet Genome Res 120:358–369 (2008)
In addition to colchicine, there are other drugs that have
been successfully applied for chromosome doubling. Herbi-
cides like trifluralin or amiprophos-methyl (APM) were
used in wheat to obtain four- and five-fold higher duplica-
tion yields than in controls, respectively (Hansen and An-
dersen, 1998). In rapeseed, trifluralin showed an even high-
er 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 ory-
zalin 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 compa-
rable duplication efficiencies for APM, oryzalin and triflu-
ralin 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 op-
posed to colchicine. Other physicochemical methods tradi-
tionally used to induce genome doubling include X- or gam-
ma-rays, heat and cold treatment and nitrous oxide (N2O)
gas (reviewed in Jensen, 1974). N2O has been used to induce
chromosome doubling in a variety of species, mostly cere-
als. However, N2O is considered to be less effective than col-
chicine in dicotyledons.
An important aspect of the use of doubling agents is their
potential effect on somaclonal variation and their contribu-
tion 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, soma-
clonal 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 andro-
genic origin. However, the authors did not exclude the pos-
sibility of variation, arguing that their DNA samples corre-
sponded to young plant stages where chromosome doubling
is not completed. In other words, they speculated with chro-
mosome doubling as a cause of somaclonal variation. Nev-
ertheless, more specific studies have also been previously
conducted in maize to test the potential effect of chromo-
some 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 an-
timitotics does not significantly affect the rates of variation,
as determined by the measurement of both qualitative and
quantitative traits.
Conclusions
Chromosome doubling is highly influenced by the envi-
ronment of in vitro cultures. During androgenesis, dou-
bling may occur through three main mechanisms: endo-
reduplication, nuclear fusion or c-mitosis. This situation
contrasts with other genome doubling events during the
plant cell cycle, where the operating mechanism is in most
cases endoreduplication. Soon after the discovery of exper-
imental induction of androgenesis, evidence led many re-
searchers to think that endoreduplication could also be the
principal mechanism during androgenesis. However, at
present much of this evidence could be reinterpreted as nu-
clear fusions. A similar situation could happen with c-mi-
tosis. Colchicine and other antimitotics have traditionally
been used to induce doubling through mitotic impairment
during androgenesis as well as in other experimental situa-
tions. But in light of recent discoveries, a role of antimitotics
in mechanisms such as nuclear fusion should also be con-
sidered. In the last decade, the use of modern and sophisti-
cated microscopic techniques together with the availability
of protocols to induce androgenesis in more species have
permitted the documentation of more nuclear fusion events.
It is likely that as more species are studied in detail, more
examples of nuclear fusion will show up. As previously pro-
posed (Kasha et al., 2006), nuclear fusion could well be the
operating mechanism during the first androgenic stages,
still under the effects of the stressing inductive treatment.

References
Aionesei T, Touraev A, Heberle BE: Pathways to mi-
crospore embryogenesis, in Palmer CE, Keller
WA, Kasha KJ (eds): Haploids in crop improve-
ment II Vol 56, pp 11–34 (Springer-Verlag, Ber-
lin Heidelberg 2005).
Baenziger PS, Keppenne VD, Morris MR, Peterson
CJ, Mattern PJ: Quantifying gametoclonal vari-
ation in wheat doubled haploids. Cereal Res
Commun 19:33–42 (1991).
Barnabas B, Pfahler PL, Kovacs G: Direct effect of
colchicine on the microspore embryogenesis to
produce dihaploid plants in wheat (Triticum
aestivum L.). Theoret Appl Genet 81: 675–678
(1991).
Barnabas B, Obert B, Kovacs G: Colchicine, an ef-
ficient genome-doubling agent for maize (Zea
mays L.) microspores cultured in anthero. Plant
Cell Rep 18:858–862 (1999).
Barret P, Brinkman M, Beckert M: A sequence re-
lated to rice Pong transposable element displays
transcriptional activation by in vitro culture
and reveals somaclonal variations in maize. Ge-
nome 49: 1399–1407 (2006).
Binarova P, Straatman KR, Hause B, Hause G, van
Lammeren AAM: Nuclear DNA synthesis dur-
ing the induction of embryogenesis in cultured
microspores and pollen of Brassica napus L.
Theoret Appl Genet 87: 9–16 (1993).
Caperta AD, Delgado M, Ressurreicao F, Meister A,
Jones RN, et al: Colchicine-induced polyploidi-
zation depends on tubulin polymerization in
c-metaphase cells. Protoplasma 227: 147–153
(2006).
Chen CC, Howarth MJ, Peterson RL, Kasha KJ: Ul-
trastructure of androgenic microspores of bar-
ley during the early stages of anther culture.
Can J Genet Cytol 26:484–491 (1984a).
Chen CC, Kasha KJ, Marsolais A: Segmentation
patterns and mechanisms of genome multipli-
cation in cultured microspores of barley. Can J
Genet Cytol 26:475–483 (1984b).
Chen ZZ, Snyder S, Fan ZG, Loh WH: Efficient pro-
duction of doubled haploid plants through
chromosome doubling of isolated microspores
in Brassica napus. Plant Breeding 113: 217–221
(1994).
Chervin D, Gawer M, Guern N, Mazliak P: Modula-
tions of the effects of choline on tobacco cells by
a solid medium and a sucrose or phosphate sup-
plementation. Plant Physiol Biochem 33: 471–
478 (1995).
Christensen CA, Gorsich SW, Brown RH, Jones LG,
Brown J, et al: Mitochondrial GFA2 is required
for synergid cell death in Arabidopsis. Plant
Cell 14:2215–2232 (2002).
Collins GB, Dunwell JM, Sunderland N: Irregular
microspore formation in Datura innoxia and its
relevance to anther culture. Protoplasma 82:
365–378 (1974).
Corbett KD, Berger JM: Emerging roles for plant
topoisomerase VI. Chem Biol 10: 107–111
(2003).
Custers JBM, Cordewener JHG, Nöllen Y, Dons JJ,
van Lookeren-Campagne MM: Temperature
controls both gametophytic and sporophytic
development in microspore cultures of Brassica
napus. Plant Cell Rep 13:267–271 (1994).
d’Amato F: Role of polyploidy in reproductive or-
gans and tissues, in Johri BM (ed): Embryology
of Angiosperms, pp 519–566 (Springer-Verlag,
New York 1984).
d’Amato F: Polyploidy in cell differentiation.
Caryologia 42: 183–211 (1989).
Datta SK: Androgenic haploids: Factors controlling
development and its application in crop im-
provement. Curr Sci India 89: 1870–1878
(2005).
Dumas de Vaulx R, Chambonnet D: Culture in vi-
tro d’anthères d’aubergine (Solanum melon-
gena L.): stimulation de la production de plan-
tes au moyen de traitements à +35 °C associés à
de faibles teneurs en substances de croissance.
Agronomie 2:983–988 (1982).
Dumas de Vaulx R, Chambonnet D, Pochard E: In
vitro culture of pepper (Capsicum annuum L.)
anthers. High-rate plant production from dif-
ferent genotypes by +35 ° C treatments. Agrono-
mie 1:859–864 (1981).
Dunwell JM, Sunderland N: Pollen ultrastructure
in anther cultures of Nicotiana tabacum I. Ear-
ly stages of culture. J Exp Bot 25: 352–361
(1974).
Dunwell JM, Sunderland N: Pollen ultrastructure
in anther cultures of Datura innoxia. II. The
generative cell wall. J Cell Sci 22: 481–491
(1976).
Duzgunes N, Wilschut J, Fraley R, Papahadjopou-
los D: Studies on the mechanism of mebrane-
fusion – role of headgroup composition in cal-
cium-induced and magnesium-induced fusion
of mixed phospholipid-vesicles. Biochim Bio-
phys Acta 642: 182–195 (1981).
Eigsti OJ, Dustin P: Colchicine in agriculture, med-
icine, biology and chemistry. (The Iowa State
College Press, Ames 1955).
Faure JE, Morgensen HL, Dumas C, Lorz H, Kranz
E: Karyogamy after electrofusion of single egg
and sperm cell protoplasts from maize – cyto-
logical evidence and time-course. Plant Cell 5:
747–755 (1993).
Finnie SJ, Forster BP, Chalmers KJ, Dyer AF, Waugh
R, Powell W: Genetic stability of microspore-
derived doubled haploids of barley – a cytolog-
ical, biochemical, and molecular study. Ge-
nome 34: 923–928 (1991).
Forster BP, Heberle-Bors E, Kasha KJ, Touraev A:
The resurgence of haploids in higher plants.
Trends Plant Sci 12:368–375 (2007).
Fowke LC, Constabel F, Gamborg OL: Fine-struc-
ture of fusion products from soybean cell-cul-
ture and pea leaf protoplasts. Planta 135: 257–
266 (1977).
Germana M: Doubled haploid production in fruit
crops. Plant Cell Tiss Org 86:131–146 (2006).
Gervais C, Newcomb W, Simmonds DH: Rear-
rangement of the actin filament and microtu-
bule cytoskeleton during induction of micro-
spore embryogenesis in Brasssica napus L. cv.
Topas. Protoplasma 213: 194–202 (2000).
Gimenez-Abian MI, Utrilla L, Canovas JL, Gi-
menez-Martin G, Navarrete MH, De la Torre C:
The positional control of mitosis and cytokine-
sis in higher-plant cells. Planta 204: 37–43
(1998).
González-Melendi P, Ramírez C, Testillano PS,
Kumlehn J, Risueño MC: Three dimensional
confocal and electron microscopy imaging de-
fine the dynamics and mechanisms of diploidi-
sation at early stages of barley microspore-de-
rived embryogenesis. Planta 222: 47–57 (2005).
Guha S, Maheshwari SC: In vitro production of em-
bryos from anthers of Datura. Nature 204: 497
(1964).
Hancock LC, Behta RP, Lopes JM: Genomic analy-
sis of the Opi(-) phenotype. Genetics 173: 621–
634 (2006).
Hansen NJP, Andersen SB: In vitro chromosome
doubling potential of colchicine, oryzalin, tri-
fluralin, and APM in Brassica napus micro-
spore culture. Euphytica 88: 159–164 (1996).
Hansen NJP, Andersen SB: Efficient production of
doubled haploid wheat plants by in vitro treat-
ment of microspores with trifluralin or APM.
Plant Breeding 117: 401–405 (1998).
Harada H, Kyo M, Imamura J: The induction of em-
bryogenesis in Nicotiana inmature pollen cul-
ture, in Bock G, Marsh J (eds): Applications of
Plant Cell and Tissue Culture, pp 59–74 (John
Wiley and Sons, Chichester 1988).
Hause B, Hause G, Pecham P, van Lammeren AAM:
Cytoskeletal changes and induction of embryo-
genesis in microspore and pollen cultures of
Brassica napus L. Cell Biol Int 17: 153–168
(1993).
Henry Y: Origin of microspore-derived dihaploid
and polyhaploid in vitro plants. Plant Tissue
Cult Biotech 4: 127–135 (1998).

Cytogenet Genome Res 120:358–369 (2008) 367

Hoekstra S, Vanzijderveld MH, Heidekamp F, Van-
dermark F: Microspore culture of Hordeum
vulgare L. – The influence of density and osmo-
lality. Plant Cell Rep 12:661–665 (1993).
Horvath I, Vanhasselt PR: Inhibition of chilling-in-
duced photooxidative damage to leaves of Cu-
cumis sativus L. by treatment with amino-alco-
hols. Planta 164: 83–88 (1985).
Hu TC, Kasha KJ: A cytological study of pretreat-
ments used to improve isolated microspore cul-
tures of wheat (Triticum aestivum L.) cv. Chris.
Genome 42: 432–441 (1999).
Inze D, De Veylder L: Cell cycle regulation in plant
development. Ann Rev Genet 40: 77–105
(2006).
Jensen CJ: Chromosome doubling techniques in
haploids, in Kasha KJ (ed): Haploids in Higher
Plants: Advances and Potential, pp 153–190
(University of Guelph, Guelph 1974).
Jensen WA: Observations on fusion of nuclei in
plants. J Cell Biol 23: 669–672 (1964).
Joubes J, Chevalier C: Endoreduplication in higher
plants. Plant Mol Biol 43: 735–745 (2000).
Jürgens G: Cytokinesis in higher plants. Ann Rev
Plant Biol 56: 281–299 (2005).
Karp A: Origins, causes and uses of variation in
plant tissue cultures, in Vasil IK, Thorpe TA
(eds): Plant Cell and Tissue Culture, pp 139–151
(Kluwer Academic Publishers, Dordrecht
1994).
Kasha KJ: Chromosome doubling and recovery of
doubled haploid plants, in Palmer CE, Keller
WA, Kasha KJ (eds): Haploids in Crop Improve-
ment II Vol 56, pp 123–152 (Springer-Verlag,
Berlin Heidelberg 2005).
Kasha KJ, Hu TC, Oro R, Simion E, Shim YS: Nu-
clear fusion leads to chromosome doubling
during mannitol pretreatment of barley (Hor-
deum vulgare L.) microspores. J Exp Bot 52:
1227–1238 (2001).
Kasha KJ, Shim YS, Simion E, Letarte J: Haploid
production and chromosome doubling, in Fari
MG, Holb I, Bisztray GD (eds): Acta Horticul-
turae Vol 725, pp 817–828 (ISHS, Debrecen
2006).
Kinney AJ, Clarkson DT, Loughman BC: The regu-
lation of phosphatidylcholine biosynthesis in
rye (Secale cereale) roots – stimulation of the
nucleotide pathway by low-temperature. Bio-
chem J 242:755–759 (1987).
Lahav R, Gammie A, Tavazoie S, Rose MD: Role of
transcription factor Kar4 in regulating down-
stream events in the Saccharomyces cerevisiae
pheromone response pathway. Mol Cell Biol 27:
818–829 (2007).
Larkins BA, Dilkes BP, Dante RA, Coelho CM, Woo
YM, Liu Y: Investigating the hows and whys of
DNA endoreduplication. J Exp Bot 52:183–192
(2001).
Lichter R, Degroot E, Fiebig D, Schweiger R, Gland
A: Glucosinolates determined by HPLC in the
seeds of microspore-derived homozygous lines
of rapeseed (Brassica napus L.). Plant Breeding
100:209–221 (1988).
Lotfi M, Alan AR, Henning MJ, Jahn MM, Earle
ED: Production of haploid and doubled haploid
plants of melon (Cucumis melo L.) for use in
breeding for multiple virus resistance. Plant
Cell Rep 21:1121–1128 (2003).
Magyar Z, De Veylder L, Atanassova A, Bako L,
Inze D, Bogre L: The role of the Arabidopsis
E2FB transcription factor in regulating auxin-
dependent cell division. Plant Cell 17: 2527–
2541 (2005).
Maraschin SF, de Priester W, Spaink HP, Wang M:
Androgenic switch: an example of plant em-
bryogenesis from the male gametophyte per-
spective. J Exp Bot 56:1711–1726 (2005).
368 Cytogenet Genome Res 120:358–369 (2008)
Meyer K: Über die Entwicklung des Pollens bei
Leontodon autumnalis L. Ber Deut Bot Ges 43:
108–114 (1925).
Nagl W: DNA endoreduplication and polyteny un-
derstood as evolutionary strategies. Nature 261:
614–615 (1976).
Nishihama R, Ishikawa M, Araki S, Soyano T, Asa-
da T, Machida Y: The NPK1 mitogen-activated
protein kinase kinase kinase is a regulator of
cell-plate formation in plant cytokinesis. Genes
Dev 15:352–363 (2001).
Ouyang JW, Liang H, Jia SE, Zhang C, Zhao TH, et
al: Studies on the chromosome doubling of
wheat pollen plants. Plant Sci 98: 209–214
(1994).
Palmer CE, Keller WA: Overview of haploidy, in
Palmer CE, Keller WA, Kasha KJ (eds): Hap-
loids in Crop Improvement II Vol 56, pp 3–9
(Springer-Verlag, Berlin Heidelberg 2005).
Park SK, Twell D: Novel patterns of ectopic cell plate
growth and lipid body distribution in the Ara-
bidopsis gemini pollen1 mutant. Plant Phys 126:
899–909 (2001).
Pauls KP, Chan J, Woronuk G, Schulze D, Brazolot
J: When microspores decide to become embry-
os – cellular and molecular changes. Can J Bot
84:668–678 (2006).
Pechan PM, Bartels D, Brown DCW, Schell J: Mes-
senger-RNA and protein-changes associated
with induction of Brassica microspore embryo-
genesis. Planta 184:161–165 (1991).
Philipp EI, Franke WW, Keenan TW, Stadler J, Ja-
rasch ED: Characterization of nuclear mem-
branes and endoplasmic reticulum isolated
from plant tissue. J Cell Biol 68: 11–29 (1976).
Pintos B, Manzanera JA, Bueno MA: Antimitotic
agents increase the production of doubled-hap-
loid embryos from cork oak anther culture. J
Plant Physiol 164: 1595–1604 (2007).
Portereiko MF, Sandaklie-Nikolova L, Lloyd A, De-
ver CA, Otsuga D, Drews GN: Nuclear fusion
defective1 encodes the Arabidopsis RPL21M
protein and is required for karyogamy during
female gametophyte development and fertiliza-
tion. Plant Physiol 141: 957–965 (2006).
Pretova A, Ruijter NC, van Lammeren A, Schel JH:
Structural observations during androgenesis
microspore culture of the 4c1 genoype of Zea
mays L. Euphytica 65: 61–69 (1993).
Rao PS, Suprasanna P: Methods to double haploid
chromosome numbers, in Jain SM, Sopory SK,
Veilleux RE (eds): In vitro Haploid Production
in Higher Plants Vol 1, pp 317–339 (Kluwer Ac-
ademic Publishers, Dordrecht 1996).
Raquin C, Amssa M, Henry Y, de Buyser J, Essad S:
Origine des plantes polyploides obtenues par
culture d’anthères. Analyse cytophotométrique
in situ et in vitro des microspores de Pétunia et
de blé tendre. Z Pflanzenzucht 89: 265–277
(1982).
Risueño MC, Giménez M, López-Saez F: Experi-
mental analysis of plant cytokinesis. Exp Cell
Res 49: 136–147 (1968).
Saisingtong S, Schmid JE, Stamp P, Buter B: Colchi-
cine-mediated chromosome doubling during
anther culture of maize (Zea mays L). Theor
Appl Genet 92: 1017–1023 (1996).
Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S,
Siniossoglou S: The yeast lipin Smp2 couples
phospholipid biosynthesis to nuclear mem-
brane growth. EMBO J 24:1931–1941 (2005).
Sato S, Katoh N, Iwai S, Hagimori M: Frequency of
spontaneous polyploidization of embryos re-
generated from cultured anthers or micro-
spores of Brassica rapa var. pekinensis L. and B.
oleracea var. capitata L. Breeding Sci 55:99–102
(2005).
Schulz P, Jensen WA: Capsella embryogenesis –
central cell. J Cell Sci 12:741–763 (1973).
Seguí-Simarro JM, Nuez F: Meiotic metaphase I to
telophase II is the most responsive stage of mi-
crospore development for induction of andro-
genesis in tomato (Solanum lycopersicum). Acta
Physiol Plant 27:675–685 (2005).
Seguí-Simarro JM, Nuez F: Embryogenesis induc-
tion, callogenesis, and plant regeneration by in
vitro culture of tomato isolated microspores
and whole anthers. J Exp Bot 58: 1119–1132
(2007).
Seguí-Simarro JM, Austin JR, White EA, Staehelin
LA: Electron tomographic analysis of somatic
cell plate formation in meristematic cells of
Arabidopsis preserved by high-pressure freez-
ing. Plant Cell 16:836–856 (2004).
Seguí-Simarro JM, Testillano PS, Risueño MC:
MAP kinases are developmentally regulated
during stress-induced microspore embryogen-
esis in Brassica napus L. Histochem Cell Biol
123: 541–551 (2005).
Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev
A: Stresses applied for the re-programming of
plant microspores towards in vitro embryogen-
esis. Acta Physiol Plant 127: 519–534 (2006).
Shim YS, Kasha KJ, Simion E, Letarte J: The rela-
tionship between induction of embryogenesis
and chromosome doubling in microspore cul-
tures. Protoplasma 228: 79–86 (2006).
Simmonds DH, Keller WA: Significance of prepro-
phase bands of microtubules in the induction of
microspore embryogenesis of Brassica napus.
Planta 208:383–391 (1999).
Sugimoto-Shirasu K, Roberts K: ‘Big it up’: endo-
reduplication and cell-size control in plants.
Curr Opin Plant Biol 6: 544–553 (2003).
Sunderland N: Anther culture as a means of haploid
induction, in Kasha KJ (ed): Haploids in Higher
Plants: Advances and Potential, pp 91–122
(University of Guelph, Guelph 1974).
Sunderland N, Evans LJ: Multicellular pollen for-
mation in cultured barley anthers. II. The A-
pathway, B-pathway and C-pathway. J Exp Bot
31:501–514 (1980).
Sunderland N, Collins GB, Dunwell JM: Role of nu-
clear fusion in pollen embryogenesis of Datura
innoxia Mill. Planta 117:227–241 (1974).
Tasseva G, Richard L, Zachowski A: Regulation of
phosphatidylcholine biosynthesis under salt
stress involves choline kinases in Arabidopsis
thaliana. Febs Lett 566:115–120 (2004).
Testillano P, Georgiev S, Mogensen HL, Coronado
MJ, Dumas C, et al: Spontaneous chromosome
doubling results from nuclear fusion during in
vitro maize induced microspore embryogene-
sis. Chromosoma 112:342–349 (2004).
Touraev A, Ilham A, Vicente O, Heberle-Bors E:
Stress-induced microspore embryogenesis in
tobacco: an optimized system for molecular
studies. Plant Cell Rep 15:561–565 (1996a).
Touraev A, Indrianto A, Wratschko I, Vicente O,
Heberle-Bors E: Efficient microspore embryo-
genesis in wheat (Triticum aestivum L.) induced
by starvation at high temperatures. Sexual
Plant Reprod 9:209–215 (1996b).
Touraev A, Pfosser M, Heberle-Bors E: The micro-
spore: A haploid multipurpose cell. Adv Bot Res
35:53–109 (2001).
Traas J, Hulskamp M, Gendreau E, Hofte H: Endo-
reduplication and development: rule without
dividing? Curr Opin Plant Biol 1: 498–503
(1998).
Valente P, Tao WH, Verbelen JP: Auxins and cyto-
kinins control DNA endoreduplication and de-
duplication in single cells of tobacco. Plant Sci
134:207–215 (1998).
Valster AH, Pierson ES, Valenta R, Hepler PK,
Emons AMC: Probing the plant actin cytoskel-
eton during cytokinesis and interphase by pro-
filin microinjection. Plant Cell 9: 1815–1824
(1997).
Wan Y, Widholm JM: Effect of chromosome-dou-
bling agents on somaclonal variation in the
progeny of doubled haploids of maize. Plant
Breeding 114: 253–255 (1995).
Wan Y, Petolino JF, Widholm JM: Efficient produc-
tion of doubled haploid plants through colchi-
cine treatment of anther-derived maize callus.
Theor Appl Genet 77: 889–892 (1989).
Wang M, Farnham MW, Nannes JSP: Ploidy of
broccoli regenerated from microspore culture
versus anther culture. Plant Breeding 118:249–
252 (1999).
West MA, Harada JJ: Embryogenesis in higher
plants: an overview. Plant Cell 5: 1361–1369
(1993).
Yasuhara H, Sonobe S, Shibaoka H: Effects of taxol
on the development of the cell plate and of the
phragmoplast in tobacco BY-2 cells. Plant Cell
Physiol 34: 21–29 (1993).
Zaki MAM, Dickinson HG: Microspore-derived
embryos in Brassica: the significance of divi-
sion symmetry in pollen mitosis I to embryo-
genic development. Sexual Plant Reprod 4: 48–
55 (1991).
Zhao JP, Simmonds DH: Application of trifluralin
to embryogenic microspore cultures to gener-
ate doubled haploid plants in Brassica napus.
Acta Physiol Plant 95: 304–309 (1995).
Zhao JP, Simmonds DH, Newcomb W: Induction of
embryogenesis with colchicine instead of heat
in microspores of Brassica napus L cv Topas.
Planta 198:433–439 (1996).
Cytogenet Genome Res 120:358–369 (2008)
Zhou WJ, Hagberg P, Tang GX: Increasing embryo-
genesis and doubling efficiency by immediate
colchicine treatment of isolated microspores in
spring Brassica napus. Euphytica 128: 27–34
(2002a).
Zhou WJ, Tang GX, Hagberg P: Efficient produc-
tion of doubled haploid plants by immediate
colchicine treatment of isolated microspores in
winter Brassica napus. Plant Growth Regul 37:
185–192 (2002b).
Zoriniants S, Tashpulatov A, Heberle BE, Touraev
A: The role of stress in the induction of haploid
microspore embryogenesis, in Palmer CE,
Keller WA, Kasha KJ (eds): Haploids in Crop
Improvement II Vol 56, pp 35–52 (Springer-
Verlag, Berlin Heidelberg 2005).
369