chromosome doubling during androgenesis

wolf.jonathan42

chromosome doubling during androgenesis

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 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

e-mail: seguisim@btc.upv.es

Fax +41 61 306 12 34

E-Mail karger@karger.ch

www.karger.com

© 2008 S. Karger AG, Basel

1424–8581/08/1204–0358$24.50/0

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:

www.karger.com/cgr


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

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 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

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


360

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,

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 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


A B

C D

E F

G

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.

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 embryogenesis

is an additional argument against endoreduplication

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 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

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 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-

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 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

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

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 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).

366

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

in dicotyledons.

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

quantitative traits.

Conclusions

Chromosome doubling is highly influenced by the environment

of in vitro cultures. During androgenesis, doubling

may occur through three main mechanisms: endoreduplication,

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 experimental

induction of androgenesis, evidence led many researchers

to think that endoreduplication could also be the

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.

Colchicine and other antimitotics have traditionally

been used to induce doubling through mitotic impairment

during androgenesis as well as in other experimental situa-

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