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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)

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-

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

Request reprints from José M. Seguí-Simarro, Instituto para laConservación y Mejora de la Agrodiversidad Valenciana (COMAV)Universidad politécnica de Valencia Ciudad Politécnica de laInnovación (CPI), Edificio 8E – Escalera 9, Camino de Vera s/nES–46022 Valencia (Spain)telephone: +34 387 7000, ext. 88472; fax: +34 96 387 9422e-mail: [email protected]

© 2008 S. Karger AG, Basel1424–8581/08/1204–0358$24.50/0

Accessible online at:www.karger.com/cgr

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

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.

Accepted in revised form for publication by M. Schmid, 14 December 2007.

Cytogenet Genome Res 120:358–369 (2008) 359

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. 1 A): endoreduplication (DNA duplica-tion 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 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. 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)360

(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. 1 B�), 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

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 endoredu-plication. ( C ) Nuclear fusion after defective cytokinesis. ( D ) Endomitosis. ( E ) C-mitosis after mitotic blockage. See text for further details.


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.,


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. 1 C) 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 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 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. 2 A, 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. 2 C, D) giving

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 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.


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. 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 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 havebeen 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).


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. 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-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-

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. 1 D) 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. 1 E) 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.


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).

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 (N 2 O) gas (reviewed in Jensen, 1974). N 2 O has been used to induce chromosome doubling in a variety of species, mostly cere-als. However, N 2 O 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


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