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The Botanical Review

The New York Botanical Garden 201010.1007/s12229-010-9056-6

Androgenesis Revisited

Jos M. Segu-Simarro 1

Instituto para la Conservacin y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politcnicade Valencia, Ciudad Politcnica de la Innovacin (CPI), Edificio 8E - Escalera I. Camino de vera, s/n,46022 Valencia, Spain

Jos M. Segu-SimarroEmail: [email protected]

Published online: 13 May 2010

Abstract

Androgenesis can be defined as the set of biological processes leading to an individual genetically

coming exclusively from a male nucleus. Androgenesis was traditionally considered as the spontaneous,

in vivo development of a male-derived haploid embryo from a fertilized egg where the female nucleus is

eliminated. However, at present it is also possible to generate androgenic haploids/doubled haploids

through in vitro microspore embryogenesis and by in vitro meiocyte-derived callogenesis. These three

androgenic alternatives clearly differ in the inducible stage, but lead to the same final haploid or doubled

haploid product. Whereas microspore embryogenesis is widely studied and applied, the other two

routes are much less known. In this paper, the evidence accounting for the existence of these three

alternative pathways is revised, as well as the mechanisms potentially involved in their induction. Their

differences and similarities are discussed from a biological perspective, leading to the notion that they

might represent an ancient survival mechanism triggered by similar factors.

Keywords Microspore Embryogenesis Meiocyte-Derived Callogenesis Male-Specific

Parthenogenesis Plant Reproduction Haploid Doubled Haploid

Resumen

La andrognesis se define como el conjunto de vas biolgicas que dan lugar a un individuo cuyo fondo



gentico proviene exclusivamente de un ncleo de origen masculino. Tradicionalmente, el concepto de

andrognesis estaba restringido al desarrollo espontneo, in vivo, de un embrin haploide o doble

haploide a partir de una clula huevo fecundada en la que el material gentico de origen femenino era

inactivado o eliminado. Sin embargo, hoy en da sabemos que existen otras vas para conseguir

haploides o doble haploides andrognicos, mediante embriognesis de microsporas y mediante la

formacin de callos derivados de meiocitos. Estas tres alternativas andrognicas difieren claramente en

la etapa en la que es posible la induccin, pero dan lugar al mismo producto haploide o doble haploide

final. Mientras que la embriognesis de microsporas es un fenmeno ampliamente estudiado y de clara

aplicacin prctica, las otras dos rutas son mucho menos conocidas. En este trabajo se revisan las

evidencias existentes al respecto de estas tres alternativas andrognicas, as como los mecanismos

potencialmente implicados en su induccin. Tambin se discuten sus diferencias y semejanzas, desde

un punto de vista biolgico, para llegar a la hiptesis de que estas rutas podran representar un

mecanismo atvico de supervivencia activado por factores similares.

Introduction

During evolution, plant cells have retained the capacity to express virtually any part of their coding

genome. This remarkable feature, called totipotency, allows for a differentiated cell to dedifferentiate

and adopt a proliferative growth pattern (callus), or to deviate towards a developmental program

different from the original. These programs include the regeneration of a new individual either through

the successive regeneration of all of its vegetative organs (organogenesis) or directly by the entry into a

new embryogenic pathway (embryogenesis). Using adequate experimental conditions, it is possible to

induce organogenesis and/or embryogenesis from cells of a variety of plant tissues, including leaves,

cotyledons, root tips, hypocotils, anthers, ovaries, etc. (Razdan, 2003; Vasil & Thorpe, 1994). From

an applied point of view, one of the most interesting cell types to regenerate individuals is haploid

(reduced) gamete precursors, due to the possibility to regenerate haploid or doubled haploid (DH)

individuals.

Haploids are interesting for fundamental research, but their main utility is to produce DH lines, which

are extremely useful tools for basic and applied research, including breeding programs (Chupeau et al.,

1998; Dunwell, 2010; Forster et al., 2007; Touraev et al., 2001). From the standpoint of plant

breeding, the DH alternative reduces the typical 78 inbreeding generations necessary to stabilize a

hybrid genotype to only one. It is therefore much faster and cheaper, and obviously this is the main

advantage of DH technology in plant breeding. Still within the context of plant breeding, DHs are also

essential for genetic mapping of complex characters such as production or quality, the most interesting



from an agronomical point of view, but also the most difficult to be addressed by other approaches.

DHs are also a powerful tool in transgenesis, to avoid hemizygotes and save time and resources in the

production of plants transformed with the transgene in both homologous chromosomes. Moreover,

from a basic scientific point of view, these lines are also very useful for basic studies of linkage and

estimation of recombination fractions. Although these studies can also be made conventionally

(backcrosses or F2), DH lines have the advantage of being auto-perpetuable, meaning that they can be

perpetuated simply by selfing. They are also an extremely useful tool for genetic selection and screening

of recessive mutants, because the phenotype of the resulting plants is not affected by the effects of

dominance, and the characters determined by recessive genes can be easily identified. Another

advantage is the ability to serve as a model system for studying embryo development in vitro, without

the interference of maternal tissue, due to the large number of similarities of microspore-derived

embryogenesis compared with zygotic embryogenesis (Segu-Simarro & Nuez, 2008a; Supena et al.,

2008).

It is possible to obtain haploid/DH individuals through different developmental pathways, involving both

the female and the male gametophytes (Dunwell, 2010). From the unpollinated female gametophyte, it

is possible to obtain haploids through a pathway known as gynogenesis (Bohanec, 2009). Gynogenesis

is used in species such as sugar-beet and onion, where other DH-inducing techniques have proven

unsuccessful. Through this route, immature ovules are cultured up to maturation of embryo sac, where a

haploid embryo is developed. For few species, this embryo is thought to be originated from antipodal

or synergid cells, but in the vast majority of cases, the gynogenic embryo is derived from the egg cell

(reviewed in Bohanec, 2009). Gynogenic embryos are predominantly haploid, which implies that in

order to obtain the desired doubled haploid, additional treatments for chromosome doubling must be

considered. A pathway similar to that induced by culturing immature ovules can be induced by wide,

interspecific or even intergeneric crosses. By crossing two sexually incompatible species, it is possible

to induce the development of a haploid plant coming exclusively from the female gamete, therefore

excluding any genetic background from the male parental. This is the case for the Hordeum

bulbosum method, initially discovered in 1970 (Kasha & Kao, 1970), and now widely used for DH

production in barley breeding programs for the delivery of commercial varieties (Devaux, 2003; Hayes

et al., 2003; Wedzony et al., 2009). In this method, a hybrid embryo is formed by the fusion of a H.

bulbosum male gamete with a H. vulgare egg cell. However, post-zigotic incompatibility barriers lead

to the progressive elimination of the chromosomes from the H. bulbosum male parental (Devaux,

2003), so that the resulting (haploid) embryo has a H. vulgare background. Incompatibility at the level

of the endosperm makes also mandatory the isolation and in vitro rescue of the haploid embryo. As for

gynogenesis, haploid embryos/plants must be exposed to chromosome doubling agents in order to

become DH. Similarly, interegeneric hybridization has also been described as capable of inducing

haploid plant development. For example, through intergeneric crosses between wheat and maize



(Zenkteler & Nitzsche, 1984). The use of maize pollen as a mentor to induce the development of

haploid embryos in other genera has been widely exploited in the last 20 years, mostly for cereals

(reviewed in Wedzony et al., 2009). Intergeneric crosses have also been used for ploidy reductions,

for example to reduce octoploids to tetrahaploids in strawberry (Janick & Hughes, 1974). Other

strategies to induce haploids of female origin include the induction of parthenogenesis with irradiated

pollen. In irradiated pollen, the sperm cells are inactivated and therefore unable to fuse with the egg

cell, but capable to trigger haploid embryo development. This approach was used to generate female-

derived parthenogenic haploids in Nicotiana rustica (Ivanov, 1938) and tomato (Ecochard et al.,

1974) among others, or to reduce tetraploids to dihaploids in blackberry (Naess et al., 1998).

Inactivation of pollen to be used as mentor for haploid parthenogenesis in Populus tremula was also

achieved by toluidine blue treatments (Illies, 1974). A similar approach, in terms of using non-fertilizing

pollen, consists of pollinating with pollen of relative species. This is the case of potato (De Maine,

2003), where dihaploids of Solanum tuberosum (4) are obtained by pollination with Solanum

phureja (2). In this case, the two sperm cells (1) from S. phureja fuse with the secondary nucleus

of S. tuberosum (4) to give rise to a 6 endosperm, which is able to support the development of a

dihaploid (2) embryo from the unfertilized S. tuberosum egg cell.

On the other hand, haploidy or doubled haploidy can also be achieved from the male gametophyte or

its precursors through a pathway known as androgenesis. According to its initial definition, the concept

androgenesis was invariably linked to fertilization. It was originally coined to define a route involving

sexual reproduction, egg fertilization, and subsequent inactivation of the female nucleus, so that a male-

derived, haploid embryo is formed within the embryo sac (Rieger et al., 1968). This androgenic

pathway, although present in nature, is extremely rare and little is known about it, likely due to its null

potential for commercial exploitation. This situation contrasts with the enormous possibilities of

microspore embryogenesis for both applied and basic research. Microspore embryogenesis, although

described more than 40 years ago, has become of great practical importance for the agronomic

industry in the last decade (Dunwell, 2010; Forster et al., 2007) due to its convenience for producing

pure, homozygous DH lines much faster than the other methods above mentioned. In this route, a male-

derived haploid individual is also obtained, but through a different developmental pathway, not involving

egg fertilization. In addition, there is a third alternative to produce a haploid/DH individual from a male

nucleus: plant regeneration from meiocyte-derived haploid callus. Examples on the existence of this

route have been published during the last 40 years in highly recalcitrant species (Bal & Abak, 2007;

Corduan & Spix, 1975; Gresshoff & Doy, 1972b, 1974; Segu-Simarro & Nuez, 2007), where other

androgenic procedures have proven unsuccessful.

In the Proceedings of the 1st International Conference on Haploids in Higher Plants, in 1974, the

basis for a broad, inclusive use of the term androgenesis were established (de Fossard, 1974b).

However, some researchers still want to preserve the original meaning, restricted only to in vivo



androgenesis, despite the limited number of studies in plants reporting examples of egg fertilization and

inactivation of the female nucleus. In addition, all of these examples date from more than 40 years ago.

In parallel, other researchers are already using androgenesis as a synonym for microspore

embryogenesis (Tai, 2005), which obviously does not include the original, in vivo pathway. And few

people know that in some species it is also possible to obtain haploids/DHs by meiocyte-derived

callogenesis. It is thus desirable to approach the different routes leading to male-derived haploidy from

an integrative point of view. In this review, I revisit the concept of androgenesis from such an integrative

perspective, focusing on these different experimental (or spontaneous) biological pathways to obtain a

haploid (or potentially DH) individual from the male gametophyte or its precursors, and exploring the

cellular and/or molecular triggers that may potentially induce each of them. Given its outstanding

potential for plant breeding and interest in economic terms, nearly all of the research on male-derived

haploidy has been focused on microspore embryogenesis. As a consequence, in the last years excellent

and comprehensive reviews and books about microspore embryogenesis have been published

elsewhere (Boutilier et al., 2005; Dunwell, 2010; Hosp et al., 2007a; Maraschin et al., 2005; Palmer

et al., 2005; Pauls et al., 2006; Segu-Simarro & Nuez, 2008a, b; Touraev et al., 2009). For this

reason, in this review I will only outline the main features of this process and the most relevant findings

of this new age in the study of microspore embryogenesis. For more detailed information I will refer

the reader to those reviews and the corresponding original research articles. However, and as a

difference with recent reviews, I will pay special attention in this review to the other two, less known

alternatives: male-derived haploid embryogenesis within the embryo sac, and meiocyte-derived

callogenesis. I revise the most relevant literature documenting these phenomena from a biological point

of view, in order to show that despite their low relevance, they constitute androgenic events. Finally, I

relate these different androgenic events mentioning their differences, but also highlighting their

commonalities. These common features strengthen the concept of androgenesis as a process whereby a

haploid/DH individual is created from a male nucleus (of a gamete, gamete precursor or gametophyte

precursor), regardless of the developmental route involved.

Male-Derived Haploid Embryogenesis in the EmbryoSac

The term androgenesis was initially confined to a male-specific form of parthenogenesis by which an

embryo is believed to originate from a fertilized egg where the female nucleus is somehow inactivated

or eliminated (Fig. 1, Route 1) and only those genes coming from the male parental are present (de

Fossard, 1974b). In nature, this seems to be a rare alternative to sexual reproduction, only used by

several clam families, a Saharan cypress tree, the Mexican axolotl Siredon mexicanum (reviewed in



McKone & Halpern, 2003; Tulecke, 1965), some interspecific hybrids between Sicilian stick insects

of the genus Bacillus (Mantovani & Scali, 1992), and in the particular case of a Drosophila mutant

(Komma & Endow, 1995). This situation contrasts with that of teleost fishes, where this route has

never been documented to occur spontaneously, but can be successfully induced and used to obtain

DHs for practical purposes (Komen & Thorgaard, 2007).

Fig. 1

The different androgenic alternatives to male gamete formation. Starting from microsporogenesis andmicrogametogenesis (route 0, purple background), a male-derived haploid or doubled haploid individual canoriginate by three ways: (1) gamete formation, egg fertilization without nuclear fusion, and dismantling ofthe maternal nucleus (route 1). (2) Deviation of the vacuolate microspore or the young pollen grain towardsembryogenesis or occasionally callogenesis followed by organogenesis (route 2). (3) Deviation of themeiocyte towards callogenesis, which may potentially lead to haploids and doubled haploids, but also toheterozygous diploids (route 3). See text for further details

In angiosperms, this spontaneous developmental pathway was described long ago, being the first

reports as old as of 1929 (Clausen & Lammerts, 1929; Kostoff, 1929). However, 80 years after,



published examples of this process are still scarce, probably due to the low rate of occurrence of this

phenomenon in nature. Besides, most of these examples date from more than 40 years ago, with very

scarce new contributions to the knowledge of this phenomenon in these four decades. In tobacco

(Nicotiana tabacum) a mean rate of one androgenic haploid every 2,500 individuals was reported by

Burk (1962). In maize (Zea mays), the highest mean frequency of spontaneous androgenesis reported

is 1/80,000 in a sample size of 400,000 individuals from ordinary strains (Chase, 1969; Goodsell,

1961). Previous studies reported even lower frequencies of 1/187,500 and 1/214,000 over

populations of 750,000 and 429,300 individuals, respectively (Gerrish, 1956; Seaney, 1955). As a

reference, natural parthenogenesis, a more common and known event, occurred also in maize at a

1/1,000 rate (Chase, 1969). This sort of natural androgenesis was also reported to occur in some

species of Capsicum (C. frutescens, Campos & Morgan, 1958), and in certain interspecific crosses

of the genus Nicotiana. In particular, in crosses N. digluta (2n=72) N. tabacum (2n=48),

where a haploid N. tabacum (2n=24) descendant was obtained (Clausen & Lammerts, 1929), in

crosses N. tabacum var. macrophylla (3n=72) N. langsdorfii (2n=18) where a small, haploid

N. langsdorfii plant (2n=9) was obtained (Kostoff, 1929), in crosses N. glutinosa (2n=24) N.

repanda (2n=48), where a haploid N. glutinosa and a haploid N. repanda were obtained, among

others (Kehr, 1951), and in crosses N. sylvestris F1 hybrids N. tabacum N. sylvestris ,

where a haploid plant expressing only characters from N. sylvestris was observed (Kostoff, 1942). In

addition to the spontaneous occurrence of this phenomenon, there are two documented cases of

induced occurrence, in Crepis tectorum and Antirrhinum majus, using an experimental approach

opposite to that above described for production of female-derived embryos with irradiated pollen. In

this case, a male-derived embryo was obtained after emasculation of the female parental, irradiation of

the pollen sac, and pollination with untreated pollen (Ehrensberger, 1948; Gerassimova, 1936). In all of

these cases, the identification of the male origin of the haploid was based on a phenotypic

characterization of the descendants expressing only those characters pertaining to the male parental. In

the case of Kermicle experiments with maize, the system used allowed also for the identification of

spontaneous genome duplication (Kermicle, 1974). Kermicle crossed a male parent heterozygous for a

nuclear restorer (Rf 1 rf 1 ) of Texas cytoplasmic male sterility (cms-T) with cms-T females (rf 1 rf 1 ).

He obtained 6 diploid, fertile plants, which gave 100% fertile descendants, indicating Rf Rf constitution,

and one plant giving only sterile offpring, indicating rf rf.

The knowledge of the genetic control of this process is still at its infancy, with the exception of maize,

where a spontaneous mutation seems to enhance the occurrence of this androgenic alternative.

According to Kermicle experiments (Kermicle, 1969, 1971, 1994), spontaneous androgenesis in

maize seems to be under genetic control. As opposed to the low frequency reported for wild type

maize plants (see above), Kermicle observed a high incidence (up to 3%) of spontaneous male-

derived, androgenic haploids in a mutant inbred line carrying a mutant allele of the indeterminate



gametophyte1 (ig1) gene. As opposed to many other mutants affecting the haploid gametophytes, the

ig1mutants are viable, being the ig mutant allele dominant and maternally sex-limited, since ig male

plants did not show any difference in the frequency of androgenesis. This mutation is located on

chromosome arm 3L and has as a direct effect an increased number of nuclear divisions before

cellularization of the embryo sac, which generates in the embryo sac an indeterminate, extra number of

micropylar and synergids cells, egg cells, central cells, and polar nuclei within central cells (Evans,

2007; Guo et al., 2004; Huang & Sheridan, 1996; Lin, 1978, 1981). This mutation also affects nuclear

migration and cellular differentiation (Lin, 1978, 1981).This array of pleiotropic effects at the level of

the female gametophyte gives rise to polyembryony and elevated ploidy levels of the endosperm after

fertilization, derived from fertilization by different pollen tubes, together with miniature endosperms,

early abortion of seeds and the occurrence of androgenic and gynogenic haploids in a 2:1 ratio

(Kermicle, 1969, 1971, 1994; Lin, 1984). Other effects include sporophytic male sterility in some

genetic backgrounds (Kermicle, 1994) or abnormal leaf morphology (Evans, 2007). Consistent with

this, it was suggested (Lin, 1981) that the ig1 locus does not control a single process but acts as a

regulator of other downstream genes involved in female gametophyte development. Some of the genes

regulated by the ig1 locus could be those involved on the switch from proliferation to differentiation in

the embryo sac, since in mutant embryo sacs the proliferative phase is prolonged (Huang & Sheridan,

1996; Lin, 1978). But most importantly, ig1 is thought to be involved in the repression of the

embryogenic program in those cells that lack one of the two parental genomes. In other words, ig1

would be preventing the uncontrolled trigger of proliferation in non-gametic cells, and of embryogenesis

in egg or sperm cells prior to fertilization. Once fertilization and karyogamy occur, ig1 would allow for

embryogenesis to take place in the unicellular zygote. Thus in ig1 mutants, the embryogenic

development of the haploid egg cell or the two haploid sperm cells after pollen tube discharge would

not be repressed. This would be consistent with the above mentioned 2:1 rate of androgenic versus

gynogenic embryo production observed by Kermicle.

The cellular aspects of this process are also largely unknown. In angiosperms, the haploid androgenic

embryo is believed to originate from a reduced sperm nucleus. Although it is clear that the genetic

material of the androgenic embryo comes from the male parental, it was generally assumed that

androgenic haploids originate exclusively from divisions of the sperm nucleus, subsequent to

fertilization. However, there does not seem to be enough supporting evidence for this. As pointed out

by Pandey (1973), it might well be the vegetative nucleus of the pollen grain that is responsible for

proliferative growth. This hypothesis is consistent with the demonstrated role that the vegetative nucleus

has in a much better studied process as is microspore embryogenesis (Maraschin et al., 2005). The

contribution of the female gametophyte remains obscure as well. Although it was shown that the

resulting haploid individual inherits cytoplasm of female origin (Chase, 1963; Goodsell, 1961), it is not

clearly established that such cytoplasm derives from the egg cell (Lacadena, 1974), so the possibility of



a female gametophytic origin other than the egg cell should not be ruled out. This possibility should be

carefully considered in the case of maize androgenic embryos derived from ig1 mutants. According to

the recent knowledge above reviewed about the role of the ig1 gene, the androgenic mechanism

triggered by the ig1 mutation would be different from that generally assumed, since the sperm-derived

haploid embryo would not imply fertilization and elimination or inactivation of the female genome. In

turn, the androgenic embryo would emerge directly from a sperm cell, without the need for fertilization

as a prerequisite, and having a cytoplasmic background contributed by the sperm cell, and/or perhaps

the central cell, but not the egg cell. Another intriguing aspect is the elimination of the maternal genome.

Similar processes of chromosome elimination occur either in a spontaneous or in an induced manner

during protoplast fusion (Liu et al., 2005), but unfortunately, as in androgenic elimination, very little is

known about the underlying mechanism. These two processes do not seem to share a common trigger,

since elimination in the fused protoplast nucleus is strongly affected by factors such as phylogenetic

distance, different ploidy levels of parents, or irradiated dosage for induced elimination (Liu et al.,

2005). Some of these factors, which may also be present in other types of haploid-inducing techniques

(gynogenesis through interspecific or intergeneric hybridization) are not present in most of the reported

cases of in vivo androgenesis. For example, it could be argued that phylogenetic distance might have an

effect in the induction of androgenic haploids by crosses between the Nicotiana species mentioned

above. However, in all of the examples of spontaneous, intraspecific crosses documented in maize,

tobacco, Capsicum, or even in the maize ig1 mutants, phylogenetic distance cannot be used as an

argument. In summary, it seems clear that in vivo androgenic plants inherit a male genetic background,

but the origin of their cytoplasm and the mechanisms by which the female genome is inactivate still await

to be unambiguously determined.

The practical application of this natural phenomenon is rather limited. In the particular case of the ig1

gene in maize, the side effect of an increased frequency of androgenic embryos has been used in

breeding to transfer germplasm (e.g., a male sterility conditioning cytoplasm) from one variety of maize

to the cytoplasm of another variety (Kindiger & Hamann, 1993). The use of the inbred line W23

(Kermicle, 1969) carrying the ig1 mutation, in combination with microsatellite (SSR) fingerprinting, has

been proposed as a strategy for maize hybrid development in tropical germplasm (Belicuas et al.,

2007). Recently, two patents have been developed in order to exploit the androgenic potential of the

ig1 mutation by introducing this mutated gene in other plant species (Evans, 2005, 2009). However,

apart from these particular examples, the low efficiency of this sort of in vivo androgenic development

has limited its practical, routine use in maize. Beyond maize, the ig1 mutation has not been described in

any other crop, and to the best of my knowledge, there is no other gene known to have a direct or

indirect role in the in vivo induction of androgenic haploids. Thus, in vivo androgenesis as a system for

producing natural androgenic haploids is not expected to have a significant impact in plant

biotechnology in the next future.



In summary, 80 years after the first reports, little new is known about the cellular, molecular, or genetic

basis of this intriguing process. There are many aspects still to be elucidated about this unusual

developmental pathway that stands as a biological rarity. Unfortunately, although this pathway has been

known for a long time, new findings are not likely to be published in the next few years. This is in part

due to the difficulties imposed by the low frequency, but mostly due to the lack of applied interest of

this spontaneous process.

Microspore/Pollen Embryogenesis

Besides those above mentioned, a male-derived haploid plant can be obtained through other different

ways. The most common and best studied route is microspore/pollen-derived haploid embryogenesis.

In this route, a male-derived haploid plant is formed when microspores, typically at the vacuolate stage,

or young pollen grains are experimentally deviated from their original gamete-producing pathway

towards embryogenesis (Fig. 1, Route 2). In 1964, Guha & Maheshwari demonstrated that this

pathway can be induced through the in vitro culture of microspore/pollen-containing anthers of Datura

innoxia. After Guha and Maheshwari, numerous researchers realized the practical significance of such

a discovery and explored this pathway in many different species and genera, and now it is considered

the most powerful tool to obtain DH plants for practical breeding purposes or genetic mapping, among

others. In addition to the method proposed by Guha and Maheshwari for anther excision and

inoculation into solid medium, further research has evidenced that microspore embryogenesis can also

be achieved through direct microspore isolation from the anther locule and inoculation into liquid

medium. This latter method is more technically demanding, which limits its application to a range of

species narrower than anther culture. However, in those species where it has been well set up, it is the

method of choice due to its higher efficiency and reduced time to obtain haploid and/or DH plants.

In 2003, successful induction of microspore/pollen embryogenesis through in vitro culture of anthers or

isolated microspores was documented for more than 250 plant species (Maluszynski et al., 2003b),

including many species of agronomic interest, from herbaceous crops such as wheat, barley, rice,

rapeseed (canola), tobacco or corn (reviewed in Maluszynski et al., 2003a), to trees such as mandarin,

bitter orange or cork, among others (reviewed in Germana, 2006; Srivastava & Chaturvedi, 2008). A

list of species and varieties of agronomic interest where protocolos for microspore embryogenesis are

published and available can be found in http://www.scri.ac.uk/assoc/COST851/DHtable2005.xls.

Among all of these species potentially inducible to microspore embryogenesis, some of them exhibit an

excellent androgenic response, namely rapeseed (Brassica napus), tobacco (Nicotiana tabacum),

and barley (Hordeum vulgare). But not all of the interesting crops respond efficiently to



embryogenesis induction. Only in few of them the androgenic potential is high enough to obtain a

number of embryos sufficient to be routinely used in DH production for breeding programs. Species

like rapeseed, tobacco, barley or wheat are responsive enough to be considered as model systems.

However, other scientifically or economically interesting species like Arabidopsis or tomato still remain

extremely recalcitrant to haploid embryogenesis induction. In between from model and extremely

recalcitrant species, many others are still far from an acceptable response. That is the case of woody

species, where induction of microspore embryogenesis would be especially advantageous for breeding

programs, but successful reports are still very limited (reviewed in Srivastava & Chaturvedi, 2008). In

gymnosperms, for example, embryos have been obtained in some instances, but no plant regeneration

has been reported (Andersen, 2005). It is thus clear that the genotype is one of the key factors

controlling the embryogenic response of the microspores. In virtually all of the species studied so far, a

constancy on the extremely different response between different cultivars has been observed. Even

within model species the genotype has a strong influence, as there exist high and null response cultivars

within the same species (Ferrie et al., 1995; Malik et al., 2008; Touraev et al., 2001). This fact

together with the demonstration that androgenic competence can be inherited by descendants reveals a

genetic basis and thus the possibility of breeding for this trait (Beckert, 1998; Rudolf et al., 1999).

As said, the late microsporeyoung pollen are the sensitive stages for embryogenesis induction. As a

general rule, it is accepted that young microspores or mature pollen cannot be induced. For most

species the only period of microspore sensitivity to inductive pretreatments revolves around the

transition from the uninucleate, vacuolate microspore to early, bicellular pollen, i.e. around the first

pollen mitosis (Maraschin et al., 2005; Raghavan, 1986; Touraev et al., 2001). In fact, there seems to

be an association between the ability to undergo embryogenesis and the polarity of the first pollen

division (Reynolds, 1997; Twell & Howden, 1998). But in non-model species, where induction is more

difficult to achieve, narrower timeframes have been reported. Most studies support the notion that the

inducible stage is the late, vacuolate microspore, where the microspore is ready to divide

asymmetrically. On the other hand, some laboratories report higher efficiencies from early, just divided

pollen. Capsicum annuum is a good example to illustrate such a discrepancy (Kim et al., 2004,

2008). In addition, in several crops, including model species such as rapeseed, induction has also been

achieved at even later stages with acceptable yields (Binarova et al., 1997). Thus, although there is not

a universal consensus about the exact start and end point between which reprogramming can be

universally achieved, it seems that vacuolate microspores and in some cases young bicellular pollen are

specially suitable to be induced. This is likely due to their proliferative, not yet fully differentiated

transcriptional status (Malik et al., 2007), as opposed to mature pollen, where a specific expression

program for pollen maturation genes is activated (Honys & Twell, 2004). It is also likely that advances

in the specific particularities of recalcitrant species will lead to a widening of the inducible range of

stages.



Besides the genotype and the developmental stage of the microspore, the third critical factor is culture

conditions, and in particular the physical and/or chemical treatment necessary to trigger microspore

embryogenesis. Once the microspore is at the right stage, an inducing treatment must be applied. At

present there is a wide consensus about the type of treatment potentially capable of induction: a

stressing treatment. Microspores must be submitted to a set of physicochemical factors that promote a

stress response, which in turn trigger the embryogenic response. Useful stressing agents are diverse.

The most used stressors are heat and starvation, but different species may require particular

combinations. For more detailed information, the reader is referred to an excellent review about the

different stressing treatments proved to be valid in the most important androgenic systems

(Shariatpanahi et al., 2006). As opposed to stress, hormones seem to be less critical for successful

induction. According to Aionesei et al. (2005), the relatively low importance of hormones in

microspore embryogenesis relies on the fact that hormonal autotrophy is a condition sine qua non for

true embryogenesis.

Once exposed to the inductive treatment, many microspores immediately arrest and/or die. Some

microspores follow a pollen-like type of development until they reach the mature pollen stage. Then,

they arrest and die, typically at the 4th5th day (Hosp et al., 2007a). Some others are effectively

induced by the stress treatment and undergo a plethora of changes at different levels, from the whole

cell architecture to gene expression. Large-scale changes include nuclear repositioning to the cell

center, rearrangements of nuclear and cytoskeletal elements and breakdown of the large central

vacuole. Gene expression changes can be grouped into three principal categories (Pauls et al., 2006):

cellular response to the stress, suppression of the gametophytic program, and expression of the

embryogenic program. An important number of genes, proteins and metabolites have been identified as

directly or indirectly related to the trigger of each of these stages. They have been the subject of recent

and excellent research articles and reviews (Boutilier et al., 2005; Dunwell, 2010; Hosp et al., 2007a,

b; Joosen et al., 2007; Malik et al., 2007, 2008; Maraschin et al., 2005, 2006; Muoz-Amatriain et

al., 2006; Pauls et al., 2006; Segu-Simarro & Nuez, 2008a; Stasolla et al., 2008). Since the amount

of information largely exceeds the scope of this review, we will only outline some of their most relevant

findings. The reader is referred to them for further details.

It is clear that in order to generate a stress response, the stress signal must be internalized first.

Although little is known in this respect, abscisic acid (Maraschin et al., 2005, 2006; Reynolds &

Crawford, 1996; Tsuwamoto et al., 2007; van Bergen et al., 1999) and some plant extracellular signal-

regulated kinase homologues (Coronado et al., 2002; Segu-Simarro et al., 2005) have been proposed

as intermediate transducers of extracellular stress signals for the activation of specific gene expression

programs (reviewed in Segu-Simarro & Nuez, 2008a). Related to the cellular response to stress,

changes in the levels of different heat-shock proteins (HSPs) have been described in several species

during stress-induced microspore embryogenesis after exposure to heat, colchicine, or starvation stress



(Cordewener et al., 1995; Segu-Simarro et al., 2003; Smykal, 2000; Zarsky et al., 1995). It is

currently believed that HSPs have a cytoprotective role related to stress tolerance (Segu-Simarro &

Nuez, 2008a). Instead, upregulation of catalase and glutathione S-transferase genes has been

interpreted as a protective response against the oxidative stress generated by the in vitro culture

conditions (Maraschin et al., 2005).

In addition to triggering the embryogenic program, the cell must cancel the gametophytic program. The

blockage of starch synthesis (a marker of pollen maturation) and the elimination of starch reserves are

key events in this process. Indeed, downregulation of a number of genes involved in starch biosynthesis

and accumulation has been observed in barley induced microspores, together with the induction of

genes for starch and sucrose breakdown (Maraschin et al., 2006). In parallel, a sort of dedifferentiation

program seems to operate in order to clean the cytoplasm from microgametogenesis-related molecules

(reviewed in Maraschin et al., 2005; Segu-Simarro & Nuez, 2008a).

The first indication for the onset of the embryogenic program is a symmetric division of the microspore

(Simmonds & Keller, 1999; Smykal, 2000; Zaki & Dickinson, 1991), as opposed to the asymmetric

division that defines the first pollen mitosis (compare pollen figures of Route 0 with the symmetrically

dividing microspores of the green rectangle of Route 2 in Fig. 1). In most androgenic systems, such

division is followed by continuous proliferative, randomly-oriented divisions, leading to an

undifferentiated, globular mass of embryonic cells, as opposed to the well ordered division pattern of

zygotic embryos (Segu-Simarro & Nuez, 2008a). From this stage on, the microspore-derived embryo

(MDE) progressively approaches the morphology of its zygotic counterpart. Exceptionally, in a model

system such as rapeseed it is possible to reproduce the ordered sequence of stages of zygotic embryos

even from the very first stages, including the transversal divisions that give rise to a filamentous

suspensor structure, and in parallel to an embryo proper at the end of the suspensor opposite to the

basal cell (Joosen et al., 2007; Malik et al., 2007; Segu-Simarro & Nuez, 2008a; Supena et al.,

2008). Other aspects of the embryogenic program are also similar between MDEs and zygotic

embryos, but not equal. That is the case for hormonal regulation. Growth regulators like ethylene, IAA

or ABA have been shown to be important for particular aspects of MDE development, as it happens

for zygotic embryos, but either the temporal profiles or the absolute levels of these plant hormones

differ from zygotic embryogenesis (Belmonte et al., 2006; Evans & Batty, 1994; Hays et al., 1999,

2000, 2001, 2002; Ramesar-Fortner & Yeung, 2006; Rudolf et al., 1999). These differences can be

attributed to the absence of endosperm as a source of regulatory cues (Segu-Simarro & Nuez,

2008a). Other substances, including arabinogalactan proteins (AGPs), hordeins and chitinases,

beneficial for the promotion of the zygotic embryo, are also synthesized by the MDE (Borderies et al.,

2004; Boutilier et al., 2005; Letarte et al., 2006; Pulido et al., 2006; Tang et al., 2006) likely to mimic

the zygotic scenario (Segu-Simarro & Nuez, 2008a), where the embryo-surrounding tissues account

for the synthesis of these substances. Thus, MDEs may adopt certain endosperm-specific functions in



order to overcome some of these deficiencies, as suggested by the upregulation of a number of

endosperm-specific genes in MDEs of different species (Magnard et al., 2000; Massonneau et al.,

2005), and by the ability of proteins secreted to the medium by maize, barley and wheat MDEs to

promote and sustain in vitro zygotic development (Holm et al., 1994; Kumlehn et al., 1998; Paire et al.,

2003).

The absence of regulatory cues coming from the endosperm or other seed tissues may also be behind

the callogenic response observed in some species where indifferentiated, callus-like proliferative

structures have been described to be formed upon in vitro culture of microspores and pollen grains.

Haploid and DH plants have been regenerated from these calli, which has made this method a source

of DHs in many species. This phenomenon has been described in a wide range of species, including

coffee (Silva et al., 2009), trees as loquat (Li et al., 2008) or different poplar species (Baldursson et

al., 1993), cereals such as rye (Ma et al., 2004) or wild barley relatives (Piccirilli & Arcioni, 1991),

and many ornamentals such as lily (Han et al., 2000), narcissus (Chen et al., 2005), coneflower (Zhao

et al., 2006), Anemone (Ari et al., 2007), Dianthus (Nontaswatsri et al., 2008) or chrysanthemum

(Yang et al., 2005). In cucurbits, callus proliferation seems the predominant or only way observed

(Gmes Juhsz & Jakse, 2005; Song et al., 2007). In oat, the genotype seems responsible for the

callus or embryo-type of response. From 38 different wild and cultivated oat genotypes, 31 of them

produced callus whereas 7 produced embryos, when exposed to the same culture conditions

(Kiviharju et al., 1998). In other species, culture conditions have been found critical for callus or

embryo generation. For example, in eggplant, the pionnering work of Dumas de Vaulx and

Chambonnet described the generation of DH plants from embryos developed within anthers cultured in

vitro (Dumas de Vaulx & Chambonnet, 1982). However, nearly 15 years after, Miyoshi described a

method for eggplant DH production based on isolated microspore culture, where microspore gave rise

exclusively to callus-like structures (Miyoshi, 1996). In our group, we have confirmed both results, but

in addition we have found that by changing some conditions in the culture medium it is possible to

induce embryo development up to a certain point. From then on, the eggplant embryo seems not to

have all the elements needed in the culture medium, and therefore reverts to a proliferative callus status

(Corral-Martnez et al., in press). Similarly, in pepper microspore cultures, different conditions may

lead to different efficiencies in embryogenesis induction, different qualities in the embryos produced, or

even the parallel production of good and bad quality embryos together with proliferating callus (Corral-

Martnez et al., in press; Kim et al., 2004, 2008; Supena et al., 2006a, b). These data strongly point

to the notion that callus proliferation is not an alternative, genotype-dependent pathway for haploid

development and regeneration. Instead, it seems the consequence of suboptimal experimental

conditions, which preclude the embryo to develop in a proper manner. It is widely known that

successful induction of embryogenesis is more complex and demanding than induction of callogenesis.

It is likely that most, if not all of the species where microspore-derived callus have been described,



would produce embryos as soon as the particular needs for embryo development in these species are

known and applied. Two evidences give further support to this notion. First, some of the species

currently considered as highly responsive, embryo-producing model systems (e.g. barley, wheat or

rice) were first considered as recalcitrant, at a time when calli were observed in cultures, the rate of

embryo production was low or null, and obviously culture conditions were far from being optimal

(Bouharmont, 1977; Gonzalez-Medina & Bouharmont, 1978; Myint & de Fossard, 1974; Picard et

al., 1974). Second, in maize ig1 mutants, in vivo male-derived androgenic haploids are though to come

from the embryogenic development of a sperm cell, a type of cell where in vitro embryogenesis

induction has never been described. It is likely that being in the best environment possible (the embryo

sac of its same species) with a perfect, in vivo control of all of the nutrients, growth factors and

regulatory cues needed, can allow for a sperm cell to proceed through embryogenesis with a high

probability of success.

In summary, there are still many different, general and specific questions to be answered about

microspore embryogenesis. Among them, the search for the factors and ultimately the genes governing

the androgenic switch has concentrated most of the research efforts. During more than 40 years,

numerous research groups have devoted their work to the search of the master key that switches

microspores towards embryogenesis. Although numerous progresses have been made due to the

development of advanced and sophisticated genomic, transcriptomic, proteomic and imaging tools, at

present the search still continues. Several candidates have been postulated during these years, but the

attempts to clearly identify the genes or gene groups unambiguously conferring the androgenic

competence have been unsuccessful to date. However, in the last decade, a common landscape of

cellular, molecular and genetic changes that define the inductive process is beginning to be depicted. It

seems that we are slowly approaching a global understanding of this process. Instead of a monogenic

genetic control, it seems that a number of inducing factors, different for each species, must concur at

the same time and place for microspore embryogenesis to initiate. The MDE expression profiles of

model species such as rapeseed (Joosen et al., 2007; Malik et al., 2007; Stasolla et al., 2008), barley

(Maraschin et al., 2006), and tobacco (Hosp et al., 2007b) is remarkably similar, which highlights the

importance of some of those common genes in this process. An interesting research line for the future

would be to find whether the genes up or downregulated during microspore embryogenesis also exhibit

expression changes in other androgenic pathways.

Meiocyte-Derived Callogenesis

Male-derived haploid and DH plants can also be regenerated from meiocyte-derived callus.



This is a rare route, less frequent and documented than microspore embryogenesis, where under the

appropriate in vitro conditions, post-recombination meiocytes are induced to proliferate into calli. Then,

haploid and DH plants can be obtained, either through indirect embryogenesis or through

organogenesis over the meiocyte-derived callus (Fig. 1, Route 3). Callus induction from immature

meiocytes has been reported for Arabidopsis thaliana, Vitis vinifera, and Digitalis purpurea

(Corduan & Spix, 1975; Gresshoff & Doy, 1972b, 1974), although these pioneering studies were

never continued. A similar situation was occasionally reported in barley, where nuclear fusion between

adjacent microspores, suggested to be meiotic products not well separated, gave rise to multicellular

structures made of diploid (potentially heterozygous), proliferating cells (Chen et al., 1984). In addition

to in vitro induction, a similar process has also been documented to occur in vivo in anthers of

Narcissus biflorus (Koul & Karihaloo, 1977) and of male-sterile interspecific hybrids (Solanum

chacoense Solanum tuberosum; Ramanna & Hermsen, 1974). Nevertheless, most of the

knowledge about this process comes from research on tomato (Solanum lycopersicum) anther

cultures. Given the outstanding importance of this crop all over the world, many laboratories have

worked in the last four decades trying to induce haploids from tomato anthers (reviewed in Bal &

Abak, 2007; Corral-Martnez et al., in press). The extreme recalcitrancy of this species has precluded

the generation of haploid or DH tomato plants by the more conventional route of microspore

embryogenesis, and only limited success has been reported in the induction of the first sporophytic

divisions from isolated microspores (Dao & Shamina, 1978; Segu-Simarro & Nuez, 2007; Varghese

& Gulshan, 1986). However, several reports have demonstrated that it is possible to regenerate

haploid and DH plants from tomato anthers under certain experimental conditions (Gresshoff & Doy,

1972a; Segu-Simarro & Nuez, 2007; Zagorska et al., 1998, 2004).

Among these conditions, the most critical seem to be the genotype and the developmental stage. As in

the other two androgenic ways to haploidy, the genotype plays an important role. In particular, male-

sterile mutant lines have been shown to be especially sensitive to being induced (Segu-Simarro &

Nuez, 2007; Zagorska et al., 1998; Zamir et al., 1980). Interestingly, male-sterile phenotypes are

usually manifested at the late meiocyte stage, which in most cases overlap with the inductive window of

meiocyte-derived callogenesis. The issue of the inducible developmental stage for tomato has been a

matter of debate for decades. Pioneering studies proposed both the meiocyte (Gresshoff & Doy,

1972a, 1974; Zamir et al., 1980) and young microspores just released from the tetrad (Dao &

Shamina, 1978; Gulshan & Sharma, 1981; Levenko et al., 1977; Varghese & Gulshan, 1986) as the

stages inducible to callogenesis. It is worth to mention that most of the works supporting the

microspore as the appropriate stage were unable to obtain fully regenerated haploid or DH plants.

However, in the last decade and coinciding with the advent of new and sophisticated molecular and

cellular techniques, all of the studies on this subject pointed to the meiocyte as the developmental stage

to interfere with (Shtereva et al., 1998; Summers et al., 1992; Zagorska et al., 1998). More recently, it



was found that for efficient induction, meiocytes must have passed meiotic prophase I, but tetrads of

microspores must not be formed yet (Segu-Simarro & Nuez, 2005). Thus, culture of tetrad and

microspore-carrying anthers exposed to the same experimental conditions consistently failed to develop

callus (Segu-Simarro & Nuez, 2007). This developmental window implies that recombination must be

successfully finished without disruption, but microspore formation (tetrad walling) has to be prevented.

After induction, meiocyte-derived calli originate by two different ways: (1) from haploid products still

enclosed within the tetrad, that stop their gametophytic program and start proliferation, or (2) when

diploid cells, coming from the fusion of two haploid products separated by defective, incomplete, or

absent cell walls, start proliferation. In the first scenario, since callus originates from a tetrad-enclosed

meiotic product (a microspore), one could argue that microspores are the true callus precursors.

Nevertheless, it has to be remarked that induction must take place prior to microspore formation, i.e.

tetrad compartmentalization. Then, haploid callus cells duplicate their haploid genome by nuclear fusion,

giving rise to true DH cells which may regenerate a DH plant (Segu-Simarro & Nuez, 2007).

The second scenario would not give rise to a DH, since fusion of two reduced meiotic products

generates new allele combinations not necessarily homozygous. Our group has recently confirmed this

notion by characterizing 19 meiocyte-derived calli with five independent microsatellite (SSR) molecular

markers proven polymorphic for the parental donor plants (J. M. Segu-Simarro, unpubl.).

Homozygosity for all of the five SSR was found in 14 out of the 19 calli tested. However, five calli

were found heterozygous for at least one SSR. These results are consistent with the notion of fusion

between two haploid, different meiotic products. In addition to meiocyte-derived calli, one cannot rule

out the possibility that some diploid calli originate from the anther connective or filament tissue induced

to proliferate. Indeed, filament tissues typically exhibit a high proliferative response when cultured in

vitro (Segu-Simarro & Nuez, 2006), and it is believed that tomato anther tissues at meiotic stages are

more sensitive to tissue culture than those of older stages (Bal & Abak, 2007). All of these collateral,

undesirable events make mandatory the analysis of every single regenerant, which clearly compromises

the usefulness of this method for practical purposes.

As mentioned above, the number of examples of this route is limited. So the question arises as to why

this process has not been extensively studied and documented in other, more interesting species. First,

it must be noted that meiosis is an extremely sensitive developmental stage where the cell purges its

cytoplasm from the expression of a diploid genome, adjusts it to the expression of a haploid genome,

and readapts for the expression of the gametophytic potential. Conceivably, any physiological

disturbance may collapse the whole process. This is why in vitro culture of meiocytes has been

historically reported as largely unsuccessful (Shivanna & Johri, 1985). In fact, we have repeatedly

tried to cultivate isolated meiocytes, but they never progressed in culture, which has precluded further

cell tracking experiments for additional support of the meiocytic origin of callus. This has been the



reason why the only way to dissect the cellular origin of haploid callus has been serial sectioning of in

vitro cultured anthers (Segu-Simarro & Nuez, 2007). Anyway, the simplest explanation relies on the

fact that most of the examples of this pathway come from some of the most recalcitrant species to be

induced towards microspore embryogenesis. Given the economic impact of these species and/or their

suitability for both fundamental and applied research, parallel efforts have been made to circumvent

their extreme recalcitrancy by exploring alternative pathways to androgenic haploidy, whereas in

species more easily inducible to microspore embryogenesis, focusing on alternatives is unnecessary in

practice, and resources can be devoted to investigate more promising ways to doubled haploidy.

Indeed, we do not exclude at all that meiocyte-derived callogenesis can also be induced in other

species, especially in those where microspore embryogenesis is easily achieved. Examination of this

possibility in a broad range of different species would provide additional support to this notion.

It could be argued that this pathway is not sufficiently different from microspore embryogenesis to be

considered as an independent one, since callus may be formed in both cases upon in vitro induction,

and it is likely that a better knowledge of the process and its particularities would make it even more

similar to microspore embryogenesis. However, the marked differences in the inducible stages in each

case have profound consequences in the final result of each process. In particular, the fact that the

meiocyte is the inducible stage implies that through this route, androgenic haploids or DHs are not the

only final product. Instead, androgenic diploids with different heterozygous combinations are also

obtained. Thus it seems reasonable to consider at present, with the current knowledge about this

process, this route as different from microspore embryogenesis. In light of current data, it is clear that

from an applied or economic point of view, the usefulness of this pathway is far away from that of

microspore embryogenesis, mostly due to its low efficiency in terms of DH yield. However, it is

relevant in biological terms, since it reflects that totipotency of male gametophytes would not be

restricted to the stage of vacuolate microspore-early bicellular pollen, as is widely accepted. Instead, it

shows that under the adequate conditions, meiocytes are also able to deviate from their original

program. This fact, together with other evidences that demonstrate the potential to produce MDEs of

older pollen stages (Binarova et al., 1997) or even sperm cells, as in the maize ig1 mutants under in

vivo conditions (see Male-Derived Haploid Embryogenesis in the Embryo Sac), suggests that further

research in this and other stages could extend the current knowledge of male germ line totipotency and

developmental plasticity.

A Revision of Pandeys Hypothesis

In this review we have dealt with three routes to haploidy, alternative to natural, zygotic embryogenesis



and male gamete development in angiosperms (Fig. 1, Route 0): (1) in vivo haploid embryogenesis in

the embryo sac (Fig. 1, Route 1), (2) microspore embryogenesis (Fig. 1, Route 2), and (3) meiocyte-

derived callogenesis (Fig. 1, Route 3). These routes differ in several aspects (summarized in Table 1),

being the most evident aspect the stage when the original program is deviated (the sperm cell/fertilized

egg, the vacuolate microspore/young pollen grain, and the post-recombination meiocyte respectively).

However, most of the differences are observed between route 1 and the other two routes. Route 1

starts with a cell type which represents the end of a developmental pathway, the male gamete-

producing pathway, whereas routes 2 and 3 are induced from cell types that represent intermediate

stages in the male gametophytic pathway. Thus, as opposed to route 1, routes 2 and 3 imply a

deviation from the original gametophytic program towards mature pollen and gamete formation. In both

cases, it seems necessary to block the original program prior to the induction of a new one. It was

previously postulated (Pandey, 1973) that the young microspore has four possible kinds of potencies:

to develop normally into a male gametophyte, to develop into a female gametophyte (giant embryo sac-

like pollen grains from certain species, see Pandey, 1973 for a review), to develop into a sporophyte

(embryo), and to dedifferentiate into a callus. According to Pandey, in normal conditions the

microspore displays a strong bias towards maleness. The strength of this bias would be a genotype-

specific, particular feature of each species, and would be determined by the molecular environment of

the cell, i.e. the developmental program being expressed at a given time. Therefore, it would account

for the different recalcitrancy of a species to be deviated from maleness. Under certain natural or

induced conditions, the balance of potencies can change by changing the molecular environment. Two

kinds of factors are compulsory for such a shift: first, factors that neutralize the original program,

reverting the cell to a totipotent status, and second, factors that provide the necessary environment to

trigger a new developmental program (e.g. microspore embryogenesis). When such a specific

environment is not present, the cell enters an undifferentiated, proliferative callus phase. Callus, in turn,

may give rise to embryos, organs, or full plantlets provided that proper conditions for indirect

embryogenesis, organogenesis, or plant regeneration are found and applied.



Table 1

Differences Between the Three Different Androgenic Routes

Route 1: male-derived haploidembryogenesis from a fertilizedegg

Route 2: microsporeembryogenesis

Route 3:meiocyte-derivedcallogenesis

Programdeviated

Zygotic embryogenesis microsporogenesis

Programtriggered

Haploid embryogenesisHaploidembryogenesis/callogenesis

Callogenesis

Sensitivestages

One-celled zygoteVacuolate microspore-young pollen

Meiocyte frommetaphase I totelophase II

Occurrence Natural Induced in vitro

Genotypedependence

High High High

Need forinductivestress

No Essential Relative

Need forhormones

No Relative Essential

Appliedrelevance

Null High Low

Documentedexamples

Few Abundant Few

The last decade has witnessed a new way to approach the study of the molecular basis of

androgenesis, focusing especially on the most useful alternative: microspore embryogenesis. The

parallel work of several laboratories has boosted the search for the gene(s) responsible for the

androgenic switch and considerable progress has been made. From a holistic point of view, these

studies (see Microspore/Pollen Embryogenesis) have confirmed the validity of Pandeys hypothesis,

more than 35 years after. These studies have proven that induction of androgenesis must be preceded

by a repression of the pollen-specific gametophytic program, thus keeping the microspore in an



undifferentiated, totipotent status. These works also confirm that Pandeys hypothesis remains useful to

explain the different alternatives observed to microsporogenesis (reviewed in Segu-Simarro & Nuez,

2008a). It seems relatively easy to reprogram towards embryogenesis in an increasing number of

species by applying stress treatments to the microspore. In other species, more reluctant to microspore

embryogenesis, microspore dedifferentiation can be achieved but conditions are not yet optimal for

embryogenesis. This would be the case of microspore cultures in cucurbits, some solanaceae and many

ornamentals, where callus proliferation has been extensively documented (see Microspore/Pollen

Embryogenesis). A third option would be that of the most recalcitrant species (tomato, grape,

Arabidopsis), where much more severe stresses have to be applied at earlier developmental stages to

promote dedifferentiation and callus proliferation, but where the button to switch direct microspore

embryogenesis on still awaits to be pushed.

Similar Triggers for All of the Androgenic Alternatives?

In spite of their specific differences, the three ways have in common a strong genotypic dependence

and the final product: an androgenic haploid or DH, i.e. an individual whose genetic background is

contributed exclusively by a male donor. Since the inducible stage in each route is markedly different,

the most logical reasoning would lead to assume that the triggering factors and the mechanisms involved

in each case are different. However, if we put the current knowledge about these three routes together,

some coincidences between them may arise in terms of inducing factors. As seen in Microspore/Pollen

Embryogenesis, it is currently believed that in order to induce a microspore towards embryogenesis, a

number of external factors must concur in a timely manner to generate metabolic alterations, which in

turn promote changes of higher order in chromatin structure (Hosp et al., 2007a). This leads to the

activation of certain genes, involved in proliferation and in the expression of a embryogenic program,

which finally gives rise to the haploid embryo. The general molecular triggers of the process of in vivo

androgenesis, supposedly from a fertilized egg, are largely unknown. Only some data are available in

the particular case of androgenesis induced by the presence of the ig1 mutant gene in maize. In this

case, it is thought that the wild type ig1 gene is a repressor of proliferation and embryogenesis in the

embryo sac (Evans, 2007). Thus, it is likely that in ig1 mutants, the non-repressed expression of these

genes generate the metabolic alterations needed to promote proliferation in cell types such as synergid

or antipodal cells, and embryogenesis in embryo-producing cells such as the egg and the sperm cells

after pollen discharge. In other words, the lack of ig1 function would create in the maize embryo sac,

and therefore in the sperm cells present after pollen discharge, an embryogenesis-promoting, molecular

environment similar to that created by the stress treatments in the case of microspore embryogenesis or

meiocyte-derived callogenesis. In support of this hypothesis counts the fact that the ig1 mutation affects



the nucleus and the microtubular cytoskeleton of the cells in the embryo sac (Huang & Sheridan, 1996)

in a way similar to that described for stress treatments in induced microspores (reviewed in Maraschin

et al., 2005; Segu-Simarro & Nuez, 2008a). For example, in both cases the nucleus fails to migrate to

the position in the cell needed to follow the normal gametophytic program. Besides, all of the

microtubular structures involved in cell division (spindle, phragmoplast) have an altered position,

orientation and behaviour in both cases. Further support for this speculation may come from the studies

of Komma and Endow in androgenic Drosophila ncd mutants (Komma & Endow, 1995), where they

found an increased androgenic frequency due to the loss of function of NCD, a microtubular motor

protein from the kinesin family.

In this context, it is tempting to speculate that in the other documented examples of in vivo

androgenesis, where no ig1 mutant genes are involved, the external factors that govern the occurrence

of androgenesis would promote effects similar to those promoted by the ig1 mutation of maize or the

ncd mutation in Drosophila. For example, among these factors one could mention wide hybridization.

The fact of hybridizing different species, as those mentioned in Male-Derived Haploid Embryogenesis

in the Embryo Sac, might have an effect similar to that described for the ig1 mutant gene in terms of

creating a similar haploid embryo-promoting molecular environment, suitable for the sperm cell to

develop as an embryo. Additionally, it might well be possible that androgenesis would be induced

directly over the sperm cell, before fertilization and karyogamy, as described for maize ig1 mutants.

Indeed, this is the best studied example of in vivo androgenesis, and as mentioned in Male-Derived

Haploid Embryogenesis in the Embryo Sac, several authors have expressed their reasonable doubts

about the assumed, but not unambiguously proven origin of the male-derived embryo in this route as

coming from a fertilized egg which losses or inactivates the female chromosomes. It was shown that

wide hybridization between two chickpea relatives (Cicer arietinum C. pinnatifidum) was able to

induce, upon exposure to zeatin, sporophytic divisions in microspores within the anthers of the hybrid,

(Mallikarjuna et al., 2005). This result would be reinforcing the notion of a role of interspecific

hybridization as a trigger for haploid embryogenesis, and would also be extending this role beyond the

sperm up to a different cell type, the microspore.

Finally, it would be interesting to pose the following question: what is the biological significance of

androgenesis? In a review from 2000, Wang et al. proposed a very attractive hypothesis: The spore

development in mosses and ferns shows clear parallels with the androgenesis process. In both

cases the haploid spore produced by the sporophyte divides mitotically and develops into a

(haploid) multicellular structure, and both processes start with an increase of the cell volume.

So we may hypothesize that during the evolution of plants, the spore development pathway into

multicellular structures was greatly shortened in favor of direct gamete formation, but that this

pathway is still present and can be activated as is shown in androgenesis. Perhaps, behind

androgenesis there is just an alternative survival mechanism based on the totipotency of plant cells and



on the capacity of primitive plants to develop a haploid multicellular structure during the extended

haploid phase of their life cycle. Perhaps, androgenesis is just a developmental pathway based on this

capacity of ancient plant relatives, and currently displaced by the evolutionary advantages of sexual

reproduction. Thus, when we block or disturb the normal process of sexual reproduction by isolating

anthers, meiocytes, microspores or pollen grains, or by hybridizing two distant species having severe

pre-zygotic or post-zygotic reproductive barriers, we may be forcing the plant to activate this ancient,

silent up to now mechanism in order to ensure reproduction by any means. However, the particular

conditions of in vivo or in vitro androgenesis would favour the development of a haploid embryo or

callus, and not a small multicellular gametophyte (protonema) as in ferns, or even a haploid plant as is

the case for mosses.

Concluding Remarks

In this review I have summarized the main aspects of the three androgenic routes known so far in

plants, in vivo haploid embryogenesis in the embryo sac, in vitro microspore/pollen embryogenesis, and

in vitro meiocyte-derived callogenesis. The three of them exhibit clear differences, being the most

important one the stage where the gamete or gamete precursor can be deviated from the original

program. But most importantly, they present a number of common features, including the essential one

in order to be considered as androgenic routes: an exclusively male-derived origin for the genome of

the androgenic plant produced.

From an applied point of view, the main limitation for practical application in DH production of the

three routes revised hereby relies on their low efficiency in terms of generation of androgenic DH

plants. Efficiency has been largely improved, mostly in the case of microspore embryogenesis, thanks

to the remarkable efforts of many research groups during the last 40 years. It is expected that in the

next future, new advances and more sophisticated technology will lead to an even greater advance in

the knowledge of this process and the increase of its efficiency. However, it has to be always kept in

mind that efficiency in these kind of routes, involving the generation of haploid and DH individuals, will

always be limited due to the unmasking of recessive lethal genes (de Fossard, 1974a), usually masked

in heterozygous individuals, and unmasked and therefore abortive in haploid or recessive homozygous

DH embryos.

From a more fundamental, biological point of view, these three routes share a number of features that

point towards a possible common origin as an atavism carried through from the ancestors of the original

plant species, and as a possible survival mechanism when the sexual way is blocked by any reason. In

addition, in light of the current knowledge they might be triggered by similar mechanisms. All of this



makes them not as different as they seem. Nevertheless, much more work is still needed to confirm

many of these attractive hypothesis. Unfortunately, such work may not be possible to be developed in

the case of in vivo androgenesis from the embryo sac, due to the difficulties imposed by its in vivo

nature, within the embryo sac. This, together with the low efficiency, has been for sure one of the main

drawbacks that has prevented an increased number of more profound studies of this process in the last

40 years.

Acknowledgements

This work was supported by grant AGL2006-06678 from the Spanish Ministry of Education and Science.

Literature Cited

Aionesei, T., A. Touraev & E. Heberle-Bors. 2005. Pathways to microspore embryogenesis. Pp

1134. In: C. E. Palmer, W. A. Keller, & K. J. Kasha (eds). Haploids in crop improvement II,

vol.56. Springer-Verlag, Berlin Heidelberg.

Andersen, S. B. 2005. Haploids in the improvement of woody species. Pp 243257. In: C. E.

Palmer, W. A. Keller, & K. J. Kasha (eds). Haploids in crop improvement II, vol. 56. Springer-

Verlag, Berlin Heidelberg.

Ari, E., S. Buyukalaca, K. Abak & S. Cetiner. 2007. Callus initiation for indirect pollen

embryogenesis in Anemone coronaria. Proceedings of the 22nd International Eucarpia Symposium

Section Ornamentals: Breeding for Beauty, Pt II 743: 8790.

Bal, U. & K. Abak. 2007. Haploidy in tomato (Lycopersicon esculentum Mill.): A critical review.

Euphytica 158: 19.

Baldursson, S., P. Krogstrup, J. V. Norgaard & S. B. Andersen. 1993. Microspore

embryogenesis in anther culture of 3 species of Populus and regeneration of dihaploid plants of

Populus trichocarpa. Canadian Journal of Forest Research-Revue Canadienne de Recherche

Forestiere 23: 18211825.

Beckert, M. 1998. Genetic analysis of in vitro androgenetic response in maize. Pp 2437. In: Y.

Chupeau, M. Caboche, & Y. Henry (eds). Androgenesis and haploid plants. Springer-Verlag, Berlin,

Heidelberg.



Belicuas, P. R., C. T. Guimaraes, L. V. Paiva, J. M. Duarte, W. R. Maluf & E. Paiva. 2007.

Androgenetic haploids and SSR markers as tools for the development of tropical maize hybrids.

Euphytica 156: 95102.

Belmonte, M. F., S. J. Ambrose, A. R. S. Ross, S. R. Abrams & C. Stasolla. 2006. Improved

development of microspore-derived embryo cultures of Brassica napus cv Topas following changes in

glutathione metabolism. Physiologia Plantarum 127: 690700.

Binarova, P., G. Hause, V. Cenklova, J. H. G. Cordewener & M. M. van Lookeren-

Campagne. 1997. A short severe heat shock is required to induce embryogenesis in late bicellular

pollen of Brassica napus L. Sexual Plant Reproduction 10: 200208.

Bohanec, B. 2009. Doubled haploids via gynogenesis. Pp 3546. In: A. Touraev, B. P. Forster, &

S. M. Jain (eds). Advances in haploid production in higher plants. Springer, Dordrecht.

Borderies, G., M. le Bechec, M. Rossignol, C. Lafitte, E. Le Deunff, M. Beckert, C. Dumas

& E. Matthys-Rochon. 2004. Characterization of proteins secreted during maize microspore culture:

Arabinogalactan proteins (AGPs) stimulate embryo development. European Journal of Cell Biology 83:

205212.

PubMed

Bouharmont, J. 1977. Cytology of microspores and calli after anther culture in Hordeum vulgare.

Caryologia 30: 351360.

Boutilier, K., M. Fiers, C. M. Liu & A. H. M. Van der Geest. 2005. Biochemical and molecular

aspects of haploid embryogenesis. Pp 7395. In: C. E. Palmer, W. A. Keller, & K. J. Kasha (eds).

Haploids in crop improvement II, vol. 56. Springer-Verlag, Berlin Heidelberg.

Burk, L. G. 1962. Haploids in genetically marked progenies of tobacco. Journal of Heredity 53: 222

225.

Campos, F. F. & D. T. J. Morgan. 1958. Haploid pepper from a sperm. Journal of Heredity 49:

135137.

Chase, S. S. 1963. AndrogenesisIts use for transfer of maize cytoplasm. Journal of Heredity 54:

152158.

. 1969. Monoploids and monoploidderivatives of maize (Zea mays L.). Botanical Review

35: 117167.



Chen, C. C., K. J. Kasha & A. Marsolais. 1984. Segmentation patterns and mechanisms of

genome multiplication in cultured microspores of barley. Canadian Journal of Genetics and Cytology

26: 475483.

Chen, L. J., X. Y. Zhu, L. Gu & B. Wu. 2005. Efficient callus induction and plant regeneration from

anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem). Plant Cell Reports 24: 401

407.

PubMed

Chupeau, Y., M. Caboche & Y. Henry. 1998. Androgenesis and haploid plants. Springer-Verlag,

Berlin, Heidelberg.

Clausen, R. E. & W. E. Lammerts. 1929. Interspecific hybridisation in Nicotiana. X. Haploid and

diploid merogony. The American Naturalist. 43: 279282.

Cordewener, J. H. G., G. Hause, E. Grgen, R. Busink, B. Hause, H. J. M. Dons, A. A. M.

van Lammeren, M. M. van Lookeren-Campagne & P. Pechan. 1995. Changes in synthesis and

localization of members of the 70-kDa class of heat-shock proteins accompany the induction of

embryogenesis in Brassica napus L. microspores. Planta 196: 747755.

Corduan, G. & C. Spix. 1975. Haploid callus and regeneration of plants from anthers of Digitalis

purpurea L. Planta 124: 111.

Coronado, M. J., P. Gonzalez-Melendi, J. M. Segui, C. Ramirez, I. Barany, P. S. Testillano &

M. C. Risueno. 2002. MAPKs entry into the nucleus at specific interchromatin domains in plant

differentiation and proliferation processes. Journal of Structural Biology 140: 200213.

PubMed

Corral-Martnez, P., F. Nuez & J. M. Segu-Simarro. In press. In: D. Thangadurai (ed).

Molecular agrobiology. Bentham Science Publishers, USA.

Dao, N. T. & Z. B. Shamina. 1978. Cultivation of isolated tomato anthers. Soviet Plant Physiology

25: 120126.

de Fossard, R. A. 1974a. Methods for producing haploids. Pp 145150. In: K. J. Kasha (ed).

Haploids in higher plants: Advances and potential. University of Guelph, Guelph, Canada.

. 1974b. Terminology in Haploid research. Pp 403410. In: K. J. Kasha (ed). Haploids in

higher plants: Advances and potential. University of Guelph, Guelph, Canada.



De Maine, M. J. 2003. Potato haploid technologies. Pp 241247. In: M. Maluszynski, K. J. Kasha,

B. P. Forster, & I. Szarejko (eds). Doubled haploid production in crop plants. A manual. Kluwer

Academic Publishers, Dordretch, the Netherlands.

Devaux, P. 2003. The Hordeum bulbosum (L.) method. Pp 1519. In: M. Maluszynski, K. J.

Kasha, B. P. Forster, & I. Szarejko (eds). Doubled haploid production in crop plants. A manual.

Kluwer Academic Publishers, Dordretch, the Netherlands.

Dumas de Vaulx, R. & D. Chambonnet. 1982. Culture in vitro danthres daubergine (Solanum

melongena L.): Stimulation de la production de plantes au moyen de traitements 35C associs de

faibles teneurs en substances de croissance. Agronomie 2: 983988.

Dunwell, J. M. 2010. Haploids in flowering plants: origins and exploitation. Plant Biotechnology

Journal 8:377424.

PubMed

Ecochard, R., G. Merkx & M. Matteoli. 1974. Parthenogenesis induced by specific

radioinactivation of the male gametes. Pp 136. In: K. J. Kasha (ed). Haploids in higher plants:

Advances and potential. University of Guelph, Guelph, Canada.

Ehrensberger, R. 1948. Versuche zur auslsung von haploidie bei Bltenpflanzen. Biologisches

Zentralblatt 67: 537546.

Evans, M. 2005. New nucleic acid molecule encoding indeterminate gametophyte 1, useful for

producing male sterility and for generating androgenetic progeny in plant species. Carnegie Inst

Washington, USA, Patent no. US2005198711-A1.

Evans, M. M. S. 2007. The indeterminate gametophyte1 gene of maize encodes a LOB domain

protein required for embryo sac and leaf development. Plant Cell 19: 4662.

PubMed

Evans, M. 2009. New isolated polypeptide or protein comprises the amino acid sequence of

indeterminate gametophyte 1 (IG1) from Zea mays, useful for identifying and isolating an ortholog of

ig1 in a non-Zea mays plant species. Carnegie Inst Washington, USA, Patent no. US2009151025-

A1.

Evans, J. M. & N. P. Batty. 1994. Ethylene precursors and antagonists increase embryogenesis of

Hordeum vulgare L anther culture. Plant Cell Reports 13: 676678.

Ferrie, A. M. R., D. J. Epp & W. A. Keller. 1995. Evaluation of Brassica rapa L. genotypes for



microspore culture response and identification of a highly embryogenic line. Plant Cell Reports 14:

580584.

Forster, B. P., E. Heberle-Bors, K. J. Kasha & A. Touraev. 2007. The resurgence of haploids in

higher plants. Trends in Plant Science 12: 368375.

PubMed

Gmes Juhsz, A. & M. Jakse. 2005. Haploids in the improvement of miscellaneous crop species

(Cucurbitaceae, Liliaceae, Asparagaceae, Chenopodiaceae, Araceae and Umbelliferae). Pp 259276.

In: C. E. Palmer, W. A. Keller, & K. J. Kasha (eds). Haploids in crop improvement II. Springer-

Verlag, Berlin Heidelberg.

Gerassimova, H. 1936. Experimentally produced haploid plant in Crepis tectorum. Biologitcheski

Journal 5: 895900.

Germana, M. 2006. Doubled haploid production in fruit crops. Plant Cell Tissue and Organ Culture

86: 131146.

Gerrish, E. E. 1956. Studies od the monoploid method of producing homozigous diploids in Zea

mays. Ph.D. Thesis. University of Minnesota, Minneapolis.

Gonzalez-Medina, M. & J. Bouharmont. 1978. Experiments on anther culture in barleyInfluence

of culture methods on cell proliferation and organ differentiation. Euphytica 27: 553559.

Goodsell, S. F. 1961. Male sterility in corn by androgenesis. Crop Science 1: 227228.

Gresshoff, P. M. & C. H. Doy. 1972a. Development and differentiation of haploid Lycopersicon

esculentum (tomato). Planta 107: 161170.

& . 1972b. Haploid Arabidopsis thaliana callus and plants from anther culture.

Australian Journal of Biological Sciences 25: 259.

& . 1974. Derivation of a haploid cell line from Vitis vinifera and importance of stage

of meiotic development of anthers for haploid culture of this and other genera. Zeitschrift Fur

Pflanzenphysiologie 73: 132141.

Guha, S. & S. C. Maheshwari. 1964. In vitro production of embryos from anthers of Datura.

Nature 204: 497.

Gulshan, T. M. V. & D. R. Sharm

androgenesis revisited - springer

Documents

the other

in vivo development

and by in vitro meiocyte

derived callogenesis

androgenesis revisitedjos

whereas microspore

discussed from

theirdifferences and