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(1)
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
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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
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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
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(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
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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
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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,
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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
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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
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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.
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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
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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.
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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
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(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
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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,
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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.
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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
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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
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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
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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.
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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
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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
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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
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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
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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.
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