Androgenesis is a form of quasi-sexual reproduction in which a male is the sole source of the nuclear genetic material in the embryo. Two types of androgenesis occur in nature. Under the first type, females produce eggs without a nucleus and the embryo develops from the male gamete following fertilization. Evolution of this type of androgenesis is poorly understood as the parent responsible for androgenesis the mother gains no benefit from it. Ultimate factors driving the evolution of the second type of androgenesis are better understood.
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Maraschin, W. Spaink, M. Embryogenesis in plants is a unique process in the sense that it can be initiated from a wide range of cells other than the zygote.
Upon stress, microspores or young pollen grains can be switched from their normal pollen development towards an embryogenic pathway, a process called androgenesis. Androgenesis represents an important tool for research in plant genetics and breeding, since androgenic embryos can germinate into completely homozygous, double haploid plants.
From a developmental point of view, androgenesis is a rewarding system for understanding the process of embryo formation from single, haploid microspores. Androgenic development can be divided into three main characteristic phases: acquisition of embryogenic potential, initiation of cell divisions, and pattern formation.
The aim of this review is to provide an overview of the main cellular and molecular events that characterize these three commitment phases. Molecular approaches such as differential screening and cDNA array have been successfully employed in the characterization of the spatiotemporal changes in gene expression during androgenesis.
These results suggest that the activation of key regulators of embryogenesis, such as the BABY BOOM transcription factor, is preceded by the stress-induced reprogramming of cellular metabolism. Reprogramming of cellular metabolism includes the repression of gene expression related to starch biosynthesis and the induction of proteolytic genes e. The combination of cell tracking systems with biochemical markers has allowed the key switches in the developmental pathway of microspores to be determined, as well as programmed cell death to be identified as a feature of successful androgenic embryo development.
The mechanisms of androgenesis induction and embryo formation are discussed, in relation to other biological systems, in special zygotic and somatic embryogenesis. Embryogenesis has evolved as a successful strategy for the reproduction of higher multicellular organisms. Zygotic embryogenesis in animals and plants starts with the fusion of the haploid female and male gametes, giving rise to a diploid zygote.
The zygote possesses the ability to initiate embryogenesis, a developmental programme that leads to the establishment of an embryo with the basic features of the adult body plan. This widely conserved mechanism of reproduction has, however, major differences between animal and plant kingdoms, as embryogenesis in flowering plants starts with two fertilization events.
The pollen grain male gametophyte is a three-celled structure composed of two generative cells encased within the vegetative cell McCormick, In the embryo sac, the double fusion of the generative cells with the egg cell and the two nuclei of the central cell give rise to the diploid zygote and the triploid endosperm, respectively Goldberg et al. Another major difference between animal and plant embryogenesis consists of the ability of plant embryos to develop in vivo or in vitro from a wide range of cell types other than the zygote Mordhorst et al.
The development of techniques and protocols to produce plant embryos asexually has had a huge technological and economical impact on agricultural systems, and nowadays these biotechnologies represent an integral part in the breeding programmes of agronomically important crops. Figure 1 provides an overview of the distinct types of cells that can undergo embryogenic development in higher plants.
During in vivo development, maternal apomixis refers to the asexual formation of a seed from the maternal tissues of the ovule, avoiding the processes of meiosis and fertilization Koltunow, Maternal apomictic embryos develop from a somatic cell within the ovule apospory or from an unreduced embryo sac derived from the megaspore mother cell diplospory.
In either case, apomictic embryo development is independent of pollination, but in some species this might be required for the initiation of endosperm development and the production of viable seeds Koltunow et al. Another type of apomictic development has been reported to occur in the gymnosperm Cupressus dupreziana , where embryos develop from unreduced pollen grains.
This type of apomixis is referred to as paternal apomixis Pichot et al. Because apomixis offers the possibility of the fixation and indefinite propagation of a desired genotype, there has been a great deal of interest in genetically engineering this ability.
Nevertheless, so far it has not been possible to manipulate the apomictic trait for clonal reproduction via seeds Bicknell and Koltunow, Clonal propagation is usually achieved via the induction of in vitro somatic embryogenesis, a process that is defined as the regeneration of a whole plant from undifferentiated somatic cells in culture. Depending on the donor tissue and the induction treatment conditions, embryos may develop either directly from single cells or indirectly through an intermediary callus phase Zimmerman, Additional routes to in vitro embryogenesis are defined by the ability of male or female gametophytes to irreversibly switch from their gametophytic pathway towards an embryogenic route.
While androgenesis refers to the development of embryos from microspores or immature pollen grains Touraev et al. By contrast to apomixis and somatic embryogenesis, which lead to clonal propagation of a specific genotype, androgenic and gynogenic plants reflect the product of meiotic segregation.
Thus, they have the remarkable characteristic of possessing only one set of chromosomes, and therefore are haploid plants. Overview of the different types of cell structures that can undergo embryogenic development in higher plants. F, fertilization; DH, double haploid; M, mitosis. For breeding purposes, the evaluation of diversity in genetic pools and the establishment of homozygous lines are of critical importance.
Homozygosity is traditionally achieved by performing time-consuming and labour-intensive backcrosses Morrison and Evans, Haploid plants derived from microspores opened a new dimension for the production of homozygous lines due to the large amount of microspores that are produced by a single plant. Due to the colchicine-induced or spontaneous process of chromosome doubling that takes place during the early stages of embryo development, fertile double haploid plants can be easily regenerated within a short period of time Wang et al.
The production of double haploids via androgenesis represents, in this context, a powerful technique both for the production of hybrid seeds and the evaluation of genetic diversity. Though androgenesis is a naturally occurring process in some species, the in vivo frequency is very low Rammana, ; Rammana and Hermsen, ; Koul and Karihaloo, Efficient androgenesis is usually induced by the application of a stress treatment to whole plants in vivo or tillers, buds, anthers, and isolated microspores in vitro Touraev et al.
Since the first description of androgenesis in in vitro- cultured anthers of Datura innoxia by Guha and Maheshwari , improvement of the conditions for androgenesis induction and microspore culture have resulted in the regeneration of double haploids of many plant species.
However, many agronomically important crops are recalcitrant to androgenesis Wang et al. Further use of this technology is largely hampered by the poor understanding of the mechanisms that render microspore cells embryogenic.
In vitro embryogenesis systems, here represented by androgenesis, are excellent model systems to study the developmental aspects of embryogenesis induction and embryo formation from single, haploid microspores. As shown by several experiments, embryogenic development during androgenesis is divided into three main characteristic, overlapping phases: in phase I, acquisition of embryogenic potential by stress involves repression of gametophytic development and leads to the dedifferentiation of the cells; in phase II, cell divisions lead to the formation of multicellular structures MCSs contained by the exine wall; in phase III, embryo-like structures ELS are released out of the exine wall and pattern formation takes place.
A time-line of the three different phases during androgenic development in the model species barley is shown in Fig. The aim of this review is to provide an overview of the main molecular and cellular events that characterize the different commitment phases of microspores into embryos, and to highlight their similarities and differences with the two most extensively studied model systems, somatic and zygotic embryogenesis.
Special emphasis is given to the initial stages of microspore embryogenic potential acquirement and the initiation of cell divisions. Cellular and molecular aspects of androgenesis. Owing to their high regeneration efficiencies, barley Hordeum vulgare L. However, with the recent advances in protocol design, molecular and morphological studies are now possible in other plant species, such as maize Zea mays ; Magnard et al.
Lessons learned from these advanced model systems suggest that androgenesis can be efficiently triggered within a relatively wide developmental window. During pollen development, the responsive period for androgenesis is represented by the stages that surround the asymmetric division of the uninucleate microspores, resulting in a polarized pollen grain containing a generative cell embedded in the large vegetative cytoplasm.
The vegetative and generative cells differ markedly, as the small condensed generative cell will undergo an additional mitotic division to produce two sperm cells, while the vegetative cell will start an intense programme of accumulation of storage products, namely starch and lipids to drive further pollen maturation Bedinger, ; McCormick, It is widely accepted that when the vegetative cytoplasm of binucleate pollen starts to accumulate starch, androgenesis can no longer be triggered Binarova et al.
Another important postulation based on practical experience is that the stress treatment, which is needed to switch efficiently the developmental fate of microspores, varies greatly depending on the plant species and the species genotype.
In barley, higher regeneration efficiencies are obtained when microspores at the mid-late to late uninucleate stage are subjected to starvation and osmotic stress, which is achieved by incubating anthers in a mannitol solution Hoekstra et al. In wheat and tobacco, higher induction rates are achieved by a period of starvation in combination with heat shock Touraev et al.
However, other types of stresses applied within the responsive developmental window have been demonstrated to trigger androgenesis at lower rates. Since so many stress factors can trigger the reprogramming of microspores into embryos, it is likely that initiation of androgenesis is induced by converging signalling pathways, although, of course, different stress signals may trigger the same downstream pathways.
An analogous situation may be found during the induction of somatic embryogenesis, where the transition of somatic cells to an embryogenic state is regulated by different classes of hormones, namely auxin, cytokinins, and abscisic acid ABA de Vries et al. During zygotic embryogenesis, however, stress per se is not directly involved with zygotic embryogenic competence.
The ability of the zygote to initiate embryogenesis appears to be related to an increase in ethylene synthesis and endogenous auxin levels after fertilization Ribnicky et al. Interestingly, reactive oxygen species ROS are second messengers during auxin- and stress-induced embryogenesis Nagata et al. Mitogen-activated protein kinase MAPK cascades may link auxin signalling to oxidative stress responses and cell cycle regulation reviewed by Hirt, , and a MAPK has been reported to be activated via stress-related ABA signalling Knetsch et al.
Thus, it is likely that downstream regulatory proteins, such as MAPKs, play an important role in bridging the gap in embryogenesis induction in different types of cells. Upon mannitol treatment to induce barley androgenesis, microspores enlarge, and this has been correlated with embryogenic potential acquisition during induction of androgenesis in many crop species Hoekstra et al.
Embryogenic microspores are characterized by the presence of a large central vacuole, and a clear cytoplasm Huang, ; Hoekstra et al. In other embryogenic systems, such as carrot Daucus carota L.
However, after the induction of somatic embryogenesis in Dactylis glomerata and Norway spruce Picea abies L. Karst , enlarged cells are not competent to become embryos. In these species, it is a subpopulation of small, cytoplasm-rich cells that become embryogenic Filonova et al. This indicates that besides cell size, other morphological markers are associated with embryogenic potential.
During androgenesis, one of these markers is the degree of cytoplasmic dedifferentiation of enlarged cells. Initiation of cell division from stressed microspores has been correlated with specific ultrastructural changes, including organelle-free regions in the cytoplasm, a significant decrease in the number and size of starch granules and lipid bodies, and an overall decline in the number of ribosomes Rashid et al.
Specifically in barley, these cytoplasmic changes are associated with the presence of a thin intine layer, contrasting to the thick intine layer displayed by pollen cells Maraschin et al.
Based on these morphological observations, it has been proposed that stress leads to the dedifferentiation of microspores by the repression of gametophytic development. There are two known pathways in eukaryotic cells that lead to cytoplasmic remodelling: the ubiquitinS proteasomal system, which is the major cellular pathway for the degradation of short- and long-lived molecules, and autophagy, which is the primary intracellular mechanism for degrading and recycling organelles via the lysosomes.
Though these pathways are developmentally regulated, they are also activated upon stress conditions, e. During the initial steps of androgenesis induction in tobacco, cytoplasmic organelles undergo programmed destruction, a process that has been shown to be mediated by the lysosomes Sunderland and Dunwell, However, not only autophagy seems to take place in cytoplasm remodelling during the dedifferentiation phase of microspores, as genes coding for enzymes involved in the ubiquitinS proteosomal pathway are induced in stressed enlarged barley microspores Maraschin et al.
Following cytoplasm dedifferentiation, the nucleus migrates towards the centre of the cell, while the large central vacuole is divided into fragments, interspersed by radially oriented cytoplasmic strands. The resulting morphology, often called star-like structure because of its radial polarity, has been described in several androgenic model systems, including barley, wheat, rapeseed, and tobacco Zaki and Dickinson, ; Touraev et al.
During pollen development, the peripheral nuclear position is maintained by microtubules and actin filaments Hause et al. One of the proposed models for the role of cytoskeleton rearrangements in androgenesis induction is related to the symmetric divisions that are observed following central positioning of the nucleus Zaki and Dickinson, According to Simmonds and Keller , this symmetric division is important in establishing consolidated cell walls via the formation of continuous pre-prophase bands, a crucial step in the formation of a multicellular organism.
These results indicate that the role of cytoskeleton inhibitors in androgenesis induction is not restricted to the induction of symmetric divisions, but it is likely to involve the induction of radial polarity in the microspores. Interestingly, heat shock leads to cytoskeleton rearrangements and central positioning of the vegetative nucleus Zhao and Simmonds, ; Binarova et al.
Although it is not yet known whether starvation leads to cytoskeleton rearrangements, starvation does lead to the displacement of the nucleus towards the centre of the cell Touraev et al. Cell tracking studies on barley and wheat revealed that star-like morphology represents the transition from a dedifferentiated state to the initiation of cell division, and therefore corresponds to the first morphological change associated with microspore embryogenic potential Indrianto et al.
Further ultrastructural studies of barley star-like structures revealed that the vegetative nucleus migrates to the middle of the structure, while the generative cell remains attached to the intine Fig. Following the central positioning of the vegetative nucleus, both generative and vegetative cells start to divide Maraschin et al.
In agreement with the hypothesis that central nuclear positioning is related to initiation of cell divisions, a star-like structure represents a characteristic morphological stage, following hormone or heat treatment to induce somatic embryogenesis in Cichorium Dubois et al. Nevertheless, star-like morphology per se does not ensure that a cell will ultimately commit to the embryogenic pathway. According to Indrianto et al.
Androgenesis: where males hijack eggs to clone themselves
Variation in general vigour of anther culture derived plants in the field. Crosses between distantly related species can bring together novel gene combinations. Anther culture androgenesis , to generate haploid plants from pollen microspores, is one way to shorten this process. It allows novel allele combinations, particularly ones involving recessive characters, to be assessed in intact plants. Useful individuals can then be developed into homozygous and fertile plants through chromosome doubling techniques, and brought into a breeding programme. We have recently been involved in a collaborative project with the Institute of Grassland and Environmental Research IGER to use this approach to improve cold-tolerance and fodder quality in grazing grasses.
Haploid plants, characterized genetically by the presence of gametic number of chromosomes in their cells i. However, there has been no reliable and reproducible method to produce haploids under field conditions. Therefore, the full potential of haploids could not be exploited in agriculture. The technique is being routinely used in crop improvement programmes and has aided development of several improved varieties. Skip to main content.