The Genetic Control of Apomixis: Asexual Seed Formation

Apomixis (asexual seed formation) is the result of a plant gaining the ability to bypass the most fundamental aspects of sexual reproduction: meiosis and fertilization. Without the need for male fertilization, the resulting seed germinates a plant that develops as a maternal clone. This dramatic shift in reproductive process has been documented in many flowering plant species, although no major seed crops have been shown to be capable of apomixis. The ability to generate maternal clones and therefore rapidly fix desirable genotypes in crop species could accelerate agricultural breeding strategies. The potential of apomixis as a next-generation breeding technology has contributed to increasing interest in the mechanisms controlling apomixis. In this review, we discuss the progress made toward understanding the genetic and molecular control of apomixis. Research is currently focused on two fronts. One aims to identify and characterize genes causing apomixis in apomictic species that have been developed as model species. The other aims to engineer or switch the sexual seed formation pathway in non-apomictic species, to one that mimics apomixis. Here we describe the major apomictic mechanisms and update knowledge concerning the loci that control them, in addition to presenting candidate genes that may be used as tools for switching the sexual pathway to an apomictic mode of reproduction in crops.     

Apomixis for crop improvement

Summary Apomixis is a genetically controlled reproductive process by which embryos and seeds develop in the ovule without female meiosis and egg cell fertilization. Apomixis produces seed progeny that are exact replicas of the mother plant. The major advantage of apomixis over sexual reproduction is the possibility to select individuals with desirable gene combinations and to propagate them as clones. In contrast to clonal propagation through somatic embryogenesis or in vitro shoot multiplication, apomixis avoids the need for costly processes, such as the production of artificial seeds and tissue culture. It simplifies the processes of commercial hybrid and cultivar production and enables a large-scale seed production economically in both seed- and vegetatively propagated crops. In vegetatively reproduced plants (e.g., potato), the main applications of apomixis are the avoidance of phytosanitary threats and the spanning of unfavorable seasons. Because of its potential for crop improvement and global agricultural production, apomixis is now receiving increasing attention from both scientific and industrial sectors. Harnessing apomixis is a major goal in applied plant genetic engineering. In this regard, efforts are focused on genetic and breeding strategies in various plant species, combined with molecular methods to analyze apomictic and sexual modes of reproduction and to identify key regulatory genes and mechanisms underlying these processes. Also, investigations on the components of apomixis, i.e., apomeiosis, parthenogenesis, and endosperm development without fertilization, genetic screens for apomictic mutants and transgenic approaches to modify sexual reproduction by using various regulatory genes are receiving a major effort. These can open new avenues for the transfer of the apomixis trait to important crop species and will have far-reaching potentials in crop improvement regarding agricultural production and the quality of the products.     

Apomixis in plant reproduction: a novel perspective on an old dilemma

Seed is one of the key factors of crop productivity. Therefore, a comprehension of the mechanisms underlying seed formation in cultivated plants is crucial for the quantitative and qualitative progress of agricultural production. In angiosperms, two pathways of reproduction through seed exist: sexual or amphimictic, and asexual or apomictic; the former is largely exploited by seed companies for breeding new varieties, whereas the latter is receiving continuously increasing attention from both scientific and industrial sectors in basic research projects. If apomixis is engineered into sexual crops in a controlled manner, its impact on agriculture will be broad and profound. In fact, apomixis will allow clonal seed production and thus enable efficient and consistent yields of high-quality seeds, fruits, and vegetables at lower costs. The development of apomixis technology is expected to have a revolutionary impact on agricultural and food production by reducing cost and breeding time, and avoiding the complications that are typical of sexual reproduction (e.g., incompatibility barriers) and vegetative propagation (e.g., viral transfer). However, the development of apomixis technology in agriculture requires a deeper knowledge of the mechanisms that regulate reproductive development in plants. This knowledge is a necessary prerequisite to understanding the genetic control of the apomictic process and its deviations from the sexual process. Our molecular understanding of apomixis will be greatly advanced when genes that are specifically or differentially expressed during embryo and embryo sac formation are discovered. In our review, we report the main findings on this subject by examining two approaches: i) analysis of the apomictic process in natural apomictic species to search for genes controlling apomixis and ii) analysis of gene mutations resembling apomixis or its components in species that normally reproduce sexually. In fact, our opinion is that a novel perspective on this old dilemma pertaining to the molecular control of apomixis can emerge from a cross-check among candidate genes in natural apomicts and a high-throughput analysis of sexual mutants.     

Sexual and apomictic reproduction in Hieracium subgenus pilosella are closely interrelated developmental pathways

Seed formation in flowering plants requires meiosis of the megaspore mother cell (MMC) inside the ovule, selection of a megaspore that undergoes mitosis to form an embryo sac, and double fertilization to initiate embryo and endosperm formation. During apomixis, or asexual seed formation, in Hieracium ovules, a somatic aposporous initial (AI) cell divides to form a structurally variable aposporous embryo sac and embryo. This entire process, including endosperm development, is fertilization independent. Introduction of reproductive tissue marker genes into sexual and apomictic Hieracium showed that AI cells do not express a MMC marker. Spatial and temporal gene expression patterns of other introduced genes were conserved commencing with the first nuclear division of the AI cell in apomicts and the mitotic initiation of embryo sac formation in sexual plants. Conservation in expression patterns also occurred during embryo and endosperm development, indicating that sexuality and apomixis are interrelated pathways that share regulatory components. The induction of a modified sexual reproduction program in AI cells may enable the manifestation of apomixis in HIERACIUM:     

Beyond promiscuity: From sexuality to apomixis in flowering plants

Summary Little is known about the genetic basis and molecular mechanisms regulating female gametogenesis in flowering plants. In many species sexuality is replaced by apomixis, a method of asexual reproduction that circumvents female meiosis and fertilization, and culminates in the formation of clonal seeds. Using a new generation of transposon based insertional mutagenesis strategies and their resulting molecular tools, we are investigating how female meiotically derived cells (megaspores) acquire their identity. We are also determining their function and interactions, and attempting the induction of apomixis initiation in the ovule of Arabidopsis. This basic knowledge will contribute to establish the transfer of apomixis into sexual crops, a major challenge faced by plant biotechnology. The introduction of apomixis as a reproductive alternative could represent a unique opportunity to simplify breeding schemes and genetically perpetuate any desired heterozygous genotype, including hybrids.     

Dynamics of callose deposition and beta-1,3-glucanase expression during reproductive events in sexual and apomictic Hieracium

Callose accumulates in the walls of cells undergoing megasporogenesis during embryo sac formation in angiosperm ovules. Deficiencies in callose deposition have been observed in apomictic plants and causal linkages between altered callose deposition and apomictic initiation proposed. In apomictic Hieracium, embryo sacs initiate by sexual and apomictic processes within an ovule, but sexual development terminates in successful apomicts. Callose deposition and the events that lead to sexual termination were examined in different Hieracium apomicts that form initials pre- and post-meiosis. In apomictic plants, callose was not detected in initial cell walls and deficiencies in callose deposition were not observed in cells undergoing megasporogenesis. Multiple initial formation pre-meiosis resulted in physical distortion of cells undergoing megasporogenesis, persistence of callose and termination of the sexual pathway. In apomictic plants, callose persistence did not correlate with altered spatial or temporal expression of a β-1,3-glucanase gene (HpGluc) encoding a putative callose-degrading enzyme. Expression analysis indicated HpGluc might function during ovule growth and embryo sac expansion in addition to callose dissolution in sexual and apomictic plants. Initial formation pre-meiosis might therefore limit the access of HpGluc protein to callose substrate while the expansion of aposporous embryo sacs is promoted. Callose deposition and dissolution during megasporogenesis were unaffected when initials formed post-meiosis, indicating other events cause sexual termination. Apomixis in Hieracium is not caused by changes in callose distribution but by events that lead to initial cell formation. The timing of initial formation can in turn influence callose dissolution. Received: 18 April 2000 / Accepted: 10 July 2000     

Identification of differentially expressed cDNA sequences in ovaries of sexual and apomictic plants of Brachiaria brizantha

The isolation of genes associated with apomixis would improve understanding of the molecular mechanism of this mode of reproduction in plants as well as open the possibility of transfer of apomixis to sexual plants, enabling cloning of crops through seeds. Brachiaria brizantha is a highly apomictic grass species with 274 tetraploid apomicts accessions and only one diploid sexual. In this study we have compared gene expression in ovaries at megasporogenesis and megagametogenesis of sexual and apomictic accessions of B. brizantha by differential display (DD-PCR), with 60 primer combinations. Specificity of 65 cloned fragments, checked by reverse northern blot analysis, showed that 11 clones were differentially expressed, 6 in apomictic ovaries, 2 in sexual and 3 in apomictic and sexual, but at different stages. Of the 6 sequences isolated that were preferentially expressed in the apomictic accession: one sequence was from ovaries at megasporogenesis stage; three were from megagametogenesis stage; two were from both stages. Of the two sequences isolated from the sexual accessions, one showed expression in ovaries at megagametogenesis, while the other sequence was shown to be specific to both stages. Three sequences were from megasporogenesis stage in apomicts but were also detected at megagametogenesis in sexual plants. Sequence analysis showed that 5 of the 11 clones had no apparent homologues in the protein database. Some of the clones identified as apomictic-specific shared homology with known genes enabling their functional annotation. The relationships of these functions to the generation of the apomictic trait are discussed.     

Specific features of early embryogenesis in apomictic Poa pratensis L

We studied the early stages of embryo formation in apomictic Poa partensis L. It was shown that during transition to parthenogenesis, at least at the initial stages of embryogenesis, the algorithm of development of the sexual embryo is preserved. This could be due to the system of genetic control of embryogenesis, common for amphimixis and apomixis. We described asynchrony of developmental processes both within the efflorescence (asynchronous maturation of ovules) and within the ovule and even gametophyte (different timing of induction of apoarchesporic initials and oospores). This feature of pseudogamous apomicts allows them to produce simultaneously both sexual and apomictic progenies.     

Apomixis technology development-virgin births in farmers' fields?

Apomixis is the process of asexual reproduction through seed, in the absence of meiosis and fertilization, generating clonal progeny of maternal origin. Major benefits to agriculture could result from harnessing apomixis in crop plants. Although >400 apomictic plant species are known, apomixis is rare among crop plants, and the transfer of apomixis to crop varieties by conventional breeding has been largely unsuccessful. Because apomictic and sexual pathways are closely related, de novo engineering of apomixis might be achieved in sexually reproducing crops. Early consideration of issues relating to biosafety and intellectual property (IP) management can facilitate the acceptance and deployment of apomixis technology in agriculture.     

Apomixis: Developmental Characteristics and Genetics

Asexual reproduction through seeds, or apomixis, is widespread in angiosperms, although does not happen frequently. It occurs in no major crop plant, but its deployment in major crops would afford advantages for breeding and maintenance of hybrid genotypes. Deployment is still a long-term goal, however, since the genetic mechanisms underlying apomixis in nature have not been determined nor has the isolation of apomictic mutants in sexual plants been achieved. Nevertheless, an increasing intensity of research toward these goals over the last decade has greatly expanded our knowledge of genome structure and gene expression in naturally occurring apomicts and female gametophyte development in sexual plants. A common working hypothesis is that apomixis is a “deregulation” of sexual processes and is increasingly supported by gene expression data. Nevertheless, the search for a unique trigger that initiates apomictic development still cannot be disqualified. Further characterization of female gametophyte-related genes and genomes of apomicts and model sexual plants will be fruitful for identifying overlaps in developmental networks.

     

Sexual reproduction development in apomictic Eulaliopsis binata (Poaceae)

Apomixis is a particular mode of reproduction that allows progeny formation without meiosis and fertilization. Eulaliopsis binata, a tetraploid apomictic species, is widely used for making paper, rope and mats. There is great potential for fixation of heterosis in E. binata due to autonomous endosperm formation in this species. Although most of its embryo sac originates from nucellus cells, termed apospory, we observed sexual reproduction initiation in 86.8 to 96.8% of the ovules, evidenced by callose deposition on the walls of cells undergoing megasporogenesis. However, only 2-3% mature polygonum-type sexual embryo sacs were confirmed by embryological investigation. Callose was not detected on aposporous initial cell walls. The aposporous initial cells differentiated during pre- and post-meiosis of the megaspore mother cell, while the sexual embryo sac degenerated at the megaspore stage. DNA content ratio of embryo and endosperm in some individuals was 2C:3C, based on flow cytometry screening of seed, similar to that of normal sexual seed. These results confirm that apomictic E. binata has conserved sexual reproduction to a certain degree, which may contribute to maintaining genetic diversity. The finding of sexual reproduction in apomictic E. binata could be useful for research on genetic mechanism of apomixis in E. binata.     

Apomixis is not developmentally conserved in related, genetically characterized Hieracium plants of varying ploidy

Apomixis is facultative in characterized members of the genus Hieracium. The three components that comprise the apomictic mechanism include apospory followed by autonomous embryo and endosperm formation. The time of aposporous embryo sac initiation and mode of embryo sac formation are different in Hieracium piloselloides (D3) and Hieracium aurantiacum (A3.4). Genetic studies have shown that a single dominant locus encodes all three components of apomixis in both species (Bicknell et al. 2000). We histologically examined a range of related, genetically characterized apomictic Hieracium plants derived from D3 and A3.4 to assess conservation of the apomictic mechanism in different genetic backgrounds. The plants varied in ploidy, and also in the amount of DNA introduced from sexual Hieracium pilosella (P4). An apomictic hybrid from a cross between the two apomicts was also examined. The developmental processes observed in the parental apomicts were not conserved in the examined plants and alterations occurred in the components of apomixis. One plant also exhibited adventitious embryony. The results show that other genetic factors can modify apomixis with respect to time of initiation, spatial location, and mode of developmental progression. Both the apomictic locus and the modifiers are essential for efficient penetrance of the trait in Hieracium. Some of the findings in Hieracium correspond with observations in Ranunculus and this is discussed in terms of models for apomictic development and the control of apomixis in crops. Received: 21 June 1999 / Revision accepted: 17 November 1999     

Apomixis in agriculture: the quest for clonal seeds

Apomixis, or asexual reproduction through seeds, is a natural trait that could have an immense positive impact on crop production. Apomictic breeding strategies could allow the fixation and indefinite propagation of any desired genotype, however complex. Apomicts display a wide variety of developmental mechanisms, which can be viewed as a short-circuiting of sexual development. Gametophytic and sporophytic apomixis are distinguished by the developmental origin of apomictically derived embryos. Genetic studies suggest that individual elements of gametophytic apomixis, such as apomeiosis and parthenogenesis, are either controlled by one or two dominant Mendelian factors. As recombination around apomeiosis loci is suppressed, it is currently not known how complex these loci are. Much less is known regarding the genetic control of sporophytic apomixis but initial studies suggest a complex genetic control. Genetic analyses of sexual reproduction in plant model systems have identified genes that, when mutated, display elements of apomixis. Such studies help in the identification of candidate genes and promoters that can be used for the de novo engineering of apomixis through biotechnology. Molecular genetic studies in apomictic and sexual systems will generate the knowledge necessary for the engineering of conditional apomixis technology. Approaches encouraging collaboration and widespread dissemination of the acquired knowledge will constitute the most innovative route to the development, deployment and acceptance of apomixis technology in agriculture.     

Sporophytic ovule tissues modulate the initiation and progression of apomixis in Hieracium

Apomixis in Hieracium subgenus Pilosella initiates in ovules when sporophytic cells termed aposporous initial (AI) cells enlarge near sexual cells undergoing meiosis. AI cells displace the sexual structures and divide by mitosis to form unreduced embryo sac(s) without meiosis (apomeiosis) that initiate fertilization-independent embryo and endosperm development. In some Hieracium subgenus Pilosella species, these events are controlled by the dominant LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) loci. In H. praealtum and H. piloselloides, which both contain the same core LOA locus, the timing and frequency of AI cell formation is altered in derived mutants exhibiting abnormal funiculus growth and in transgenic plants expressing rolB which alters cellular sensitivity to auxin. The impact on apomictic and sexual reproduction was examined here when a chimeric RNAse gene was targeted to the funiculus and basal portions of the ovule, and also when polar auxin transport was inhibited during ovule development following N-1-naphthylphthalamic acid (NPA) application. Both treatments led to ovule deformity in the funiculus and distal parts of the ovule and LOA-dependent alterations in the timing, position, and frequency of AI cell formation. In the case of NPA treatment, this correlated with increased expression of DR5:GFP in the ovule, which marks the accumulation of the plant hormone auxin. Our results show that sporophytic information potentiated by funiculus growth and polar auxin transport influences ovule development, the initiation of apomixis, and the progression of embryo sac development in Hieracium. Signals associated with ovule pattern formation and auxin distribution or perception may influence the capacity of sporophytic ovule cells to respond to LOA.     

Apomixis technology and the paradox of sex

Most plant species produce genetically variable seeds by the fusion of meiotically reduced egg cells and pollen grains. However, a small proportion of seed plants produces clonal, asexual seeds by the process of apomixis. The fixation of heterosis by apomixis is of great interest for plant breeding. The prospect of changing sexual crop species into apomictic crop species by genetic engineering--apomixis technology--has recently caused a boom in apomixis research. According to evolutionary biological theories, a dominant apomixis gene will rapidly become fixed in an outcrossing sexual population. Therefore, in theory, apomixis transgenes could have unconditional advantages that could result in the uncontrollable spread of the transgenes. By contrast, 'classic' transgenes might only have conditional advantages. Paradoxically, sexual reproduction and not apomixis is common in nature. However, this is no guarantee that apomixis transgenes will be ecologically safe because there could be essential differences between natural and transgenic apomicts.     

Apomixis in Coix aquatica Roxb

When plants of Coix aquatica Roxb. were grown in isolation orbagged, with removal of staminate spikelets several producedone or two seeds, and one plant formed several seeds. Thesewere presumably formed through apomixis, of the autogamous type.Apomixis occurs side by side with sexual reproduction, and istherefore facultative. The fact that one of the plants grownunder the same conditions had higher apomictic seed set thanothers, and both its apomictic and selfed progeny also showedhigher apomictic seed formation suggests that these have greaterapomictic potentialities than others. Genetic analysis of apomixissuggests that it is recessive to sexuality, and is probablygoverned by a number of genes. A few triploids tested did notshow any apomictic seed set indicating that polyploidy per semay not be responsible for initiation of apomixis. Except thatit is a diploid, C. aquatica seems to fulfil the criteria forapomixis, yet it reproduces predominantly by sexual means.     

Heterochronic features of the female germline among several sexual diploid <Emphasis Type="Italic">Tripsacum</Emphasis> L. (Andropogoneae,Poaceae)

One element of gametophytic apomixis is unreduced embryo sac (ES) formation, which often occurs precociously displacing or replacing meiosis and causing apospory or diplospory, respectively. This study evaluated a premise that apomixis may evolve in hybridogenous plants that contain duplicate sets of allelically divergent ovule development heterochrony genes. The duplicate sets of genes would belong to duplicate genomic regions that are recombinationally isolated from each other (no gene flow) by allopolyploidy or paleopolyploidy, and this isolation would genetically stabilize apomixis. For apomixis to evolve, the ancestral donors of the duplicate regions must have differed from each other in timing of megasporogenesis, ES formation and embryony such that epigenetic misexpressions, or competitions in expression, of the duplicate heterochrony genes in hybridogenous derivatives would cause apomixis. Herein, we report substantial heterochrony in onset timing of germline stages among several sexual diploid Tripsacum genotypes, which may have been progenitors of apomictic polyploid Tripsacum. Tripsacum floridanum and Tripsacum zopilotense genotypes entered meiosis early. The former advanced rapidly through ES formation, but the latter entered a lengthy lag phase prior to ES formation. In two Tripsacum dactyloides var. dactyloides genotypes, meiosis occurred late and was followed by a distinct lag phase prior to ES formation. Likewise, the T. dactyloides var. meridonale genotype entered meiosis late, but the lag phase was brief. These differences appear to reflect allelic diversity at loci responsible for onset timing of different germline development stages within and across species and possibly across the recombinationally isolated duplicate chromosome regions in the Tripsacum paleopolyploid haplome (x = 18). Unique combinations of divergent alleles in hybridogenous plants coupled with polyploidy induced gene misexpressions may be required for apomixis to evolve. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.