An <Emphasis Type="Italic">Hieracium</Emphasis> mutant, <Emphasis Type="Italic">loss of</Emphasis><Emphasis Type="Italic">apomeiosis 1</Emphasis> (<Emphasis Type="Italic">loa1</Emphasis>) is defective in the initiation of apomixis

Asexual seed formation (apomixis) in Hieracium aurantiacum occurs by mitotic embryo sac formation without prior meiosis in ovules (apomeiosis), followed by fertilization-independent embryo and endosperm development. Sexual reproduction begins first in Hieracium ovules with megaspore mother cell (MMC) formation. Apomixis initiates with the enlargement of somatic cells, termed aposporous initial (AI) cells, near sexual cells. AI cells grow towards sexually programmed cells undergoing meiosis, which degrade as the dividing nuclei of AIs obscure and displace them. Following Agrobacterium-mediated transformation of an aneuploid Hieracium aurantiacum apomict, a somaclonal mutant designated “loss of apomeiosis 1” (loa1) was recovered, which had significantly lost the ability to form apomictic seed. Maternal apomictic progeny were rare and low levels of germinable seedlings were primarily derived from meiotically derived eggs. Cytological analysis revealed defects in AI formation and function in loa1. Somatic cells enlarged some distance away from sexual cells and unlike AI cells, these expanded away from sexual cells without nuclear division. Surprisingly, many accumulated callose in the walls, a marker associated with meiotically specified cells. These defective AI (DAI) cells only had partial sexual identity as they failed to express a marker reflecting entry to meiosis that was easily detected in MMCs and they ultimately degraded. DAI cell formation did not lead to a compensatory increase in functional sexual embryo sacs, as collapse of meiotic embryo sacs was prevalent in the aneuploid somaclonal mutant. Positional cues that are important for AI cell differentiation, growth and fate may have been disrupted in the loa1 mutant and this is discussed. The authors Takashi Okada, Andrew S. Catanach and Susan D. Johnson made equal contributions to the data.     

Ploidy level selection during germination and early stages of seedling growth in the progeny of allohexaploid facultative apomict,Hieracium rubrum (Asteraceae)

Selection within progeny of a facultative apomict,Hieracium rubrum, was studied using flow cytometry of embryos in seeds (a modified method of Flow Cytometric Seed Screen) and seedlings. Flow cytometric screening of particular progeny classes was based on distinct ploidy categories, reflecting the way of their origin. The results of both estimations of progeny composition significantly differed, which makes direct comparison of proportions detected in seeds or seedling stage impossible. The results suggest that progeny originating from reduced egg cells have higher mortality during germination and the early establishment phase than that from unreduced egg cells. Within the progeny of emasculated plants, the proportion of polyhaploid progeny decreased in favor of apomictically derived plants. Within the progeny of plants pollinated byH. pilosella, the proportion of polyhaploid progeny decreased significantly in favor of apomictically derived plants and 2n + n hybrids. It is argued that at least a proportion of the sexually derived progeny of this facultatively apomictic maternal parent has a lower survival rate than apomictically derived progeny.     

Genetic diversity and reproductive biology in ecotypes of the facultative apomict Hypericum perforatum L

Apomixis is a mode of asexual reproduction through seed. Progeny produced by apomixis are clonal replicas of a mother plant. The essential feature of apomixis is that embryo sacs and embryos are produced in ovules without meiotic reduction or egg cell fertilisation. Thus, apomixis fixes successful gene combinations and propagates high fitness genotypes across generations. A more profound knowledge of the mechanisms that regulate reproductive events in plants would contribute fundamentally to understanding the evolution and genetic control of apomixis. Molecular markers were used to determine levels of genetic variation within and relationship among ecotypes of the facultative apomict Hypericum perforatum L. (2n = 4x = 32). All ecotypes were polyclonal, being not dominated by a single genotype, and characterised by different levels of differentiation among multilocus genotypes. Flow cytometric analysis of seeds indicated that all ecotypes were facultatively apomictic, with varying degrees of apomixis and sexuality. Seeds set by haploid parthenogenesis and/or by fertilisation of aposporic egg cells were detected in most populations. The occurrence of both dihaploids and hexaploids indicates that apospory and parthenogenesis may be developmentally uncoupled and supports two distinct genetic factors controlling apospory and parthenogenesis in this species. Cyto-embryological analysis showed that meiotic and aposporic processes do initiate within the same ovule: the aposporic initial often appeared evident at the time of megaspore mother cell differentiation. Our observations suggest that the egg cell exists in an active metabolic state before pollination, and that its parthenogenetic activation leading to embryo formation may occur before fertilisation and endosperm initiation.     

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:     

Apomixis in hawkweed: Mendel's experimental nemesis

Mendel used hawkweeds and other plants to verify the laws of inheritance he discovered using Pisum. Trait segregation was not evident in hawkweeds because many form seeds asexually by apomixis. Meiosis does not occur during female gametophyte formation and the mitotically formed embryo sacs do not require fertilization for seed development. The resulting progeny retain a maternal genotype. Hawkweeds in Hieracium subgenus Pilosella form mitotic embryo sacs by apospory. The initiation of sexual reproduction is required to stimulate apospory in ovules and to promote the function of the dominant locus, LOSS OF APOMEIOSIS, which stimulates the differentiation of somatic aposporous initial (AI) cells near sexually programmed cells. As AI cells undergo nuclear mitosis the sexual pathway terminates. The function of the dominant locus LOSS OF PARTHENOGENESIS in aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion of either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in reversion to sexual development. In these apomicts, sexual reproduction is therefore the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode factors critical for sexual reproduction but might recruit the sexual pathway to enable apomixis. Incomplete functional penetrance of these dominant loci is likely to lead to the generation of rare sexual progeny also derived from these facultative apomicts.     

Sexual devolution in plants: apomixis uncloaked?

There are a growing number of examples where naturally occurring mutations disrupt an established physiological or developmental pathway to yield a new condition that is evolutionary favored. Asexual reproduction by seed in plants, or apomixis, occurs in a diversity of taxa and has evolved from sexual ancestors. One form of apomixis, diplospory, is a multi-step development process that is initiated when meiosis is altered to produce an unreduced rather than a reduced egg cell. Subsequent parthenogenetic development of the unreduced egg yields genetically maternal progeny. While it has long been apparent from cytological data that meiosis in apomicts was malfunctional or completely bypassed, the genetic basis of the phenomenon has been a long-standing mystery. New data from genetic analysis of Arabidopsis mutants in combination with more sophisticated molecular understanding of meiosis in plants indicate that a weak mutation of the gene SWI, called DYAD, interferes with sister chromatid cohesion in meiosis I, causes synapsis to fail in female meiosis and yields two unreduced cells. The new work shows that a low percentage of DYAD ovules produce functional unreduced egg cells (2n) that can be fertilized by haploid pollen (1n) to give rise to triploid (3n) progeny. While the DYAD mutants differ in some aspects from naturally occurring apomicts, the work establishes that mutation to a single gene can effectively initiate apomictic development and, furthermore, focuses efforts to isolate apomixis genes on a narrowed set of developmental events. Profitable manipulation of meiosis and recombination in agronomically important crops may be on the horizon.     

The influence of genotype and environment on the fecundity and facultative expression of apomixis inHieracium pilosella

The potential for sexual reproduction in the facultative apomictHieracium pilosella was determined by pollinating with the closely related but morphologically distinctH. aurantiacum. The hybrid characteristics of the sexually produced progeny were used as markers for sex. Crossing was carried out under a range of environmental treatments to test the influence of (1) field versus glasshouse conditions, (2) nutrient level and the presence of a pathogen, and (3) photoperiod on the frequency of sex in facultative apomicticH. pilosella. We found significantly more sexual reproduction in the glasshouse than in the field for the two populations tested, but no influence of nutrient level, presence of the pathogen, or individual genotype. Photoperiod was not a significant factor for the single population tested. In several of the experiments seed production was significantly different between the treatments, despite the absence of an effect on the frequency of sex, indicating greater plasticity in this trait. In all three experiments a positive association was found between fecundity and the frequency of sex, with crosses producing sexual progeny having significantly more offspring than those that produced progeny exclusively via apomixis. We hypothesise that the cost of sex in this species may be offset by an increase in fecundity, with the cost of meiosis “diluted” by an increase in total progeny production.     

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     

Possible pathways of the gene flow inTaraxacum sect.Ruderalia

Reproductive behaviour and the pathways of gene flow among ploidy levels were studied experimentally inTaraxacum sect.Ruderalia. Diploid, triploid and tetraploid individuals were sampled from mixed diploid — polyploid natural populations. 136 experimental hybridizations between the plants of different ploidy levels were performed. Seeds resulting from these crosses, those obtained from isolated anthodia as well as from open pollinated anthodia (both from cultivated and wild plants) were subjected to the flow-cytometric seed screening (FCSS) to determine ploidy levels in the progeny and to infer breeding behaviour of maternal plants. Three possible pathways of the gene flow were studied: (A) fertilization of sexuals by pollen of apomicts, (B) B_III hybrid formation, (C) facultative apomixis. Diploid maternal plants when experimentally crossed with triploid pollen donors produced diploids and polyploid progeny, while when pollinated with a mixture of the pollen of diploids and triploids or insect pollinated, no polyploids were discovered. It seems that in the mixture with the pollen of diploids, the pollen of triploids is ineffective. Tetraploids produce hybrids much easier with diploid mothers and their role in wild populations requires further study. Triploid mothers, even those with subregular pollen did not show traces of facultative apomixis. B_III hybrids were present in the progeny of both triploids and tetraploids, in tetraploids in quite high percentages (up to 50% of the progeny in some crosses).     

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.     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.     

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 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 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.     

Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency

The effects of the Agrobacterium rhizogenes rolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.     

Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum

Some angiosperms reproduce by apomixis, a natural way of cloning through seeds. Apomictic plants bypass both meiosis and egg cell fertilization, producing progeny that are genetic replicas of the mother plant. In this report, we analyze reproductive development in Tripsacum dactyloides, an apomictic relative of maize, and in experimental apomictic hybrids between maize and Tripsacum. We show that apomictic reproduction is characterized by an alteration of developmental timing of both sporogenesis and early embryo development. The absence of female meiosis in apomictic Tripsacum results from an early termination of female meiosis. Similarly, parthenogenetic development of a maternal embryo in apomicts results from precocious induction of early embryogenesis events. We also show that male meiosis in apomicts is characterized by comparable asynchronous expression of developmental stages. Apomixis thus results in an array of possible phenotypes, including wild-type sexual development. Overall, our observations suggest that apomixis in Tripsacum is a heterochronic phenotype; i.e., it relies on a deregulation of the timing of reproductive events, rather than on the alteration of a specific component of the reproductive pathway.     

Developmental genetics of gametophytic apomixis

Some higher plants reproduce asexually by apomixis, a natural way of cloning through seeds. Apomictic plants produce progeny that are an exact genetic replica of the mother plant. The replication is achieved through changes in the female reproductive pathway such that female gametes develop without meiosis and embryos develop without fertilization. Although apomixis is a complex developmental process, genetic evidence suggests that it might be inherited as a simple mendelian trait - a paradox that could be explained by recent data derived from apomictic species and model sexual organisms. The data suggest that apomixis might rely more on a global deregulation of sexual reproductive development than on truly new functions, and molecular mechanisms for such a global deregulation can be proposed. This new understanding has direct consequences for the engineering of apomixis in sexual crop species, an application that could have an immense impact on agriculture.     

外来入侵植物胜红蓟的胚胎学观察及繁殖系统研究

以采自我国广东江门和广州两个种群的胜红蓟(Ageratum conyzoides L.)种子为材料,采用流式细胞种子筛选技术(FCSS)和人工控制授粉实验,对胜红蓟的繁殖系统进行研究,并结合整体透明技术和微分干涉差(DIC)显微镜观察法,对其胚珠发育过程和花药结构进行细胞胚胎学观察。种子筛选结果显示,胜红蓟的种子既可以通过有性生殖产生,又可以通过不需要假受精的无融合生殖产生,属于兼性无融合生殖类型。开放性授粉和套袋处理之间的结实率均较高,分别为88%±1.2%和86.2%±1.2%,两者之间无显著差异;而去雄处理的结实率和开放性授粉、套袋这两种处理之间差异显著。胚珠的细胞胚胎学观察结果发现,胜红蓟的有性生殖胚囊发育方式为蓼型,无融合生殖胚囊的发育方式为山柳菊型。胜红蓟的花粉粒在花药内就开始萌发出花粉管,具有闭花受精特性。研究结果表明闭花受精和兼性无融合生殖等繁殖特性保证了胜红蓟在各种生存环境下的结实量,提高其在新生境中归化和入侵的可能性。     

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.     

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.