Sexual reproduction is the default mode in apomictic Hieracium subgenus Pilosella, in which two dominant loci function to enable apomixis

Asexual seed formation, or apomixis, in the Hieracium subgenus Pilosella is controlled by two dominant independent genetic loci, LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP). We examined apomixis mutants that had lost function in one or both loci to establish their developmental roles during seed formation. In apomicts, sexual reproduction is initiated first. Somatic aposporous initial (AI) cells differentiate near meiotic cells, and the sexual pathway is terminated as AI cells undergo mitotic embryo sac formation. Seed initiation is fertilization-independent. Using a partially penetrant cytotoxic reporter to inhibit meioisis, we showed that developmental events leading to the completion of meiotic tetrad formation are required for AI cell formation. Sexual initiation may therefore stimulate activity of the LOA locus, which was found to be required for AI cell formation and subsequent suppression of the sexual pathway. AI cells undergo nuclear division to form embryo sacs, in which LOP functions gametophytically to stimulate fertilization-independent embryo and endosperm formation. Loss of function in either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in total reversion to sexual reproduction. Therefore, in these apomicts, sexual reproduction is the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode genes essential for sexual reproduction, but may function to recruit the sexual machinery at specific time points to enable apomixis.     

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.     

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.     

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 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.     

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.     

Embryological Studies on Apomixis in Pennisetum squamulatum

Studies on the formation and development of the embryo sac of the apomictic material of Pennisetum squamulatum Fresen indicated that normal archesporial cell did form with consequent development of a megaspore mother cell and later meiotic division to give rise to a triad. But invariably the megaspore mother cell and the triad underwent degeneration after formation. During the period of formation or degeneration of the megaspore or the triad a number of nucellar cells around the degenerated sexual cell became much enlarged. Frequently, one of the enlarging nucellar cells near the micropylar end became vacuolated and then developed into an aposporous uninucleate embryo sac, which underwent two further mitotic divisions to form an aposporous four-nucleate embryo sac, where the four nuclei remained in the micropylar end. Thus in the mature aposporous embryo sac there were one egg cell, one synergid and one central cell (containing two polar nuclei). Antipodal cells were completely lacking. The pattern of development of the aposporous embryo sac resembles the panicum type. There were two types of embryo formed during apomictic development namely ( 1 ) The pre-genesis embryo--embryo formed without fertilization, 1 to 2 days before anthesis, and (2) The late-genesis embryo--derived from the unfertilized egg cells, 3 to 4 days after anthesis. In the late-genesis embryo type, the egg cell divided after the secondary nucleus has undergone division to form the endosperm nuclei. All egg cells developed vacuoles before they differentiated into embryos. The development of the aposporous embryo followed the sequence of the formation of globular, pearshaped embryo and full stages of differentiation. The unfertilized secondary nucleus divides to form free endosperm nuclei after being stimulated by pollination. The development of the endosperm belongs to the nuclear-type.     

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     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsis binata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料.龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%.胚囊发育属大黍型.多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%.胚珠内没有有性胚囊的发育.胚的发生有两种类型:(1)早发生胚(74%),开花前1～2 d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2～3 d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚.存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%.胚乳由极核不经受精自发分裂产生.     

水蔗草兼性无融合生殖胚胎学研究

对水蔗草 (ApludamuticaL .)的生殖方式进行研究 ,结果表明水蔗草进行兼性无融合生殖。胚囊发育分为两种类型 ,即有性生殖的蓼型和无孢子生殖的大黍型。无融合生殖胚囊频率为 6 0 .74%。在大孢子母细胞发育至四分体后 ,珠孔端的 3个大孢子解体。合点端的大孢子未解体时 ,邻近大孢子的 1个珠心细胞开始特化 ,形成无融合生殖的原始细胞 ,由该原始细胞发育形成有 1个卵细胞、1个助细胞和 2个极核的四核胚囊。     

水蔗草兼性无融合生殖胚胎学研究

对水蔗草(Apluda mutica L.)的生殖方式进行研究,结果表明水蔗草进行兼性无融合生殖.胚囊发育分为两种类型,即有性生殖的蓼型和无孢子生殖的大黍型.无融合生殖胚囊频率为60.74%.在大孢子母细胞发育至四分体后,珠孔端的3个大孢子解体.合点端的大孢子未解体时,邻近大孢子的1个珠心细胞开始特化,形成无融合生殖的原始细胞,由该原始细胞发育形成有1个卵细胞、1个助细胞和2个极核的四核胚囊.     

In search of female sterility causes in the tetraploid and pentaploid cytotype of Pilosella brzovecensis (Asteraceae)

Within the agamic Pilosella complex, apomixis (asexual reproduction through seed) involves apospory, parthenogenesis, and autonomous endosperm development. Observations of reproductive biology in P. brzovecensis throughout four growing seasons in the garden have shown that both tetraploid and pentaploid plants of this species do not produce viable seeds and reproduce exclusively vegetatively by underground stolons. The reasons for the seed development failure were unknown, therefore our research focused on the analysis of reproductive events in the ovules of this taxon. We found that apospory was initiated in the ovules of both cytotypes. Multiple aposporous initial (AI) cells differentiated in close proximity to the megaspore mother cell (MMC) and suppressed megasporogenesis at the stage of early prophase I. However, none of the AI cells was able to further develop into a multi-nucleate aposporous embryo sac (AES) due to the inhibition of mitotic divisions. It was unusual that callose was accumulated in the walls of AI cells and its synthesis was most likely associated with a response to the dysfunction of these cells. Callose is regarded as the isolating factor and its surprising deposition in the ovules of P. brzovecensis may signal disruption of reproductive processes that cause premature termination of the aposporous development pathway and ultimately lead to ovule sterility. The results of our embryological analysis may be the basis for undertaking advanced molecular studies aimed at fully understanding of the causes of female sterility in P. brzovecensis.

     

Sexual and apomictic development in Hieracium

 Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis. Received: 11 February 1998 / Revision accepted: 23 July 1998     

草地早熟禾胚胎学研究 Ⅲ．多胚囊及多胚现象

报道了草地早熟禾中多胚囊的起源、发育和结构。在１个胚珠中,大孢子母细胞周围可以有一到多个起源于珠心细胞的胚囊原始细胞,并可以发育成为多胚囊,其中具有两个胚囊的可以发育成为成熟胚囊。起源于珠心的体细胞无孢子生殖胚囊的发育属于山柳菊型。两个成熟胚囊中,都可以形成胚和胚乳,因而形成了具假多胚的种子。位于中部的胚来源于珠心还囊,属于无融合生殖形成的胚。两个以上的多胚囊不能形成成熟胚囊。     

Ultrastructural characterization of apospory in Panicum maximum

The nucellar ultrastructure of apomictic Panicum maximum was analyzed during the meiocytic stage and during aposporous embryo sac formation. At pachytene the megameiocyte shows a random cell organelle distribution and sometimes only an incomplete micropylar callose wall. The chalazal nucellar cells are meristematic until the tetrad stage. They can turn into initial cells of aposporous embryo sacs. The aposporous initials can be recognized by their increased cell size, large nucleus, and the presence of many vesicles. The cell wall is thin with few plasmodesmata. If only a sexual embryo sac is formed, the nucellar cells retain their meristematic character. The aposporous initial cell is somewhat comparable to a vacuolated functional megaspore. It shows large vacuoles around the central nucleus and is surrounded by a thick cell wall without plasmodesmata. In the mature aposporous embryo sac the structure of the cells of the egg apparatus is similar to each other. In the chalazal part of the egg apparatus the cell walls are thin and do not hamper the transfer of sperm cells. Structural and functional aspects of nucellar cell differentiation and aposporous and sexual embryo sac development are discussed.     

Apomixis and amphimixis in flowering plants

The objective of the present article is to compare apomictic and sexual reproduction (amphimixis) in flowering plants. Light-optical and ultrastructural aspects of the cytoembryological processes in apomicts, beginning with the early stages of development of the ovule and concluding with the newly formed seed, are considered. In the overwhelming majority of apomicts, an inability to develop an autonomous endosperm or to form viable seeds without the involvement of the process of fertilization of the nuclei of the central cell of the embryo sac is observed. Characteristic features of the ultrastructural differentiation of the megasporocytes in diplospory, of aposporous initial cells in apospory, of embryocytes in adventive embryony, and of ovicells in parthenogenesis and synergids in apogamety are identified and are compared to the generative structures of amphimicts. The hypothesis is made that the mechanisms of genetic regulation in the formation and development of the generative structures in apomixis and amphimixis are similar at the cellular level. The present study is not a survey of apomixis in general. Previously published original results that have been obtained by the present author as a result of many years of research in the area of apomixis have served as a basis for the preparation of the study.     

Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

Apomictic seed development is a complex process including formation of unreduced embryo sac, parthenogenetic embryo development from the egg cell, and endosperm formation either autonomously, or due to fertilization of polar nuclei by the sperm (under pseudogamous form of apomixis). In the latter case, an obstacle to the normal endosperm development is disturbance of maternal (m) -to-paternal (p) genomic ratio 2m: 1p that occurs in the cases of pollination of unreduced embryo sac with haploid sperms. Usage of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m: 2p that provides formation of kernels with plump endosperm. Using tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum accessions. These kernels had hybrid endosperm and diploid maternaltype embryo or hybrid embryo with different ploidy level (2n, 3n, 4n). The frequencies of plump kernels on the ear ranged from 0.2-0.3% to 5.7-6.2% counting from the number of ovaries. Maternal-type plants were found in two maize lines, their frequency varying from 10.7 to 37.5% of the progeny plants. In CMS-lines of sorghum pollinated with tetraploid sorghum accessions, the frequency of plump kernels ranged from 0.6 to 14.0% counting from the number of ovaries; the frequency of maternal-type plants varied from 33.0 up to 96.1%. The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm proteins. These data testify to variable modes of seed formation under diploid × tetraploid crosses in maize and sorghum both by amphi- and by apomixis. Therefore, usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.     

水蔗草胚珠附器的研究

本文对水蔗草的胚珠附器进行研究,结果表明：在功能大孢子时期,珠孔端的1～3个珠心细胞开始特化,发育成胚珠附器;胚珠附器发生时,有些胚珠同时出现无孢子生殖原始细 胞;有性生殖和无孢子生殖的胚囊中均有胚珠附器存在;但在无孢子生殖的胚囊中,胚珠附器一般很大,长约是宽的1～３倍;而有性生殖胚囊的胚珠附器的长约是宽的1～２倍;和有性生殖胚囊相比,无孢子生殖胚囊的胚珠附器更加发达;存在发达的胚珠附器是水蔗草无孢子生殖胚囊的特点之一。     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsisbinata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料。龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%。胚囊发育属大黍型。多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%。胚珠内没有有性胚囊的发育。胚的发生有两种类型:(1)早发生胚(74%),开花前1~2d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2~3d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚。存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%。胚乳由极核不经受精自发分裂产生。