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 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.     

Apomixis and sexuality in diploid and tetraploid accessions of Brachiaria decumbens

 Meiotic and aposporous embryo sacs and the initial steps of parthenogenetic embryogenesis and endosperm formation were investigated in diploid and tetraploid accessions of Brachiaria decumbens in two environments, differing mainly in day length: early summer and late autumn. Both diploid and tetraploid accessions were facultative apomicts. Di(ha)ploids showed a much lower level of apomixis (10% to15%) than tetraploids (80% to 95%). No obligate sexual diploids were found; thus, their occurrence in natural populations is obscure. It is suggested that reproduction in B. decumbens, as in other agamic complexes of the Paniceae tribe, in general, approximates a diploid-tetraploid-(di)haploid reproductive cycle which does not involve triploids. The dihaploids were fertile and survived in nature. Development of the reproductive structures depended on the environment. In autumn, in contrast to early summer, many meiotic and aposporous embryo sacs degenerated during development, leading to a significant reduction in the proportion of parthenogenetic embryos. Whether this effect can be attributed to day length or simply to age remains to be investigated. The ratio of aposporous to sexual embryo sacs was relatively stable over the two seasons. Received: 15 April 1998 / Revision accepted: 13 October 1998     

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:     

Tripsacum dactyloides (Poaceae): a natural model system to study parthenogenesis

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.352 larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (B_III hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin::#-glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.     

Genetic and embryological evidences of apomixis at the diploid level in <Emphasis Type="Italic">Paspalum rufum</Emphasis> support recurrent auto-polyploidization in the species

Gametophytic apomixis is an asexual mode of reproduction by seeds. This trait is present in several plant families and is strongly associated with polyploidy. Paspalum rufum is a forage grass with sexual self-incompatible diploids (2n = 2x = 20) and aposporous-apomictic pseudogamous tetraploids (2n = 4x = 40). In previous work embryological observations of the diploid genotype Q3754 showed 8.8–26.8% of the ovaries having one meiotic plus an aposporous-like embryo sac, suggesting some capability for apomictic reproduction. The objective of this work was to characterize progenies derived from Q3754 to determine if aposporous sacs were functional and generated progenies via apomixis at the diploid level. Re-examination of Q3754 ovaries showed that 12.5% of them contained one sexual plus an aposporous sac confirming previous results. Progeny tests were carried out on two experimental families (H₁ and S₁) employing heterozygous RAPD marker loci. Family H₁ was obtained crossing Q3754 with a natural diploid genotype (Q3861) and S₁ derived from the induced self-pollination of Q3754. Genetic analysis of H₁ showed that all individuals derived from sexual reproduction. However, 5 out of 95 plants from S₁ showed the same heterozygous state as the mother plant for 14 RAPD loci suggesting a clonal origin. Further experiments, designed to test the functionality of aposporous sacs by flow cytometric analyses, were carried out on a third family (M₁) obtained by crossing Q3754 with the tetraploid plant Q3785. Histograms of 20 M₁ plants showed 15 diploids (75%), 4 triploids (20%) and 1 tetraploid (5%). Triploids and the tetraploid may have originated from functional aposporous embryo sacs. Likewise, the reconstruction of the developmental route of 40 individual seeds demonstrated that 11 of them (27.5%) derived from fertilized aposporic sacs. The results presented in this work indicate that gametophytic apomixis is effectively expressed at the diploid level in Paspalum rufum and could be the foundation of a recurrent auto-polyploidization process in the species.     

Apomixis in <Emphasis Type="Italic">Eulaliopsis binata</Emphasis>: characterization of reproductive mode and endosperm development

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds with essentially clonal embryos, stimulating wide interest from biologists and plant breeders for its ability to fix heterosis. Eulaliopsis binata (Poaceae), is identified here as a new apomictic species. Embryological investigation indicates that the developmental pattern of embryo sac formation in E. binata represents gametophytic apospory, the embryo originating from an unreduced cell, without fertilization and the mode of endosperm development was autonomous. Sexual embryo sacs were found with a frequency of 1–4% depending on the biotype. The DNA content of nuclei (C-value) in mature seeds was screened by flow cytometry (FCSS) and demonstrated that the endosperm was derived autonomously without fertilization and the three biotypes of E. binata showed varying degrees of apomixis. The Wide-leaf type showed obligate apomixis whereas the Slender-leaf and the Red-haulm type displayed facultative apomixis. In addition, adventitious embryos were observed on the wall of ovary, integument and nucellus cells, indicating that E. binata produces embryos via a mixture of apospory and adventitious embryony.     

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     

Apomixis and ploidy barrier suppress pollen-mediated gene flow in field grown transgenic turf and forage grass (Paspalum notatum Flüggé)

Bahiagrass (Paspalum notatum Flüggé) is the predominant forage grass in the southeastern US. The commercially important bahiagrass cultivar ‘Argentine’ is preferred for genetic transformation over sexual diploid cytotypes, since it produces uniform seed progeny through apomixis. Pseudogamous apomictic seed production in Argentine bahiagrass may contribute to transgene confinement. It is characterized by embryo development which is independent of fertilization of the egg cell, but requires fertilization with compatible pollen to produce the endosperm. Pollen-mediated gene transfer from transgenic, glufosinate-resistant apomictic bahiagrass as pollen donor at close proximity (0.5–3.5 m) with non-transgenic sexual or apomictic bahiagrass cultivars as pollen receptors was evaluated under field conditions. Hybridization frequency was evaluated by glufosinate herbicide resistance in >23,300 seedlings derived from open-pollinated (OP) pollen receptor plants. Average gene transfer between transgenic apomictic, tetraploid and sexual diploid bahiagrass was 0.03%. Herbicide-resistant hybrids confirmed by immuno-chromatographic detection of the PAT protein displayed a single copy bar gene identical to the pollen parent. Hybrids resulting from diploid pollen receptors were confirmed as triploids or aneu-triploids with significantly reduced vigor and seed set as compared to the parents. Transmission of transgenes to sexual bahiagrass is severely restricted by the ploidy difference between tetraploid apomicts and diploid sexual bahiagrass. Average gene transfer between transgenic apomictic tetraploid and non-transgenic, apomictic tetraploid bahiagrass was 0.17%, confirming a very low frequency of amphimixis in apomictic bahiagrass cultivars. While not providing complete transgene containment, gene transfer between transgenic apomictic and non-transgenic bahiagrass occurs at a much lower frequency than reported for other cross-pollinating or facultative apomictic grasses.     

Reproduction pathway analysis of several Hypericum perforatum L. somaclonal families

Flow cytometry seed screen of mature seeds originating from several in vitro regenerated Hypericum perforatum L. somaclones and their seed progenies were used to screen the ways of reproduction of 4 subsequent generations of several somaclonal families and to search for the relation between the ploidy and prevalent mode of reproduction. The prevalent reproduction pathway of diploid plants was sexual reproduction. Seed samples of plants with higher ploidy levels showed an extensive variation in the mode of reproduction: BII and BIII hybrid formation and/or aposporous pseudogamy including parthenogenetic development of a reduced embryo sac.     

四倍体双穗雀稗兼性无孢子生殖的研究

研究了四倍体双穗雀稗(Paspalum distichum L)无孢子生殖胚囊、胚胎发育以及假受精特点。当其大孢子母细胞发育至四分体阶段时,大多数情况下会发生四分体退化,同时有多个特化珠心细胞发育为1—3个无孢子生殖胚囊的现象。成熟无孢子生殖胚囊一般3核,包括1个卵细胞和2个极核。卵细胞在抽穗前就能自发分裂形成原胚团,而极核则在抽穗和传粉后参与假受精形成胚乳。当胚珠内存在多个无孢子生殖胚囊时,只是靠近珠孔端的1个无孢子生殖胚囊内的极核与精核结合,而其它的并不参与。种子成熟后出现很低频率的二胚苗。此外,还能观察到少量的有性生殖胚囊的发育以及有性生殖胚囊和无孢子生殖胚囊在同一胚珠中的发育现象,因此判断该类群为兼性无孢子生殖体。     

An efficient screen for reproductive pathways using mature seeds of monocots and dicots

Seed samples of 32 species (obligate and facultative sexuals and apomicts of monocots and dicots) were investigated by flow cytometry to reveal the pathway of reproduction. Ten different pathways of seed formation could be reconstructed considering whether the female and/or male gametes were reduced or unreduced, the embryos arose via the zygotic or parthenogenetic route and the endosperm via the pseudogamous or autonomous route. The screen is suited to select sporophytic or gametophytic mutants in sexual species, to identify pure sexual or obligate apomictic genotypes from facultative apomictic species, and to analyze the inheritance of the individual reproductive processes. Corresponding unique results are presented for Arabidopsis, Arabis, Hypericum and Poa. The screen of mature seeds by flow cytometry yielded more information about the reproductive behavior of individual plants than any other available test, and is very useful both in basic research and plant breeding.     

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.     

Mendelian segregation for two-factor apomixis in Erigeron annuus (Asteraceae)

The inheritance of asexual seed development (apomixis) in Erigeron annuus (Asteraceae) was evaluated in a triploid (2n=3x=27) population resulting from a cross between an apomictic tetraploid (2n=4x=36) pollen parent and a sexual diploid (2n=2x=18) seed parent. Diplospory (unreduced female gametophyte formation) and autonomous development (embryo and endosperm together) segregated independently in the population yielding four distinct phenotype classes: (1) apomictic plants combining diplospory and autonomous development, (2) diplosporous plants lacking autonomous development, (3) meiotic plants with autonomous (though abortive) development and (4) meiotic plants lacking autonomous development. Each class was represented by approximately one-quarter of the population (n=117), thus corresponding to a two-factor genetic model with no linkage (chi(2)=2.59, P=0.11). Observations demonstrate that autonomous embryo and endosperm development (jointly) may occur in either reduced or unreduced egg cells. The cosegregation of the traits is attributed to tight linkage or pleiotropy. The data are consistent with the hypothesis that autonomous development in E. annuus is regulated by a single fertilization factor, F, which initiates development of both the embryo and the endosperm in the absence of fertilization.     

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.     

Characterization of mode of reproduction in Commiphora wightii [(Arnot) Bhandari] reveals novel pollen–pistil interaction and occurrence of obligate sexual female plants

Guggul [Commiphora wightii (Arnot) Bhandari], a polygamous woody tree valued for its medicinal oleoresin gum rich in guggulsterone, is reported to reproduce via sporophytic apomixis. Details about its natural diversity, and mode and extent of sexual reproduction are, however, scanty. Therefore, a comprehensive investigation of guggul reproduction was made employing histology, controlled pollination, flow cytometry and RAPD analyses of progeny to assess the occurrence and extent of sexual reproduction. We report the discovery of obligate sexual female plants of guggul through these studies. Also, we document a unique pollen–pistil incompatibility that prevents all but one pollen tube growth into the style to effect fertilization. Consequently, obligate sexual female plants produced single-seeded fruit although each flower contains four ovules. In apomictic plants bearing more than one seed per fruit, at most only one seed was of sexual origin. Further, flow cytometric analysis conclusively demonstrated that endosperm development occurs either autonomously or following triple fusion. Autonomous endosperm development was invariably associated with endoreduplication, a unique feature of apomixis in guggul. Despite predominance of apomixis, a low frequency of sexual reproduction was found to persist in apomictic plants yielding new genetic variation. RAPD analysis clearly distinguished accessions and was useful in identifying sexual progenies. The implications of the novel pollen–pistil interaction on establishment and spread of apomixis in guggul are discussed. The study has not only revealed novel features of guggul reproduction but also opened new opportunities for molecular genetic analysis of sporophytic apomixis and breeding improvement of guggul.     

Quantitative variation for apomictic reproduction in the genus Boechera (Brassicaceae)

? Premise of the study: The evolution of asexual seed production (apomixis) from sexual relatives is a great enigma of plant biology. The genus Boechera is ideal for studying apomixis because of its close relation to Arabidopsis and the occurrence of sexual and apomictic species at low ploidy levels (diploid and triploid). Apomixis is characterized by three components: unreduced embryo-sac formation (apomeiosis), fertilization-independent embryogenesis (parthenogenesis), and functional endosperm formation (pseudogamy or autonomous endosperm formation). Understanding the variation in these traits within and between species has been hindered by the laborious histological analyses required to analyze large numbers of samples. ? Methods: To quantify variability for the different components of apomictic seed development, we developed a high-throughput flow cytometric seed screen technique to measure embryo:endosperm ploidy in over 22000 single seeds derived from 71 accessions of diploid and triploid Boechera. ? Key results: Three interrelated features were identified within and among Boechera species: (1) variation for most traits associated with apomictic seed formation, (2) three levels of apomeiosis expression (low, high, obligate), and (3) correlations between apomeiosis and parthenogenesis/pseudogamy. ? Conclusions: The data presented here provide a framework for choosing specific genotypes for correlations with large "omics" data sets being collected for Boechera to study population structure, gene flow, and evolution of specific traits. We hypothesize that low levels of apomeiosis represent an ancestral condition of Boechera, whereas high apomeiosis levels may have been induced by global gene regulatory changes associated with hybridization.     

Residual sexuality and its seasonal variation in natural apomictic Paspalum notatum accessions

Traditionally, tetraploid Paspalum notatum was considered an obligate or a facultative apomict according to cytoembryological analyses. The degree of facultativeness was usually determined by the relative amount of mature ovules bearing aposporous or meiotic (sexual) embryo sacs, or both together. We established, through progeny tests conducted with the aid of AFLP markers, the degree of residual sexuality expressed in four selected biotypes. The results showed it to be substantially and significantly lower than predicted by previous embryological analyses for the same biotypes. Moreover, the lowest expression of residual sexuality was coincident with maximum flowering period. Seed development in facultative apomictic P. notatum shows a definite bias against meiotic embryo sacs.