Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B. decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants.     

The best of both worlds: Apomixis and sexuality co-occur in species of Microlicia,Melastomataceae

Apomixis, the asexual formation of embryos and seeds, occurs in approximately 18% of angiosperm families. Melastomataceae exhibits a remarkable number of apomictic species, distributed among different tribes. This mode of reproduction has been elucidated in Miconieae, but remains unclarified for other groups, such as Microlicieae. Although apomixis has been previously described for Microlicieae species, the cytological basis for this phenomenon is entirely unknown in this group. Thus, populations of Microlicia fasciculata and M. polystemma were used in order to (a) investigate the presence of autonomous apomixis; (b) verify if this mode of reproduction leads to polyembryony; and (c) investigate whether apomixis may occur in parallel with the sexual process. We tested these species for autonomous fruit set and polyembryony, and pollen viability, and analyzed pollen tube growth. Anatomical techniques were used to elucidate the micro- and megasporogenesis and gametogenesis. The species showed autonomous fruit and seed formation and exhibited polyembryony. Apospory and adventitious embryony were the developmental mechanisms of apomixis in M. fasciculata and M. polystemma, respectively. Both species exhibited low pollen viability. However, some viable pollen, reduced embryo sac formation, natural pollination and pollen tube growth enable sexual reproduction and characterize these species as facultative apomicts. The independence of pollinators for fruit set, uniparental reproduction and the possibility of sexual reproduction, confer reproductive assurance and flexibility, bringing together advantages of sexual and asexual reproduction. In this sense, apomixis may have played an important role in the evolution and diversification of Microlicia, a widely distributed genus in the Brazilian Cerrado.     

Comparative cyto-embryological investigations of sexual and apomictic dandelions (Taraxacum) and their apomictic hybrids

. In the autonomous apomictic Taraxacum officinale (common dandelion), parthenogenetic egg cells develop into embryos and central cells into endosperm without prior fertilisation. Unreduced (2n) megaspores are formed via meiotic diplospory, a nonreductional type of meiosis. In this paper, we describe the normal developmental pathways of sexual and apomictic reproduction and compare these with the development observed in the apomictic hybrids. In sexual diploids, a standard type of megasporogenesis and embryo sac development is synchronised between florets in individual capitula. In contrast, we observed that megasporogenesis and gametogenesis proceeded asynchronously between florets within a single capitulum of natural triploid apomicts. In addition, autonomous endosperm and embryo development initiated independently within individual florets. Parthenogenetic initiation of embryo development in outdoor apomicts was found to be temperature-dependent. Egg cells produced in natural apomicts were not fertilised after pollination with haploid pollen grains although pollen tubes were observed to grow into their embryo sacs. Both reductional and diplosporous megasporogenesis were observed in individual inflorescences of triploid apomictic hybrids. Embryo and endosperm development initiated independently in natural and hybrid apomicts.     

Influence of environmental,geographic, and seasonal variations in the chemical composition of Miconia species from Cerrado

Miconia albicans (Sw.) Triana and Miconia chamissois Naudin, commonly known as “pixirica” and “pixirica-açu”, two Melastomataceae species, are commonly found in Cerrado areas. M. albicans species grows in dry and humid habitats, so it has adapted to both conditions and M. chamissois species grows in humid environment only. In this work, we have investigated the content of triterpenes, flavonols and flavanones in three different Cerrado fragments of São Paulo State, involving distinct environments (dry and humid) and seasons (dry and rainy) to comprehend the complex interactions among plants and the seasonal, environmental conditions and geographic locations. The leaves plants materials were harvest in August 2016 (dry season) and November/December 2016 (rainy season) in Pirassununga, Pedregulho and Luíz Antônio in dry and humid environments. The contents of the standards rutin (R), quercetin (Q), miconioside B (B), matteucinol 7-O-β-apiofuranosyl(1 → 6)-β-glucopyranoside (matt), ursolic acid (AU), and oleanolic acid (AO) were determined by HPLC-DAD. The data were analyzed using nonparametric tests, Pearson's linear correlation, principal component analysis and hierarchical cluster analysis. The results demonstrated similar flavanone and triterpene production during the dry period, and similar flavonol and flavanone production in the rainy season, thus confirming a seasonal variation in the content of the compounds in the evaluated specimens. Our results also demonstrated intra- and inter-population variations in compounds patterns, M. albicans contained major amounts of the flavonols R and Q; their concentrations were higher in the rainy season. B and matt were the major compounds in M. chamissois; the matt concentration decreased in the rainy season. The triterpenes AU and AO occurred in both M. albicans and M. chamissois, but their production dropped during the rainy periods. Thus corroborating the adaptation of M. albicans and M. chamissonis to the high stress and low levels of resources presented in Cerrado.     

Unexpectedly high genetic diversity and divergence among populations of the apomictic Neotropical tree Miconia albicans

• Since tropical trees often have long generation times and relatively small reproductive populations, breeding systems and genetic variation are important for population viability and have consequences for conservation. Miconia albicans is an obligate, diplosporous, apomictic species widespread in the Brazilian Cerrado, the savanna areas in central Brazil and elsewhere in the Neotropics. The genetic variability would be, theoretically, low within these male‐sterile and possibly clonal populations, although some variation would be expected due to recombination during restitutional meiosis.
• We used ISSR markers to assess genetic diversity of M. albicans and to compare with other tropical trees, including invasive species of Melastomataceae. A total of 120 individuals from six populations were analysed using ten ISSR primers, which produced 153 fully reproducible fragments.
• The populations of M. albicans presented mean Shannon's information index (I) of 0.244 and expected heterozygosity (H_e) of 0.168. Only two pairs of apparently clonal trees were identified, and genetic diversity was relatively high. A hierarchical amova for all ISSR datasets showed that 74% of the variance was found among populations, while only 26% of the variance was found within populations of this species. Multivariate and Bayesian analyses indicated marked separation between the studied populations.
• The genetic diversity generated by restitutional meiosis, polyploidy and possibly other genome changes may explain the morpho‐physiological plasticity and the ability of these plants to differentiate and occupy such a wide territory and different environmental conditions. Producing enormous amounts of bird‐dispersed fruits, M. albicans possess weedy potential that may rival other Melastomataceae alien invaders.

     

Introgression of apomixis into sexual species is inhibited by mentor effects and ploidy barriers in the Ranunculus auricomus complex

Background and Aims

Apomictic plants maintain functional pollen, and via pollination the genetic factors controlling apomixis can be potentially transferred to congeneric sexual populations. In contrast, the sexual individuals do not fertilize apomictic plants which produce seeds without fertilization of the egg cells. This unidirectional introgressive hybridization is expected finally to replace sexuality by apomixis and is thought to be a causal factor for the wide geographical distribution of apomictic complexes. Nevertheless, this process may be inhibited by induced selfing (mentor effects) of otherwise self-incompatible sexual individuals. Here whether mentor effects or actual cross-fertilization takes place between diploid sexual and polyploid apomictic cytotypes in the Ranunculus auricomus complex was tested via experimental crosses.

Methods

Diploid sexual mother plants were pollinated with tetra- and hexaploid apomictic pollen donators by hand, and the amount of well-developed seed compared with aborted seed was evaluated. The reproductive pathways were assessed in the well-developed seed via flow cytometric seed screen (FCSS).

Key Results

The majority of seed was aborted; the well-developed seeds have resulted from both mentor effects and cross-fertilization at very low frequencies (1·3 and 1·6 % of achenes, respectively). Pollination by 4x apomictic pollen plants results more frequently in cross-fertilization, whereas pollen from 6x plants more frequently induced mentor effects.

Conclusions

It is concluded that introgression of apomixis into sexual populations is limited by ploidy barriers in the R. auricomus complex, and to a minor extent by mentor effects. In mixed populations, sexuality cannot be replaced by apomixis because the higher fertility of sexual populations still compensates the low frequencies of potential introgression of apomixis.Key words: Apomixis, Ranunculus auricomus, evolution, geographical parthenogenesis, crossing experiments, flow cytometry     

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     

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 versus sexuality in blackberries (Rubus subgen.Rubus,Rosaceae)

InRubus L. a connection seems to exist between the degree of meiotic disturbances on the one hand, and the production of unreduced embryo sacs, pollen fertility and relative seed set on the other hand. Severe meiotic disturbances commonly encountered in apomictic taxa decrease pollen fertility and thereby seed set since pollen is necessary for endosperm development. By contrast interspecific hybrids between apomictic taxa appear to be sexual and exhibit high pollen fertilities, probably due to an improved meiosis. Thus, apomixis leads to a decreased fertility inRubus, not the opposite, as often discussed.     

APOMIXIS IN THE POA SECUNDA COMPLEX

Observation of cleared ovules of Poa secunda Presl showed that plants produce aposporous embryo sacs, and emasculation studies showed that they do not set seed without pollen. The ratio of sexual to asexual embryo sacs on a given plant does not correlate with percent stainable pollen, nor does pollen stainability correlate with meiotic regularity or with chromosome number. Percent apomictic ovules, percent pollen stainability, and regularity of meiosis all vary within a given population; they also vary if the same plant is moved from one site to another. Although there is morphological variation in the species, none of the reproductive features correlates with morphology. Pollen from any morph will stimulate seed set in any other morph, and hybrids have been produced in low frequency. Plants will also set seed using their own pollen. In its reproductive biology, P. secunda exhibits many similarities with P. pratensis L.     

Reproductive development in apomictic populations of <Emphasis Type="Italic">Arabis holboellii</Emphasis> (Brassicaceae)

Megasporogenesis, megagametogenesis and seed formation were analyzed cytologically in populations of Arabis holboellii originating from North America (Colorado) and Greenland. The Colorado population contained only triploid plants, while the Greenland population consisted of diploid and triploid plants. The penetrance of the apomictic trait was assessed at the level of embryo sac development. All populations showed facultative apomeiotic embryo sac development; however the penetrance of this trait differed between the populations. Apomeiotic and meiotic embryo sac development were characterized by diplosporous dyad formation (Taraxacum-type) and meiotic tetrad formation (Polygonum-type), respectively. Flow cytometric analyses of single mature seeds from all three populations suggest that only unreduced gametes participate in viable seed development. Pseudogamy was the predominant mode of endosperm formation; however, autonomous endosperm development was also observed. The fertilization of unreduced egg cells with unreduced pollen was observed at a low frequency in the Greenland populations. The mechanisms of apomictic reproduction in A. holboellii are discussed.     

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     

Megagametogenesis in Halophila johnsonii, a threatened seagrass with no known seeds,and the seed-producing Halophila decipiens (Hydrocharitaceae)

Megagametogenesis, the development of a megaspore into an embryo sac, has been identified in the seagrass Halophila johnsonii, a threatened species with no known sexual reproduction or seeds. Megagametogenesis in H. johnsonii was compared with megagametophyte development in Halophila decipiens, a related species known to readily produce viable seeds. In both species, ovules were structurally similar, megaspore mother cells were seen in premeiotic ovules, and linear tetrads and megagametophytes with two to eight nuclei were present in postmeiotic ovules. However, H. decipiens postmeiotic ovules had a chalazal pouch that was absent in the postmeiotic ovules of H. johnsonii. Late-stage H. decipiens ovules also contained embryos, indicating that they had been fertilized, whereas all late-stage H. johnsonii ovules were degrading and showed no signs of fertilization. These observations suggest that meiosis does occur in H. johnsonii megasporocytes, leading to the formation of viable megagametophytes and egg cells that could be fertilized if pollination occurred. Thus, the lack of seed set is due to a lack of pollination rather than any loss of capacity to produce seeds in this species.     

A pattern of unique embryogenesis occurring via apomixis in Carya cathayensis

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds that have essentially clonal embryos. In this report, hickory (Carya cathayensis Sarg.), which is an important oil tree, is identified as a new apomictic species. The ovary has a chamber containing one ovule that is unitegmic and orthotropous. Embryological investigations indicated that the developmental pattern of embryo sac formation is typical polygonum-type. Zygote embryos were not found during numerous histological investigations, and the embryo originated from nucellar cells. Nucellar embryo initials were found both at the micropylar and chalazal ends of the embryo sac, but the mature embryo developed only at the nucellar beak region. The mass of the nucellar embryo initial at the nucellar beak region developed into a nucellar embryo or split into two nucellar proembryos. The later development of the nucellar embryo was similar to the zygotic embryo and progressed from globular embryo to heart-shape embryo and to cotyledon embryo.     

Sexual and apomictic seed formation in Hieracium requires the plant polycomb-group gene FERTILIZATION INDEPENDENT ENDOSPERM

A Polycomb-Group (PcG) complex, FERTILIZATION INDEPENDENT SEED (FIS), represses endosperm development in Arabidopsis thaliana until fertilization occurs. The Hieracium genus contains apomictic species that form viable seeds asexually. To investigate FIS function during apomictic seed formation, FERTILIZATION INDEPENDENT ENDOSPERM (FIE), encoding a WD-repeat member of the FIS complex, was isolated and downregulated in sexual and apomictic Hieracium species. General downregulation led to defects in leaf and seed development, consistent with a role in developmental transitions and cell fate. PcG-like activity of Hieracium FIE was also supported by its interaction in vitro with the Arabidopsis CURLY LEAF PcG protein. By contrast, specific downregulation of FIE in developing seeds of sexual Hieracium did not result in autonomous endosperm proliferation but led to seed abortion after cross-pollination. Furthermore, in apomictic Hieracium, specific FIE downregulation inhibited autonomous embryo and endosperm initiation, and most autonomous seeds displayed defective embryo and endosperm growth. Therefore, FIE is required for both apomictic and fertilization-induced seed initiation in Hieracium. Since Hieracium FIE failed to interact with FIS class proteins in vitro, its partner proteins might differ from those in the FIS complex of Arabidopsis. These differences in protein interaction were attributed to structural modifications predicted from comparisons of Arabidopsis and Hieracium FIE molecular models.     

Consistent variation in seed germination across an environmental gradient in a Neotropical savanna

Environmental conditions experienced by mother plants determine germination patterns. Here, we investigated the germination ecology of Miconia albicans (Melastomataceae), a widespread apomictic shrub, along a soil fertility gradient in the Brazilian Cerrado (a Neotropical savanna). The mosaic of vegetations in the Cerrado ranges from grasslands to woodlands, which present different conditions for seedling establishment. Cerrado grasslands are a more unpredictable environment because they are more prone to disturbances such as fires and prolonged droughts than closed woodlands. We expect lower reproductive investment but increasing germinability, germination speed and synchrony in areas with more dense vegetation, due to prolonged droughts in grasslands and higher soil properties in woodlands. Seeds of M. albicans were collected in grasslands, shrublands and woodlands for three years in a Cerrado area in southeastern Brazil. Seeds collected in all vegetations were set to germinate under identical experimental conditions. According to our prediction, we found a consistent pattern of increasing germinability, mean germination time and synchrony in areas with more closed vegetation. Both slower and asynchronous germination in seeds from grassland plants are likely to increase seedling survival under a more unpredictable environment. On the other hand, seeds from plants in the woodlands germinated at higher rates and germination was more synchronous, thus increases the chances of survival under a more competitive light-limited environment. This pattern was found in all three years of study. Our study suggests that stressful environmental conditions experienced by mother plants in the Cerrado may act as cues signalizing unfavourable conditions for seed germination and help explain functional divergence in germination traits within species. In conclusion, we showed for the first time a temporally consistent pattern of intraspecific variation in the germination traits of M. albicans, with different germination strategies at the extremes of the ecological gradient.     

Polyembryony and apomixis in Eriotheca pubescens (Malvaceae - Bombacoideae)

Apomixis and adventitious polyembryony have been reported for several species of Bombacoideae, including Eriotheca pubescens, a tree species of the Neotropical savanna (Cerrado) areas in Brazil. However, the origin of polyembryonic seeds and their importance for the reproduction of the species remained to be shown. Here, we analyzed the early embryology of this species to establish the apomictic origin of extranumerary embryos. We also observed the geographic distribution of polyembryony in E. pubescens, and tested if apomixis was related to the source of pollen (self or cross) and population density. Moreover, we tested if polyembryonic apomictic embryos would develop normally into seedlings. In the observed seed primordia, after a relatively long quiescent period, the zygote developed into a sexual embryo concurrently with adventitious apomictic embryos which developed from nucellus cells. Adventitious embryos develop faster than sexual ones and are morphologically similar, so that 44 days after anthesis it was virtually impossible to distinguish and trace the fate of the sexual embryo. Polyembryony is widely distributed in populations some 400 km distant, and only one strictly monoembryonic individual was observed during the study. The number of embryos per seed varied between fruits and individuals but was significantly higher in seeds from cross-pollinations than from selfs, although fruit and seed set after crosses were much lower than after selfs. Embryo development into seedlings depended on their weight at germination, but polyembryonic seeds germinated and produced up to seven seedlings per seed in greenhouse conditions. Adventitious embryony and apomictic seedlings would explain the mostly clonal populations suggested by molecular studies.