Apomictic Embryo Sacs Research Articles

Eragrostis curvula, a Model Species for Diplosporous Apomixis.

Eragrostis curvula (Schrad.) Ness is a grass with a particular apomictic embryo sac development called Eragrostis type. Apomixis is a type of asexual reproduction that produces seeds without fertilization in which the resulting progeny is genetically identical to the mother plant and with the potential to fix the hybrid vigour from more than one generation, among other advantages. The absence of meiosis and the occurrence of only two rounds of mitosis instead of three during embryo sac development make this model unique and suitable to be transferred to economically important crops. Throughout this review, we highlight the advances in the knowledge of apomixis in E. curvula using different techniques such as cytoembryology, DNA methylation analyses, small-RNA-seq, RNA-seq, genome assembly, and genotyping by sequencing. The main bulk of evidence points out that apomixis is inherited as a single Mendelian factor, and it is regulated by genetic and epigenetic mechanisms controlled by a complex network. With all this information, we propose a model of the mechanisms involved in diplosporous apomixis in this grass. All the genetic and epigenetic resources generated in E. curvula to study the reproductive mode changed its status from an orphan to a well-characterised species.

SERK genes identification and expression analysis during somatic embryogenesis and sporogenesis of sexual and apomictic Brachiaria brizantha (Syn. Urochloa brizantha).

In Brachiaria brizantha BbrizSERK1, BbrizSERK2 and BbrizSERK3 were identified. SERK expression marks somatic embryogenesis, sexual MMC, and sexual and apomictic PMC. BbrizSERK3 might have a regulatory role in reproductive development. Somatic embryogenesis receptor-like kinase (SERK) consists of plasma membrane receptor genes that have been characterized in various species, associated with several aspects of plant development, including reproduction. SERK genes are involved in anther development and in early embryo development in sexual and asexual seed formation. To comprehend the complexity of the SERK genes and their function in Brachiaria reproduction, we performed a homology-based search in a genomic database of a sexual B. brizantha and identified sequences of three SERK genes, BbrizSERK1, BbrizSERK2, and BbrizSERK3. RNASeq data showed equivalent abundance of BbrizSERK1 and BbrizSERK2 transcripts in ovaries at early megasporogenesis of sexuals and apomicts, while BbrizSERK3 transcripts were more abundant in ovaries of sexuals than in apomicts. BbrizSERK3 results in three coding sequences due to alternative splicing, among them Variant 1 results in a protein with all the predicted domains of a SERK. BbrizSERK transcripts were detected in male reproductive tissues of both sexual and apomictic plants, suggesting a role in controlling anther development. BbrizSERK transcripts were detected early in ovule development, in the integuments, and in the megaspore mother cell of the sexual plant, butnot in the cells that give rise to apomictic embryo sacs, suggesting a role in female reproductive development of sexuals. This paper provides evidences that SERK genes plays a role in the onset and establishment of somatic embryogenesis and in the reproductive development of B. brizantha and suggests a distinct role of BbrizSERK in apomixis initiation.

New apomictic pathway in Myrtaceae inferred from Psidium cattleyanum female gametophyte ontogeny

Adventitious embryony and aposporous apomixis have been described in Myrtaceae. Psidium cattleyanum Sabine is a South American species with commercial potential as a fruit tree in natural areas. Interestingly the species is listed among the 100 world’s worst invasive alien species. Morphological and molecular uniformity of the offspring obtained from seeds suggests an apomictic pathway of reproduction. The aim of this study was to describe the ovule ontogeny and embryology of selected materials of Psidium cattleyanum for two infraspecific entities Psidium cattleyanum f. cattleyanum and Psidium cattleyanum f. lucidum O. Deg. and to identify possible ontogenetic evidence of apomictic embryo sac development. We collected and analyzed anatomical sections of flowers from very early stages of development until fruit formation started. Both taxa showed the typical ontogenetic pattern of Myrtaceae. Ovules are anatropous, bitegmic and crassinucelate and they show a zig-zag micropyle. As the megaspore mother cell enlarges in the nucellus no evidence of meiosis was registered. A megaspore-like cell was observed in the following stages of development. The embryo sac is Polygonum type with persistent antipodals. Immature seeds have nuclear endosperm. Development patterns of embryo sac and non-meiotic origin of the megaspore lead us to reject the adventitious embryony and apospory cited for other taxa in the family and, instead, a possible disploporic origin of the embryo sac is proposed for the first time for Myrtaceae.

Synergids and filiform apparatus in the sexual and apomictic dandelions from section Palustria (Taraxacum, Asteraceae).

An evolutionary trend to reduce “unnecessary costs” associated with the sexual reproduction of their amphimictic ancestors, which may result in greater reproductive success, has been observed among the obligatory apomicts. However, in the case of the female gametophyte, knowledge about this trend in apomicts is not sufficient because most of the ultrastructural studies of the female gametophyte have dealt with amphimictic angiosperms. In this paper, we tested the hypothesis that, in contrast to amphimictic plants, synergids in apomictic embryo sacs do not form a filiform apparatus. We compared the synergid structure in two dandelions from sect. Palustria: the amphimictic diploid Taraxacum tenuifolium and the apomictic tetraploid, male-sterile Taraxacum brandenburgicum. Synergids in both species possessed a filiform apparatus. In T. brandenburgicum, both synergids persisted for a long time without any degeneration, in spite of the presence of an embryo and endosperm. We propose that the persistent synergids in apomicts may play a role in the transport of nutrients to the embryo.

Unusual microtubular cytoskeleton of apomictic embryo sac of Chondrilla juncea L.

The mature apomictic embryo sac of Chondrilla juncea is highly vacuolated and demonstrates a polarization similar to that of the amphimictic gametophyte. The microtubule cytoskeleton of this embryo sac is uncharacteristic and relatively weak. The microtubules are positioned along cell walls and resemble cortical microtubules of somatic cells. They do not form the parallel, brushlike structures observed around the filiform apparatus of synergids in the amphimictic embryo sac. In the apomictic embryo sac, the microtubules of both the egg cell and the central cell develop a cortical-like structure, which is entirely different from the radial arrangement observed around the nuclei in the amphimictic embryo sac.

Co‐segregation of AFLP and RAPD markers to apospory in Guineagrass (Panicum maximum Jacq.)

SynopsisA genetic linkage map of guinea grass (Panicum maximum Jacq.) was generated with nine of the AFLP markers found to be associated with apospory. These apospory‐associated markers were assigned to a linkage group having previous association with microsporogenesis of the aposporous guineagrass cultivar ‘Natsukaze’. An aposporous linkage group was constructed utilizing 38 AFLP markers. Embryo sac analysis revealed that sexual and apomictic embryo sacs occurred at a frequency of 1 : 1, indicating simple inheritance of a single major gene controlling apospory in guineagrass. In addition, utilizing 56 AFLP primer combinations and 41 RAPD primers, 39 linkage groups and 360 simplex marker loci were assigned to the genetic map of the ‘Natsukaze’ cultivar. These markers covered 1703.5 cM of the autotetraploid guineagrass genome (2n = 4x = 32), with an average spacing of 4.7 cM. These tightly linked markers to apospory locus in guineagrass could be a powerful tool for marker‐assisted selection of apospory and map‐based cloning of the apospory gene.

The Mechanisms of Appearance of Aposporous Initial Cell and Apomictic Embryo Sac Formation in Paspalum notatum.

The Mechanisms of Appearance of Aposporous Initial Cell and Apomictic Embryo Sac Formation in Paspalum notatum.

Isolation and Characterization of a Gene Expressed during Early Embryo Sac Development in Apomictic Guinea Grass ( Panicum maximum)

A novel classification method, based on ovary length, for sampling different developmental stages of embryo sac formation in obligate sexual and facultatively apomictic genotypes of guinea grass (Panicum maximum) has been employed to make a cDNA library and to isolate a stage-specific cDNA clone, probably representing a full-length gene. This A2-134 cDNA, designated ASG-1 (Apomixis Specific Gene), was found to be expressed in flower buds of the apomictic but not of the sexual accession. Furthermore, the gene was also not expressed in buds of apomicts until completion of megasporogenesis but could only be found during a phase characterized by the. appearance of aposporous initial cells (AICs) of the embryo sac. The appearance of these cells is strictly limited to apomictic genotypes. Sequence analysis revealed that the A2-134 cDNA (1,177bp) codes for a protein of 305 amino acids with a molecular mass of 34.2 kDa. The amino acid sequence of A2-134 is related to RD22, a seed-specific and drought-induced gene of Arabidopsis thaliana, to USP, an unknown seed protein precursor of Vicia faba, to a polygalaturonase 1 beta chain precursor (Polygl) of Lycopersicon esculentum and to ADR6, an auxin down-regulated gene of Glycine max. Southern blot analysis showed a different hybridization pattern at the genomic level when A2134 cDNA was hybridized with total DNAs isolated from leaves of sexual as well as apomictic plants, indicating that the gene (or related family members) exists in both types. The cDNA library and the ASG-1 gene should be valuable tools toward understanding the molecular mechanism of apomixis, particularly apospory, and toward the transfer of apomixis to important crops. To our knowledge, A2-134 represents the first case of the isolation and characterization of a cDNA related to apomictic embryo sac development.

Cytological Evidence of Seed-forming Embryo Development in Polyembryonic Ovules of Facultatively Apomictic Guineagrass(Panicum maximum Jacq.).

Seven facultatively apomictic accessions of guineagrass (Panicum maximum) were studied cytologically for clarifying the mechanisms of seed-forming embryo development in poly-embryonic ovules. The continuous observations of ovules before and after anthesis under the condition of open-polination, indicate that 1) The first AESIC appeared is located dominantly in micropylar end (Chen and Kozono 1994), and the percentage of mature embryo sacs in micropylar end was higher than that in the other end; 2) The rates of the ovules contained developed embryo sacs in micropylar end at 4-days after anthesis were 33%-91% comparable to 0%-2% of that in the other ends; 3) The embryo of the developed sac in micropylar end, in final, became a seed-forming embryo, and in contrast, the other sacs, were crowded out to chalazal end and degenerated at 10 or more days after anthesis. From the above results, it can be concluded that the AESIC-derived embryo sac in micropylar end, in most of the cases, has temporal dominant in embryo sac mature, and has the positional dominant in fertilization and subsequent development of embryo sac when compared with the sacs in the other end in polyembryonic ovules. We, therefore, suggest that the degrees of sexuality or apomixis can be estimated based on the frequency of sexual and apomictic embryo sacs in the micropylar end by means of embryo sac analysis in present generation, even without progeny test. The other types of embryo sac development were also investigated and discussed.

Cytology and Quantitative Analysis of Aposporous Embryo Sac Development in Guineagrass (Panicum maximum Jacq.).

Seven facultatively apomictic accessions and three obligately sexual accessions of guineagrass (Panicum maximum) were studied cytologically and quantitatively for clarifying the mechanisms of appearance of aposporous embryo sac initial cell (AESIC) and apomictic embryo sac formation by using ovary length as an index. The observations of AESIC appearance and their development indicates that most of ovules in apomictic accessions contain several AESICs, and the number of AESICs increased as the ovary grew before anthesis. That is, several AESICs in the same ovule did not differentiate synchronously, but instead, they seemed following a continuous course and appeared one by one during the period from after megasporegenesis to the first AESIC-derived embryo sac maturity. It was also found that the higher the frequency of apospory was, the greater the number of AESICs was, and the longer the duration of AESIC appearance should be. This result will produce the information on sampling of the materials used for the apomixis gene isolation program. The type of embryo sacs in same ovules of apomictic plants were also distinguished and discussed.

Cytology and reproductive behavior of hybrids between Paspalum urvillei and two hexaploid P. dilatatum biotypes

Sexual, tetraploid (2n = 4x = 40) vaseygrass (Paspalum urvillei Steud.) was crossed with two apomictic, hexaploid (2n = 6x = 60) dallisgrass (P. dilatatum Poir.) biotypes (Uruguayan and Uruguaiana) to determine their relationships and the reproductive behavior of the hybrids. One vaseygrass × Uruguayan and two vaseygrass × Uruguaiana hybrids were studied and all had 2n = 5x = 50 chromosomes. The mean pairing meiotic association for the vaseygrass × Uruguayan hybrid was 10.18 I and 19.91 II, and for the two vaseygrass × Uruguaiana hybrids was 10.28 I, 19.82 II, 0.006 III, and 0.01 IV. These findings indicate that the parents of both hybrids have two homologous genomes. Because vaseygrass and Uruguayan dallisgrass have the genome formulas IIJJ and IIJJXX, respectively, their hybrid has the formula IIJJX and the two homologous genomes are the I and J genomes. Likewise, chromosome pairing in the vaseygrass × Uruguaiana hybrids demonstrates that both species have two similar genomes, I and J. The Uruguaiana biotype usually has 8–10 univalents per cell and these result from the failure of some members of the third genome to pair. Thus the Uruguaiana biotype has different forms of the X genome, and its genome formula may be designated as IIJJXX2. Cytologically all three hybrids were facultative apomicts but their uniform F2 progeny indicates that only the apomictic embryo sacs are functional. Seed fertility ranged from 10.8 to 16.2%.Key words: Interspecific hybrids, genomes, chromosome pairing, species relationships, dallisgrass, vaseygrass.

Cytogenetics of double cross hybrids between Pennisetum americanum — P. purpureum amphiploids and P. americanum x Pennisetum squamulatum interspecific hybrids

Pearl millet, Pennisetum americanum L. Leeke-napiergrass, Pennisetum purpureum Schum. amphiploids (2n=42) were crossed with pearl millet X Pennisetum squamulatum Fresen. interspecific hybrids (2n=41) to study the potential of germplasm transfer from wild Pennisetum species to pearl millet. These two interspecific hybrids were highly cross-compatible and more than two thousand trispecific progenies were produced from 17 double crosses. All doublecross hybrids were perennial and showed a wide range of morphological variations intermediate to both parents in vegetative and inflorescence characteristics. Some crosses resulted in sublethal progenies. Chromosomes paired mainly as bivalents (¯x15.88) or remained as univalents. At metaphase I, trivalents, quadrivalents, an occasional hexavalent and a high frequency of bivalents indicated some homeology among the genomes of the three species. Delayed separation of bivalents, unequal segregation of multivalents, lagging chromosomes, and chromatin bridges were observed at anaphase I. Although approximately 93% of the double-cross hybrids were male-sterile, pollen stainability in male-fertile plants ranged up to 94%. Seed set ranged from 0 to 37 seed per inflorescence in 71 plants under open-pollinated conditions. Apomictic embryo sac development was observed in double-cross progenies when crosses involved a pearl millet x P. squamulatum apomictic hybrid as pollen parent. These new double-cross hybrids may serve as 'bridging hybrids' to transfer genes controlling apomixis and other plant characteristics from the wild Pennisetum species to pearl millet.

In vitro sexual and apomictic embryo sac development in Cenchrus ciliaris

Sexual and aposporous apomictic embryo sac development in three Cenchrus ciliaris genotypes was evaluated in situ and in vitro. An aseptic procedure was developed to culture involucres in which the pistils contained archesporial cells. Development of sexual embryo sacs or differentiation and development of nucellar cells continued, in vitro, through mature embryo sac formation in the three buffelgrass lines. A numerical assessment system was devised to evaluate the stage of embryo sac development in vitro or in situ. Theculture medium contained Murashige and Skoog mineral salts supplemented with 1 × 10−4 M FeEDTA, 5.6 × 10−4 M myo-inositol, and 8.8 × 10−2 M sucrose in 1% agar. The best culture conditions were a 14 h light: 10 h dark photoperiod at a 25:19 °C (light:dark) temperature. Generally, 50% or more of the cultured involucres contained mature embryo sacs in good to excellent condition, and these sacs were indistinguishable from field-grown material. The in vitro culture technique provides a method to study embryo sac development and physiologic regulation of apomixis in Cenchrus ciliaris.

Microsporogenesis, reproductive behavior, and fertility in five Pennisetum species

Microsporogenesis, reproductive behavior, pollen fertility and seed set were studied in Pennisetum basedowii Summerhayes and C. E. Hubbard, 2n = 54; P. macrostachyum (Brough.) Trin., 2n = 54; P. macrourum Trin., 2n = 36; P. polystachion (L.) Schult, 2n = 54; and P. squamulatum Fresen 2n = 54. Meiosis was regular in P. basedowii with primarily bivalent pairing. As many as 54 univalents were observed at metaphase I in P. macrostachyum. A high frequency of univalents at metaphase I in P. macrourum resulted in lagging chromosomes and micronuclei at anaphase I and telophase I, respectively. Pennisetum polystachion and P. squamulatum showed frequent multivalent chromosome associations. Studies of megasporogenesis and embryo sac development in P. basedowii showed sexual reproduction. Pennisetum macrostachyum was highly male sterile with predominantly aposporous apomictic embryo sac development. Pennisetum macrourum, P. polystachion, and P. squamulatum had only aposporous embryo sac development. Seed propagated progenies of these latter three species were uniform and matromorphic, confirming the obligate apomixis nature.

Meiotic and Reproductive Behavior of Facultative Apomictic BC1 Offspring Derived from Pennisetum americanum‐P. orientale Interspecific Hybrids1

This study reports on the chromosome numbers, meiotic behavior, method of reproduction and fertility of BC1 progenies from Penniserum americanum L. Leeke, pearl millet ✕ P. orientale L.C. Rich. interspecific hybrids backcrossed to P. americanum. This information would be useful for future studies on transfer of genes controlling apomixis from the tertiary gene pool to P. americanum. Two facultatively apomictic interspecific hybrids between Pennisetum americanum, (A. chromosomes) and P. orientale (O chromosomes), 2n = 25, were pollinated with P. americanum. Sixteen backcross progenies were obtainewhich were of three cytotypes: 32‐(14 A + 18 O), 23‐ (14 A + 9 O)and 27‐(7 A + 20 O) chromosomes. They resulted from fertilization of unreduced gametes or partially reduced gametes by a 7 A chromosome gamete, or by development or unreduced aposporic embryo sacs, respectively. In 23 chromosome plants, the 14 A chromosomes paired mainly as blvalents or remained as univalents while the 9 O chromosomes appeared as univalents, Intergenomal pairing between P. americanum and P. orientale also were observed and could make segmental exchange possible. In the 27 chromosome progeny, the 20 O chromosomes paired, while the 7 A chromosomes remained as univalents. Meiotic behavior in 32‐chromosome plants was regular with 7 A bivalents plus 9 O bivalents. The backcross progenies were male sterile but partially female fertile and produced a few seeds when pollinated with P. americanum pollen. The 23‐chromosome, BC1 progeny were reconstituted in BC2 progenies of 32‐ chromosome plants ✕ pearl millet. All BC1 progenies had some degree of apomictic embryo sac development and the 23‐chromosome plants showed apomictic development even though the O chromosomes on which the genes controlling apomixis are located were in the simplex condition. Both the 23‐and 32‐chromosome plants showed facultative apomictic development whereas the 27‐chromosome plants showed only obligate apomictic development, Intergenome pairing, chromosome instability, female fertility in 32‐chromosome plants, and the expression of apomixis genes in both the simplex and duplex condition indicates that genes controlling apomixis may be transferred from the tertiary gene pool to pearl millet using various gene transfer and recombination techniques.

Facultative Apomixis in Buffelgrass1

We studied embryo sac morphology, chromosome number, reproductive behavior, and phenotype of buffelgrass (Cenchrus ciliaris L.) line 18‐35 to obtain further evidence regarding the occurrence of facultative apomixis in the species. Line 18‐35 is an F1 llne derived from a cross of obligately sexual TAM‐CRD B‐Is with an apomictic plant from Africa. Within line 18‐35, pistils with single sexual embryo sacs of the Polygonum type occurred at a low frequency in the same inflorescences as pistils with aposporous apomictic embryo sacs of the Oenothera type. Some pistils contained a sexual sac and one or more aposporous sacs. A sample of 2,057 pistils from three field plantings and one greenhouse planting had 85.8% apomictic pistils, 5.8% sexual pistils, and 8.4% mixed pistils. Frequency of sexual pistils differed between sampling dates of one field, indicating that environment may have influenced embryo sac type.In a spaced planting of 1,292 open‐pollinated progeny of 18‐35, 1,252 progeny resembled the maternal parent, and 40 progeny were offtype. Line 18‐35 and 21 of the offtype progeny were tetraploid (2n = 36). Four of these offtype tetraplold progeny produced primarily or exclusively sexual pistils. The 21 offtype tetraplold progeny probably originated by fertilization of reduced eggs. Fifteen offtype progeny were hexaploid and were believed to originate by fertilization of unreduced eggs. We concluded that line 18‐35 is facultatively apomictic.

Effects of temperature and photoperiod on growth and reproductive development in Hyparrhenia hirta

Hyparrhenia hirta occurs naturally in the Mediterranean region and in the winter rainfall areas of South Africa, in association with temperate festucoid grasses. However, the temperature response curves, flowering behaviour, and widespread apomixis found in this species are more typical of other tropical members of the Andropogoneae. This residual tropical adaptation provides an explanation for the sensitivity of H. hirta to low temperature (below 15�C) during seed germination, and especially during the seedling stage, which makes autumn establishment difficult in temperate climates. Also, the low seed yield is attributable to the high proportion (c. 50%) of male florets in the inflorescence, and the breakdown of both sexual and apomictic embryo sacs during development. A possible genetic approach to the improvement of both these characters, utilizing facultative apomixis, is discussed.

A Contribution to the Embryology of <i>Rhynchelytrum repens</i> (Willd) C.E. Hubbard

The present investigation deals with morphological and embryological studies of Rhynchelytrum repens (Willd) C.E. Hubbard. The development of anther walls are found to be Monocotyledonous type. The tapetal cells are substantially large, glandular and uninucleate. The middle layer is ephemeral and their cells are small in size. It is sandwiched between endothecial and tapetal layer. The endothecial cells are large and develop fibrous thickenings. The microspore mother cell undergoes two successive reduction divisions, giving rise to isobilateral microspore tetrad. The tetrad separates and give rise to four pollen grains. Occasionally, the anther show degenerating pollen grains before dehiscence. Formation of Ubisch's bodies has also been observed. The pollen grains shed at three celled stage. The exine is thick while intine is thin. The ovule is anatropous, bitegmic and crassinucellate. The female archesporial cell becomes large with dense cytoplasm. It directly functions as megaspore mother cell and undergoes two meiotic divisions to produce a linear megaspore tetrad. The micropylar three cells degenerate and chalazal one becomes functional. The chalazal functional megaspore undergoes three mitotic divisions without wall formation and produces 8-nucleate embryosac. Such 8-nucleate embryosac organizes into Polygonum type of embryosac. It is interesting to note that some somatic cells of the ovule undergo nuclear divisions and give rise to facultative apomictic embryosacs. Key Words: Eldoret; Microsporangium; Ubisch's bodies; Facultative apomixis; Female gametophyte. DOI: 10.3126/sw.v7i7.3822 Scientific World Vol.7(7) 2009 pp.37-40

REPRODUCTION IN SOME POA SPECIES

The writer has demonstrated through breeding experiments repeated over three years, that Poa arctica R. Br., Poa alpigena Fr. Lindm., Poa alpina L. from Greenland as well as Poa alpina L. from Georgian Bay, Ontario, and a race of Poa pratensis L. from Gaspé, Quebec, are all apomictic (parthenogenic) and pseudogamous. In pseudogamy the pollen tube enters the stigma and activates the apomictic embryo sac to development, but it does not as a rule enter the ovarian tissue to achieve fertilization of the ovule. Pollen from any one species germinated readily and with a high percentage on the stigmas of any one of the others. Pollen sterility did not exceed 2%.