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.     

Structure and function of the microtubular cytoskeleton during megasporogenesis and embryo sac development in Gasteria verrucosa (Mill.) H. Duval

Summary Using immunocytochemical techniques, tubulin distribution in various stages of meiosis and embryo sac development was studied. In the archespore cell some microtubules appeared to be randomly oriented. During zygotene and pachytene, when the cell volume increases, a large number of microtubules in dispersed configurations and bundles were observed. During this stage the nucellar cells divide, and their parallel cortical microtubules play an important role in preparing the direction of cell enlargement. The protoderm cells show anticlinal-directed cortical microtubules. It can be concluded that the enlargement of the meiocyte during these early meiotic stages is influenced both by its own cytoskeleton and by growth of the nucellus. Thereafter, the microtubules function directly in meiosis and disappear for the greater part until the two-nucleate coenocyte is formed. In a four-nucleate coenocyte microtubules reappear around the nucleus; in a young synergid, randomly oriented microtubules are involved in cell shaping during the formation of the filiform apparatus; in the synergids of the mature embryo sac, many parallel arrays of microtubules are present. Microtubules are less abundant in other cells. It is concluded that the cytomorphogenesis of the developing coenocyte and embryo sac are due to cell growth of the nucellar cells together with vacuolation of the coenocyte.     

Apomixis in plants: structural and functional aspects of diplospory inPoa nemoralis andP. palustris

Summary Data on structural and functional aspects of mitotic diplospory and later stages of apomictic seed formation are reported forPoa palustris andP. nemoralis. In this study, the plant material of two Russian populations ofP. nemoralis andP. palustris were used for transmission electron microscope observations. Seed formation was investigated by phase contrast microscopy in two populations ofP. nemoralis collected in The Netherlands. The processes of transformation of the megasporocytes to the megaspore mother cells of diplosporous embryo sacs, and thereafter to one- and two-nucleate diplosporous embryo sacs (Antennaria type) were characterized by an increase of cell size, structural and functional reorganization of the nucleus, nucleolus, and cytoplasm, and cell isolation as a result of thickening of the cell wall. These were accompanied by an increase in the cell metabolic activity inferred from visual evidence of the activation of nucleus, nucleolus, endoplasmic reticulum, dictyosomes, mitochondria, and from the appearance of a dense population of ribosomes and polysomes. The diplosporous embryo sac of the Antennaria type was characteristic for bothP. nemoralis andP. palustris. No signs of the presence of synaptonemal complexes were observed during the process of diplosporous-embryo-sac megaspore mother cell differentiation and division. About 90–95% of the diploid egg cells of diplosporous embryo sacs were able to produce apomictic embryos. These embryos developed before anthesis. However, many of them degenerated at the globular stage because of lack of endosperm. The ultrastructural events occurring during the process of diplospory of apomictic species, and meiosis and megagametogenesis of sexually reproduced plants are discussed.Abbreviations DMC megaspore mother cell of diplosporous embryo sac - TEM transmission electron microscopy - ER endoplasmic reticulum     

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.     

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     

Activity and localisation of sucrose synthase and invertase in ovules of sexual and apomicticBrachiaria decumbens

Summary Brachiaria decumbens has sexual and apomictic reproduction. Apomixis is facultative and of the aposporic type. In early stages of ovule development, differences in the pattern of callose deposition between sexual and apomictic plants were observed which points to possible differences in carbohydrate metabolism. Therefore, a comparative study on carbohydrate metabolism between a sexual diploid ecotype and an apomictic tetraploidB. decumbens was made. A histochemical determination of two enzymes responsible for sucrose degradation, sucrose synthase and invertase, was performed for all stages of ovule development. In addition, the concentrations of sucrose, glucose, and fructose were measured for each stage of ovule development, both for sexual and apomictic plants. The enzymes were localised by immunohistochemistry with heterologous antibodies. A distinct difference between sexual and apomictic plants was observed in the localisation of sucrose synthase activity as well as in the amount of activity, especially in the early stages of ovular development. Invertase activity localisation was comparable between ovules of the sexual and apomictic plants, but its activity is clearly higher in ovules of sexual plants. The localisation of the enzymes coincided with the place of activity. For both sexual and apomictic plants the amount of sucrose in the ovaries increased with the stage of ovule development. Differences in the amount of sucrose between sexual and apomictic plants in ovaries with ovules in comparable stages of development were detected. A delay in the onset of carbohydrate metabolism during early stages of ovule development characterises the apomictic plant.Abbreviations MMC megaspore mother cell - MC meiocyte - MS megaspore - AI apospore initial - CO coenocyte - MES mature embryo sac - SuSy sucrose synthase - INV invertase - BMM buthylmethyl methacrylate - DTT dithiothreitol - DAPI 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline     

高粱SSA-1无融合生殖胚胎学研究

经常规石蜡切片法,在光学水平观察了高粱（Ｓｏｒｇｈｕｍ ｂｉｃｏｌｏｒ （Ｌ．） Ｍｏｅｎｃｈ） ＳＳＡ－１ 无融合生殖的胚胎发生。高粱ＳＳＡ－１ 的无融合生殖为无孢子生殖和二倍体孢子生殖两种类型。两种生殖类型的单核胚囊经３ 次有丝分裂形成７ 细胞（８ 核）的成熟胚囊,由卵细胞、２ 个助细胞、２ 个极核和３ 个反足细胞组成。反足细胞迅速分裂增殖,形成由２０—３０ 个细胞组成的细胞团。此外,还具有一定频率的无孢子生殖多孢原和多胚囊现象。在未授粉情况下,卵细胞自发分裂形成典型的禾本科类型单子叶胚。经切片统计表明,ＳＳＡ－１ 的无融合生殖频率为４２％ ,证明该系为一兼性无融合生殖系。文中还讨论了ＳＳＡ－１ 无融合生殖过程的特点。     

EMBRYO SAC LACKING ANTIPODAL CELLS IN ARABIDOPSIS THALIANA (BRASSICACEAE)

Ultrastructure of the embryo sac lacking antipodals in prefertilization stages in Arabidopsis thaliana has been examined 2 hr before and 5 hr after manual cross pollination. The cytoplasm of both synergids before fertilization is rich in ribosomes, mitochondria, and rough endoplasmic reticulum, and also contains several microbodies and spherosomes. The filiform apparatus includes electron-dense material and a fibrous part. Many cortical microtubules appear in the filiform apparatus area. One of the two synergids degenerates before fertilization. The synergids, the egg cell, and central cell have a rich cytoskeleton of microtubules; only the synergids appear to contain microfilaments. At the chalazal end, the antipodals are initially present but degenerate by the time of pollination in most embryo sacs in the starchless line studied. The embryo sac is completely surrounded by a wall containing an electron-dense layer, separating it from the nucellus, including the chalazal end. When the antipodals have degenerated, the electron-dense layer disappears at the chalazal end only, and the wall between the central cell and the nucellus is homogeneous. Between the central cell and nucellar cells no plasmodesmata are found. The membranes of both antipodal cells at the chalazal end of the embryo sac appear sinuous, like those of transfer cells. The central cell has plastids preferentially distributed around the nucleus, but the other organelles are randomly distributed. The central cell in the embryo sac and the adjacent chalazal nucellar cells show a transfer-cell function in the embryo sac after the antipodals degenerate.     

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     

Embryological Study on Apomixis in a Sorghum Line SSA-1

The Developmental process of apomictic embryo sac and embryo in a sorghum (Sorghum bicolor (L.) Moench. ) line SSA-1 was observed under light microscope, using the method of conventional paraffin sectioning. The result showed that the apomictic development conforms apospory and diplospory. The uninucleate embryo sac underwent mitotic divisions for three times to form a seven-celled or eight-nuclei mature embryo sac including an egg, two synergids, two polar nuclei and three antipodals. The antipodals divided and multiplicated to form an antipodal mass. Moreover, aposporous multiarchesporial cells and multiple embryo sacs were infrequently observed. Without pollination, the egg divided autonomously to form a typical graminaceous mature embryo. The authors counted the apomictic sections in the whole sections and the result showed that the frequency of apomixis was 42%, indicating the facultative apomictic property in the line SSA-1. The characteristics of apomictic process in the line SSA-1 is also discussed.     

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     

Discovery of highly apomictic and highly amphimictic dihaploids in Allium tuberosum

 To discover highly apomictic and amphimictic Allium tuberosum diploids, we evaluated the degree of apomixis in three dihaploids (2n=16, 2x), KaD2, TeD1 and GMD1, derived from highly apomictic tetraploids. The degree of apomixis, calculated as the percentage of diploid seedlings in the progeny obtained after cross-pollination with tetraploid cultivars, was 96% in KaD2, 7% in TeD1 and 39% in GMD1. In addition to this general index of apomictic nature, two analytical indices were evaluated in KaD2 and TeD1. The degree of diplospory, calculated as the percentage of endoreduplicated embryo-sac mother cells, was 96% in KaD2 and 2% in TeD1. The degree of parthenogenesis, calculated as the percentage of ovules with the egg cell developing parthenogenetically, was 98% in KaD2 and 10% in TeD1. Among angiosperms with gametophytic apomixis, KaD2 is the first diploid apomict whose reproductive mode has been fully described by these three quantitative indices of apomictic nature. And TeD1 is the first highly amphimictic plant found in the A. tuberosum complex. Although TeD1 is poorly fertile, the present results encourage further screening trials for highly fertile, highly amphimictic dihaploids, which may be effective counterparts to KaD2 in diploid-level cross experiments to genetically analyze apomixis in A. tuberosum. Received: 4 December 1995 / Revision accepted: 8 May 1996     

多花地宝兰胚囊和胚发育的细胞学研究

为探讨多花地宝兰(Geodorum recurvum)胚胎发育的系统分类学意义,采用石蜡制片法对多花地宝兰胚囊和胚的发育进行解剖学观察。结果表明,在开花前,多花地宝兰胚珠原基发育缓慢,开花授粉后胚珠原基快速发育成"树状二杈分枝结构",随后在"分枝结构"末端形成孢原细胞,开始胚囊发育。多花地宝兰的胚囊发育属于单孢蓼型胚囊,胚珠具有双层珠被。孢原细胞形成后,经过细胞膨大延长发育形成胚囊母细胞,胚囊母细胞经过减数分裂形成线性四分体,在珠孔端形成1个功能大孢子,功能大孢子经过3次有丝分裂形成8核胚囊。多花地宝兰的胚发育具有藜型和紫苑型两种方式。双受精完成后,多花地宝兰合子进行一次橫裂后形成基细胞和顶细胞;基细胞经过多次分裂形成细胞团,细胞团中的细胞向不同方向膨大延长形成多个胚柄细胞;顶细胞有两种分裂方式,一种是横裂形成藜型胚,一种是纵裂形成紫苑型胚。因此,推测多花地宝兰在兰科植物系统分类学上属于较为原始种。     

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.     

Embryo sac development in Arabidopsis thaliana II. The cytoskeleton during megagametogenesis

The microtubular and actin cytoskeletons have been investigated during megagametogenesis in Arabidopsis thaliana using immunofluorescence labelling of isolated coenocytic and mature embryo sacs. We found both actin and microtubules (MTs) to occur in abundance throughout megagametogenesis and in all constituent cells of the mature embryo sac. During many stages, the patterns of distribution of these cytoskeletal elements are congruent and may prove to be co-aligned. Many changes in the arrays of MTs and microfilaments take place and indicate varying roles of the cytoskeleton in the different stages and cell types of megagametogenesis. Two major populations of MTs recur throughout embryo sac formation: (1) Elaborate nuclear-based networks are found during the two-nucleate and four-nucleate developmental stages as well as in the egg cell. These arrays may function in positioning the nuclei. (2) Cytoplasmic MTs in longitudinal orientation in the two-nucleate embryo sac, synergids and part of the egg cell, or in a reticulate pattern in the four-nucleate embryo sac, egg and central cell probably participate in organization of the cytoplasm. Synergid MTs converge at the filiform apparatus. Preprophase bands of MTs are absent throughout megagametogenesis but phragmoplast arrays occur during cellularization of the embryo sac. Well developed arrays of cortical MTs are restricted to the antipodal cells. A large concentration of MTs in the part of the egg cell adjacent to the synergids is well placed for being involved with sperm cell movement within the degenerative synergid. On the basis of the morphology of the cytoskeleton, we concur with views that the shape of megagametophyte is largely determined by the surrounding tissues, including the integumentary tapetum.     

Phylogenetic relationships of coexisting Heterocypris (Crustacea, Ostracoda) lineages with different reproductive modes from Lampedusa Island (Italy)

Coexisting bisexual and unisexual populations of individuals belonging to the genus Heterocypris are found in ephemeral freshwater ponds on the island of Lampedusa (Pelagie Islands, Italy). Different reproductive modes were associated with a peculiar morphological trait: a lamella hyalina on the posterior margin of the left valve was observed in amphimictic females, a feature missing in apomictic females. In order to clarify the phylogenetic relationships among taxa with different morphological traits and reproductive modes, we used four polymorphic enzyme loci (GPI, ICD2, MPI and PGM) and mitochondrial DNA 16S ribosomal sequences. We identified three main evolutionary units that showed a combination of morphological and reproductive characteristics: (1) amphimictic females of H. barbara with a lamella hyalina, according to the typical feature of the species, and apomictic females of H. barbara without lamella that are sympatric in one temporary pond; (2) apomictic females of H. incongruens without a lamella, as typical of the species; (3) apomictic females without a lamella, living in sympatry with H. barbara, but characterised by a high genetic diversity from both H. incongruens and H. barbara. We discuss the possible origin of apomictic lineages as a result of independent transition episodes to apomixis from different sexual ancestors. Time of divergence reflected the genetic differentiation within and among multiple ancestors and different possible routes to parthenogenesis.     

鹤顶兰胚囊发育过程中微管变化的共焦显微镜观察

光镜的观察确定了鹤顶兰（Ｐｈａｉｕｓ ｔａｎｋｅｒｖｉｌｌｉａｅ （Ａｉｔｏｎ） Ｂｌ．）胚囊发育属单孢子蓼型。应用免疫荧光标记技术及共焦镜观察了胚囊发育过程中微管分布的变化。当孢原细胞初形成时,细胞内的微管呈网状分布。之后,孢原细胞体积增大发育为大孢子母细胞。大孢子母细胞延长,进入减数分裂Ⅰ。微管由分裂前的网状分布变为辐射状排列。二分体的两个细胞内的微管分布一样,呈辐射状。四分体的近珠孔端的３ 个大孢子解体,细胞内的微管消失。靠合点端的功能大孢子内有许多微管呈网状分布。当功能大孢子进入第一次有丝分裂时,细胞内的微管由网状变为辐射状,从核膜伸展至周质。再经两次有丝分裂形成八核胚囊。在核分裂之前微管一般是呈网状分布并紧包围着核。在分裂期间二核和四核胚囊都呈极性现象,微管系统也呈极性分布。微管在八核胚囊内的分布变化情形特别复杂。首先,八核分别作不同程度的移动,其中两个核移向胚囊中央,珠孔端和合点端的３ 个核分别互相靠拢,形成３ 个区,即中央区、反足区和卵器区。胚囊未形成区时,８ 个核都被网状分布的微管包围着。当胚囊明显分成区时,反足区内的微管仍作网状分布。中央区的微管分布则趋疏松,形成篮形结构,包围着液泡和两个极核。在     

Microtubular configurations during the cellularization of coenocytic endosperm in Ranunculus sceleratus L.

Summary Endosperm cellularization in Ranunculus sceleratus was studied in terms of the initiation of cell-wall formation in the coenocytic endosperm. The first endosperm cell walls were in an anticlinal position relative to the cell wall of the embryo sac and originated from the cell plates and not from wall ingrowths from the embryo-sac wall itself. Alveolar endosperm was formed 3 days after pollination. Microtubules were associated with the freely growing wall ends of the anticlinal walls and were observed in various orientations that generally ranged from angles of 45 ° to 90 ° to the plane of the wall. They were absent in the regions where vesicles had already fused. These microtubules may function in maintaining the growth and the direction of growth of the anticlinal wall until cellularization is completed. At the site where three neighbouring alveoli share their freely growing wall ends, remarkable configurations of microtubules were observed: in each alveolus, microtubules ran predominantly parallel to the bisector of the angle formed by the common walls. These microtubules may form a physically stable framework and maintain the direction of growth of the wall edges. It is concluded that the growing edge of the anticlinal endosperm wall and its associated microtubules are a special continuum of the original phragmoplast that gave rise to the anticlinal wall.     

Confocal Microscopic Observations on Microtubular Cytoskeleton Changes During Megasporogenesis and Megagametogenesis in Phaius tankervilliae (Aiton) Bl.

In nun orchid (Phaius tankervilliae (Alton) B1. ) embryo sac development follows the monosporic pattern. Changes in the pattern of organization of the microtubular cytoskeleton during megasporogenesis and megagametogenesis in this orchid were studied using the immunofluorescence technique and eonfocal microscopy. At the initial stage of development the microtubules in the arehesporium were randomly oriented into a network. Later the archesporial cell elongated to form the megasporocyte. The cytoskeleton in the elongated megasporoeyte was radially organized in which microtubules extending from the nuclear envelope to the peripheral region of the cell. The megasporoeyte then underwent meiosis 1 to form a dyad. The dyad cell at the chalazal end was larger than the cell at the micropylar end. Microtubules in the dyad cell were radially oriented. The dyad underwent meiosis to give rise to a linear array of four megaspores (i. e. tetrad formation). The chalazal-far most megaspore survived and became the functional megaspore, which contained a set of randomly oriented microtubules. The microtubules in the other 3 megaspore disappeared as the cells degenerated. The functional megaspore then underwent mitotic division giveing rise to a 2 nucleate embryo sac. The nuclei of the 2-nucleate embryo sac were separated by a set of longitudinally oriented microtubules which ran parallel to the long axis of the embryo sac. Each nucleus in the embryo sac was surrounded by a set of perinuelear microtubules. The gnucleate embryo sac again underwent mitotic division to form a 4-nucleate embryo sac. The division of the two nuclei was synchronous. But the orientation of the division plan of the two spindles was different (i. e. the spindle microtubules at the chalazal end ran parallel with the long axis of the embryo sac and those at the mieropylar end ran at right angle to the axis of the embryo sac). The 4 nuclei of the 4-nucleate embryo sac were all tightly surrounded by randomly oriented microtubules. Later the paired nuclei at the micropylr end and at the chalazal end as well underwent mitotic division in seguence. At this time when the embryo sac had reached the 8-nucleate embryo sac stage. The pattern of organization of the microtubules was very complex. Initially the nuclei were surrounded by a set of randomly oriented microtubules, but after the two polar nuclei had moved to the central region of the embryo sac, three different organizational zones of microtubules appeared, viz: a randomly oriented set of microtubules surrounding each nucleus in the chalazal zone: a set (in the form of a basket) of cortical microtubules which surrounded the vacuoles and the two polar nuclei in the central zone and a loosely knitted network of microtubules surrounding the nucleus that later became the egg cell nucleus in the micropylar zone. The two nuclei that would become the nuclei of the synergids were surrounded by a set of more densely packed mierotubules. Towards far the most micropylar end some microtubules formed thick bundles. The site of appearance of these thick bundles coincided with the site of development of the filiform apparatus. The pattern of microtubule organization after cellularization (i. e. at the beginning of embryo sac maturation) did not change much. The author's results indicated that various patterns of microtubule organization observed in the developing embryo sac of nun orchid reflected the complexity and dynamism of the embryo sac.     

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.