Calcium changes during megasporogenesis and megaspore degeneration in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.)

Potassium pyroantimonate was used to localize loosely-bound calcium in young ovules of lettuce (Lactuca sativa L.) during megasporogenesis to investigate the relationship between ionically available calcium and megaspore degeneration. At the megasporocyte (megaspore mother cell) stage, few calcium precipitates were located in the ovule. Following meiosis in the megasporocyte, a linear tetrad of four megaspores is formed, with three of the four megaspores degenerating from the micropylar end inward. Only the chalazal-most megaspore continues to develop, becoming the functional megaspore. A decrease in amount of calcium precipitates in the megaspore, particularly in the nucleus, precedes the breakdown of the micropylar megaspores, which subsequently undergo structural disintegration and loss of recognizable cellular features. A partial recovery of calcium precipitates occurs during later degeneration. The functional megaspore retains a consistently higher concentration of calcium precipitates during development, which is retained in the developing embryo sac. This, to our knowledge, is the first report related to calcium dynamics during megaspore degeneration, and may facilitate future research aimed at elucidating the mechanisms of megasporogenesis.     

A NOMARSKI INTERFERENCE STUDY OF MEGASPOROGENESIS AND MEGAGAMETOGENESIS IN SMELOWSKIA CALYCINA (CRUCIFERAE)

Ovule development, megasporogenesis and megagametogenesis in an aneuploid population of the arctic-alpine crucifer, Smelowskia calycina, were examined to test for the possibility of apomictic seed production. Whole mounts of ovules cleared in Herr's “4½” clearing fluid were examined using Nomarski differential interference microscopy. The campylotropous ovule was bitegmic, with a micropyle formed by both integuments. The single archesporial cell of a crassinucellar nucellus functioned directly as a megasporocyte, dividing to form a linear tetrad of megaspores. The chalazal megaspore divided to form an 8-nucleate, 7-celled gametophyte of the Polygonum type, having hooked synergids with a well-developed filiform apparatus and polar nuclei that fused prior to fertilization. In the absence of any anomalous development indicative of agamospermy, seed production was assumed to be sexual.     

五唇兰未受精胚珠离体培养及雌配子体发育的研究

五唇兰(Doritispulcherrima Lindl.)的胚珠属于倒生型,具薄珠心,两层珠被.胚囊发育类型为双孢子葱型,授粉后约45 d形成七细胞八核的成熟胚囊.五唇兰未受精胚珠在离体培养初期对外源激素的依赖性很小,在没有外源激素的培养基上,大孢子母细胞也能经过减数分裂发育为二核胚囊.在培养后期,外源激素对胚囊发育的影响很大.在培养基无外源激素或仅含生长素或细胞分裂素时,雌配子体的发生过程不能顺利完成;在改良VW培养基上添加0.5 mg/L BA和0.1 mg/LNAA时,形成成熟胚囊.     

濒危植物香木莲的胚胎学研究

对香木莲（Manglietia aromatica）的大、小孢子发生以及雌、雄配子体发育过程进行了研究,并结合已有的资料归纳出木链属的胚胎学特征。香木链花药四囊型,腺质绒毡层有1-2层细胞,小孢子形成时胞质分裂方式为修饰性同时型,小孢子四分体排列方式为交叉型,有时为左右对型,成熟花粉粒为二细胞型。胚珠倒生,厚珠心,双珠被,大孢子四分体呈直线排列,功能大孢子位于合点端。胚囊发育属于蓼型。木莲属的胚胎学特征与木兰属、含笑属、鹅掌揪属等植物的胚胎学特征基本相同,都属于较原始的被子植物胚胎学类型。     

Megasporogenesis,Microsporogenesis and Development of Gametophytes in Eleutherococcus senticosus (Araliaceae)

This paper describes megasporogenesis, microsporogenesis, and development of female and male gametophytes in Eleutherococcus senticosus. The main results are as follows: Flowers of E. senticosus are epigynous, pentamerous. Anthers are 4 -microsporangiate. An ovary has 5 loculi. Each ovary loculus has 2 ovules: the upper ovule and the lower ovule. The upper one is orthotropous and degenerates after the formation of archesporial cell, while the lower one is anatropous, unitegmic and crassinucellar, and able to continue developing. In male plants, microsporogenesis and development of male gametophytes took place in regular way, but a series of abnormal phenomena were found in megasporogenesis and development of female gametophytes. The microspore mother cells gave rise to tetrahedral tetrads by meiosis. Cytokinesis was of the simultaneous type. The mature pollen was 3-celled and shed singly. The anther wall formation belonged to the dicotyledonous type. At the stage of microspore mother cell, the anther wall consisted of four layers, i.e. epidermis, endothecium, middle layer, and tapetum. The tapetum was of glandular type and its most cells were binucleate. When microspores were at the uninucleate stage, the tapetum began to degenerate in situ. When microspores developed into 3-celled pollen grains, the tapetum had fully degenerates. In the lower ovule of male flower, the megaspore mother cell gave rise to a linear or “T” -shaped tetrad. In some cases, a new archesporial cell over the tetrad or two tetrads parallel or in a series were observed. Furthermore, the position of functional megaspore was variable; any one or two megaspores might be functional, or one megaspore gave rise to a uninucleate embryo sac, but two other megaspores also had a potentiality of developing into the embryo sac. In generally, on the day when flowers opened, female gametophytes contained only 4 cells: a central cell, two irregular synergids and one unusual egg cell. In female plants, microspore mother cells and secondary sporogenous cells were observed. But at the stage of secondary sporogenous cell, the newly differentiated tapetum took the appearance of degeneration. Later, during the whole stage of meiosis, the trace of degenerative tapetum could be seen. At last, the microsporangium degenerated and no tetrad formed. On the blossom day, all anthers shriveled without pollen grains. In female flowers, megasporogenesis and development of female gametophytes were normal: the tetrad of megaspores was linear or “T”-shaped; the chalazal megaspore was usually functional; the development of embryo sac was of the Polygonum type. On the blossom day, most embryo sacs consisted of 7 cells with 8 nuclei or 7 cells with 7 nuclei; but the egg apparatus was not fully developed. In hermaphroditic plants, microsporogenesis was normal but the development of male gametophytes was partially abnormal. When the hermaphroditic flowers blossomed, there were more or less empty pollen grains in the microsporangium and these pollen grains were quite different in size. The development of most gynoecia was normal but numerous abnormal embryo sacs could be seen. On the blossom day, female gametophytes were mainly 7-celled with 8-nuclei or with 7-nuclei or 4-celled with antipodal cells degenerated; the egg apparatus wasnot fully developed either.     

羊草大、小孢子发生与雌、雄配子体发育的观察

利用常规石蜡制片技术研究了羊草大、小孢子发生及雌、雄配子体发育过程。主要结果是：(1)花药壁由4层结构组成,最外层为表皮,其次为药室内壁,1层中层,最内层绒毡层为分泌型;(2)小孢子母细胞减数分裂过程中的胞质分裂为连续型,四分孢子为左右对称型;(3)成熟花粉粒为3细胞型,具单萌发孔;(4)羊草为单子房、单胚珠,双珠被、薄珠心、倒生型胚珠,大孢子母细胞减数分裂形成线型或T型排列的4个大孢子;合点端大孢子具功能;(5)具有双孢原,双大孢子母细胞、双大孢子四分体和双胚囊的情况;(6)胚囊发育为蓼型,反足细胞经无丝分裂形成4～6个细胞的反足细胞群;(7)同一朵花中,前期雄蕊的发育早于雌蕊的发育,后期当花粉成熟时,雌配子体也达到成熟,雌雄蕊发育趋于同步。     

OVULE ONTOGENY,MEGASPOROGENESIS, AND EARLY GAMETOGENESIS IN TRIFOLIUM REPENS (PAPILIONACEAE)

To aid in understanding of the early events in seed development, surface topography observations with the scanning electron microscope can be coupled with new methods of clearing tissues for light microscopy study. These techniques reveal that two to four ovules begin development along the placental ridge as conduplication of the carpel proceeds in Trifolium repens L. A multicellular archesporium may develop giving rise to several sporogenous cells and ultimately to more than one megasporocyte. However, meiosis is completed in only one megasporocyte to give rise to a single linear tetrad of megaspores. The chalazal megaspore functions in megagametogenesis. Megasporogenesis and megagametogenesis progress as ovule ontogeny proceeds. The outer integument develops more rapidly than the inner and contributes to the final form of the campylotropous ovule. The most dramatic change in ovule form occurs as the tetrad develops and the functional spore enlarges and divides mitotically to produce the two-nucleate megagametophyte. It can be demonstrated that this early gametophyte develops faster than it is allowed to expand in the nucellar mass. This may in part explain why there is gametophyte failure and reduced seed set in clovers.     

埃及白睡莲的胚胎学研究

对埃及白睡莲的大、小孢子的发生,雌、雄配子体的发育,以及胚和胚乳的发育进行了观察研究.结果表明埃及白睡莲的花药壁由5层细胞组成,绒毡层细胞具双核,属于分泌型.小孢子母细胞减数分裂时,胞质分裂属于同时型,小孢子四分体呈四面体型.成熟花粉为三细胞类型,花粉粒表面具有环沟.胚珠为倒生型、双珠被、厚珠心,珠孔仅由内珠被形成.大胞子母细胞减数分裂形成三分体,合点端2个细胞退化,珠孔端1个细胞发育为功能性大胞子.成熟雌配子体由4细胞组成,即2助细胞,1卵细胞和1中央细胞.合子的第一次分裂是横向的,形成的基细胞不再分裂,体积增大后成为一个大的胚柄细胞.而顶细胞进行一系列分裂形成胚.在此基础上,还比较了睡莲目不同属的胚胎学特征.     

Localisation pattern of homogalacturonan and arabinogalactan proteins in developing ovules of the gymnosperm plant Larix decidua Mill

We have identified and characterised the temporal and spatial distribution of the homogalacturonan (HG) and arabinogalactan proteins (AGP) epitopes that are recognised by the antibodies JIM5, JIM7, LM2, JIM4, JIM8 and JIM13 during ovule differentiation in Larix decidua Mill. The results obtained clearly show differences in the pattern of localisation of specific HG epitopes between generative and somatic cells of the ovule. Immunocytochemical studies revealed that the presence of low-esterified HG is characteristic only of the wall of megasporocyte and megaspores. In maturing female gametophytes, highly esterified HG was the main form present, and the central vacuole of free nuclear gametophytes was particularly rich in this category of HG. This pool will probably be used in cell wall building during cellularisation. The selective labelling obtained with AGP antibodies indicates that some AGPs can be used as markers for gametophytic and sporophytic cells differentiation. Our results demonstrated that the AGPs recognised by JIM4 may constitute molecules determining changes in ovule cell development programs. Just after the end of meiosis, the signal detected with JIM4 labelling appeared only in functional and degenerating megaspores. This suggests that the antigens bound by JIM4 are involved in the initiation of female gametogenesis in L. decidua. Moreover, the analysis of AGPs distribution showed that differentiation of the nucellus cells occurs in the very young ovule stage before megasporogenesis. Throughout the period of ovule development, the pattern of localisation of the studied AGPs was different both in tapetum cells surrounding the gametophyte and in nucellus cells. Changes in the distribution of AGPs were also observed in the nucellus of the mature ovule, and they could represent an indicator of tissue arrangement to interact with the growing pollen tube. The possible role of AGPs in fertilisation is also discussed.     

Microtubular configurations during meiosis and megasporogenesis in Gasteria verrucosa and Chamaenerion angustifolium

Summary In Gasteria and Chamaenerion, microtubular configurations were visualized immunocytochemically during meiosis and megasporogenesis in order to study their relationship to cell development, meiotic divisions and selection of the functional megaspore. In Chamaenerion, the intensity of the fluorescence found in megaspores was weaker than that found in Gasteria. Both plants exhibited concentrations of microtubules around the meiocyte nuclei during pachytene-diplotene. Preprophase bands were not observed. In Chamaenerion, cytoplasmic microtubules radiating from meiocyte nuclei were found at late prophase, the dyad stage and in the functional megaspore; in Gasteria, they were observed only at the dyad stage and in the functional megaspore. During the second meiotic division of Gasteria, dividing cells and their nuclei exhibited differences in volumes. Also, the two microtubular spindles of the dyad cells had different widths. Fluorescence indicating the presence of the cytoskeleton diminished during maturation of the large functional megaspores in both plants, whereas in the three degenerating smaller megaspores, fluorescence intensity persisted. Our conclusion is that only an indirect relationship exists between the organization of the microtubular cytoskeleton and selection of the functional megaspore.     

The microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae

This study examines the microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae . The subepidermal cell located at the terminal end of the nucellar filament differentiates first into an archesporial cell and then enlarges to become the megasporocyte. The megasporocyte undergoes the first meiotic division, giving rise to two dyad cells of unequal size. Immunostaining reveals that microtubules become more abundant as the megasporocyte increases in size. Microtubules congregate around the nucleus forming a distinct perinuclear array and many microtubules radiate directly from the nuclear envelope. In the megasporocyte, prominent microtubules are readily detected at the chalazal end of the cell cytoplasm. After meiosis I, the chalazal dyad cell expands in size at the expense of the micropylar dyad cell. At this stage, new microtubule organizing centres can be found at the corners of the cells. The appearance of these structures is stage-specific and they are not found at any other stages of megasporogenesis. The functional dyad cell undergoes the second meiotic division, resulting in the formation of two megaspores of unequal size. The chalazal megaspore enlarges and eventually gives rise to the embryo sac. As the functional megaspore expands, the microtubules again form a distinct perinuclear array with many microtubules radiating from the nuclear envelope. A defined cortical array of microtubules has not been found in P. tankervilliae during the course of megasporogenesis.     

Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.     

莴苣大孢子发生过程中钙的分布动态

莴苣胚囊发育为蓼型,减数分裂形成的4个大孢子中只有合点端的一个大孢子可继续发育,其余3个大孢子从珠孔端依次退化.大孢子母细胞中的钙沉淀颗粒很少,减数分裂后的四分体中的钙沉淀颗粒稍有增加.以后,4个大孢子中的钙沉淀颗粒在数量上有明显差异:即将退化的大孢子中钙明显减少,而未退化大孢子细胞质中则保持有较多的细小钙沉淀颗粒.大孢子的退化是一种细胞程序死亡现象,细胞中的钙浓度降低时可能启动了大孢子细胞的程序性死亡过程,而细胞中的钙浓度高时则保持大孢子细胞的继续发育.文章首次揭示了大孢子发生过程中钙的分布特征.     

珍稀濒危植物巴东木莲胚胎学研究

对巴东木莲(Manglietia patungensis Hu)的花发育以及胚胎发育过程进行了系统研究。巴东木莲花顶生,花器官头年年底开始分化到第二年3月分化出花被、雌雄蕊群直至6月发育成熟。雌蕊成熟时胚珠倒生,双珠被,厚珠心,大孢子四分体线形排列,合点端发育成功能大孢子,珠孔端的3个退化,大孢子为单孢子发生型,胚囊发育方式属蓼型;雄蕊花药外侧壁玫瑰红色,内侧有4个白色花粉囊,绒毡层有1层多核细胞,小孢子四分体排列方式多为左右对称形和交叉形,四面体形,偶为T字形和线形,成熟花粉粒为二细胞型。在巴东木莲花发育和大、小孢子发生以及雌、雄配子体形成过程中未见异常现象,因此笔者认为该物种的花器官发育以及雌、雄配子体发育并不构成导致该物种濒危的因素。     

Ultrastructure and development of the megagametophyte in Eleusine tristachya (Lam.) Lam. (Poaceae)

Eleusine tristachya (Lam.) Lam. is native from subtropical South American climates. Widespread in Argentina and Uruguay, it is frequently found in landscape prairies of the province of Buenos Aires. Megasporogenesis and megagametogenesis in this species were studied using light and transmission microscopy. The ovule is hemitropous, bitegmic and tenuinucellate. The megaspore mother cell enlarges and undergoes meiosis division resulting in a T-shaped tetrad of megaspores. The three micropylar megaspores degenerate, and the chalazal one develops into the Polygonum-type megagametophyte. The synergid cells have the cytoplasm very electron dense because it has got a rich complement of organelles. The synergid wall is strongly thickened at the micropylar pole, developing the filiform apparatus. At maturity, the antipodals originate a wall with large projections into the cytoplasm, acquiring transfer cells characteristics. The antipodals cytoplasm, enriched with organelles shows a high metabolic activity, and it is suggested that these cells perform as an efficient system for metabolites transport.     

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:     

Development and ultrastructure of the megagametophyte in Passiflora caerulea L. (Passifloraceae)

Megasporogenesis and megagametogenesis of Passiflora caerulea L. were studied using light and transmission electron microscopy. The archesporial tissue is generally formed by one cell. The megaspore mother cell gives rise to a linear tetrad of megaspores. The chalazal megaspore is the functional one, and originates a Polygonum -type female gametophyte. The antipodals are ephemeral. Abundant starch is found in the nucellar cells, specially the ones adjacent to the megagametophyte. The two synergids show ultrastructural differences, involving the filiform apparatus, the nucleolus and the endoplasmic reticulum; these differences suggest a functional differentiation, probably related to the reception of the pollen tube. This is the first report in angiosperms of substantial morphological differences between the two synergids.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 73–81.     

濒危植物南方红豆杉大孢子发生和雌配子体发育

南方红豆杉(Taxus chinensis var.mairei)为第三纪孑遗树种,也是我国Ⅰ级保护植物。本文通过对其大孢子发生及雌配子发育过程进行细胞学观察发现,南方红豆杉胚珠多为直立单生,偶见1个苞片内着生2个胚珠;大孢子母细胞减数分裂形成4个大孢子,其中3个退化,仅位于合点端的大孢子发育为功能性大孢子;功能性大孢子经过有丝分裂形成256个游离核;颈卵器单生,2~6个;从传粉到受精约2个月;有16个原胚自由核,原胚为标准型;简单多胚和裂生多胚并存,胚发育不同步。因南方红豆杉胚珠直立单生、游离核数目、传粉到受精间隔期、原胚游离核数目等特征与松科差异较大,而与广义柏科具有许多相似之处,故我们支持将红豆杉科置于广义柏科之下的观点。此外,本研究结果还表明,南方红豆杉大孢子发生与雌配子体发育正常,不是致其濒危的主要原因。     

Megasporogenesis, female gametophyte development and embryonic development of Ambrosia L. in China

The megasporogenesis, female gametophyte development and embryonic development of Ambrosia artemisiifolia L. and Ambrosia trifida L. of genus Ambrosia L. in China were studied using conventional paraffin section technology and optical microscopy. The results show that both A. artemisiifolia L. and A. trifida L. have a bilobed pistil stigma, two carpels, one chamber, basal placenta, unitegmic, tenuinucellate, anatropous ovule, and well-developed integumentary tapetum. Megaspore mother cells are directly developed from archesporial cells originated from the nucellar cells under the nucellar epidermis and further undergo meiosis to form linear tetrads. The megaspore at the chalazal end develops into a functional megaspore and the other three megaspores are degraded. The development of embryo sac is monosporic type. After three consecutive mitosis, mononucleate embryo sac becomes a mature embryo sac with two synergids and one egg cell at the micropylar end, a central cell at the center and three antipodal cells at the chalazal end. Most antipodal cells are mononucleate or binucleate, only few are trinucleate. The embryonic development process contains four stages: globular embryo, heart-stage embryo, torpedo-stage embryo and mature embryo. The development of endosperm is cellular type.