Ultrastructure of microsporogenesis and microgametogenesis in Campsis radicans (L.) Seem. (Bignoniaceae)

In the present study, microsporogenesis, microgametogenesis and pollen wall ontogeny in Campsis radicans (L.) Seem. were studied from sporogenous cell stage to mature pollen using transmission electron microscopy. To observe the ultrastructural changes that occur in sporogenous cells, microspores and pollen through progressive developmental stages, anthers at different stages of development were fixed and embedded in Araldite. Microspore and pollen development in C. radicans follows the basic scheme in angiosperms. Microsporocytes secrete callose wall before meiotic division. Meiocytes undergo meiosis and simultaneous cytokinesis which result in the formation of tetrads mostly with a tetrahedral arrangement. After the development of free and vacuolated microspores, respectively, first mitotic division occurs and two-celled pollen grain is produced. Pollen grains are shed from the anther at two-celled stage. Pollen wall formation in C. radicans starts at tetrad stage by the formation of exine template called primexine. By the accumulation of electron dense material, produced by microspore, in the special places of the primexine, first of all protectum then columellae of exine elements are formed on the reticulate-patterned plasma membrane. After free microspore stage, exine development is completed by the addition of sporopollenin from tapetum. Formation of intine layer of pollen wall starts at the late vacuolated stage of pollen development and continue through the bicellular pollen stage.     

Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell-wall components in wild-type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild-type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild-type. Enzymatic removal of callose from wild-type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild-type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild-type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type-specific pectin degradation to separate microspores.     

巴戟天花药发育过程中多糖和脂滴分布特征

巴戟天花药发育中多糖和脂滴类物质的分布呈现一定的规律：减数分裂之前,花药壁的绒毡层细胞中有少量脂滴,其他细胞中脂滴和淀粉粒都很少。四分体时期,四分体小孢子中开始出现脂滴,绒毡层细胞中的脂滴较以前增加,其他细胞中的脂滴和淀粉粒仍然很少。小孢子早期,游离小孢子在其表面形成了花粉外壁,靠外壁下方有一层周缘分布的多糖物质。绒毡层细胞中的脂滴明显减少。发育晚期的小孢子中形成一个大液泡,细胞质中出现淀粉粒;同时在药壁和药隔组织中也出现了淀粉粒。此时绒毡层退化。在二胞花粉早期,花粉中积累了大量淀粉粒和一些脂滴。但在成熟的花粉中（二胞花粉晚期）,淀粉粒消失,只有一定数量的脂滴保留。巴戟天成熟花粉中积累的营养物质主要为脂滴。     

POLLEN WALL AND TAPETAL ORBICULAR WALL DEVELOPMENT IN SORGHUM BICOLOR (GRAMINEAE)

Pollen wall development in Sorghum bicolor is morphologically and temporally paralleled by the formation of a prominent orbicular wall on the inner tangential surface of the tapetum. In the late tetrad stage, a thin, nearly uniform primexine forms around each microspore (except at the pore site) beneath the intact callose; concurrently, small spherical bodies (pro-orbicules) appear between the undulate tapetal plasmalemma and the disappearing tapetal primary wall. Within the primexine, differentially staining loci appear, which only develop into young bacula as the callose disappears. Thus, microspore walls are devoid of a visible exine pattern when released from tetrads. Afterwards, sporopollenin accumulates simultaneously on the primexine and bacula, forming the exine, and on the pro-orbicules, forming orbicules. Channels develop in the tectum and nexine, and both layers thicken to complete the microspore exine. Channeled sporopollenin also accumulates on the orbicules. A prominent sporopollenin reticulum interconnects the individual orbicules to produce an orbicular wall; this wall persists even after the tapetal protoplasts degenerate and after anthesis. While the pollen grains become engorged with reserves, a thick intine, containing conspicuous cytoplasmic channels, forms beneath the exine. Fibrous material collects beneath the orbicular wall. The parallel development and morphological similarities between the tapetal and pollen walls are discussed.     

Studies on the Ontogeny of the Pollinium of Goodyera Procera

Using light, transmission and scanning electron microscopy, the development of the pollinium of Goodyera procera (Ker-Gawler) Hooker. was investigated. At the early stage, sporogenous cells inside the microsporangium were seen grouping together into small aggregates each containing few cells. After the aggregates have formed the sporogenous cells inside the aggregates (which could now be called massulae) divide to form numerous pollen mother cells. Later, the pollen mother cells undergo meiosis to form tetrads. The pattern of formation of the exine of tetrads varies according to the location of the tetrads inside the micro- sporangium. Those tetrads that are situated near the outer region of the massulae can form: exine with well developed tectum, bacula and foot layer; and the sequence of events leading to the formation of this type of well developed exine is as follows the original wall and the cyto- plasmic channels associated with the wall become surrounded by a thick layer of callose thus isolating the wall from the plasmalemma. Near the plasmalemma a layer of primexine containing callose and cellulose begins to form. Later, the primexine develops into exine and between the exine and plasmalemma a layer of intine is laid down. Similar type of exine with well developed tectum, bacula and foot layer, is also present in tetrads facing the tapetum. But in this case the original wall of the tedtrad is not retained but undergoes dissolution and in its place a new exine formed. The pattern of formation of exine in the region between tetrads is even more different. Here the original wall also undergoes dissolution but instead of forming a proper exine it only forms a thin foot layer with bulges at places. The pattern of formation of the exine in the cells inside the tetrad is even more different. Here the original wall of the cells only undergoes partial dissolution. The loose fibrils of the partially dissolved wall then become mixed with the callose layer surrounding the cell. Inside this wall-fibril/callose mixture thin sheets of exine appear, but these thin sheets of exine do not develop further into tectum or bacula. In Goodyera a quite substantial amount of callose is retained in the regions between massulae and tetrads, and we believe that it is this callose which is holding the massulae and tetrads together to form pollinium.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

Microsporogenesis and exine structure in Trochodendron aralioides Siebold and Zuccarini (Trochodendraceae)

This study aimed to elucidate the anther wall development, pollen wall development, and exine structure of Trochodendron aralioides Siebold and Zuccarini, a tree with primitive vessels but long considered to lack vessel elements in its wood. The anther wall is the basic type: epidermis, endothecium layer, three middle layers, and tapetum. The anther tapetum is glandular and cells are uniseriate. Microspore mother cells undergo meiosis with simultaneous cytokinesis to produce tetrahedral tetrads enclosed within a callose wall. Before development of the protectum, primexine is inserted against the callose, and the plasma membrane is invaginated. Then, the probacula are elongated under the protectum and arise basally from the plasma membrane. The foot layer formation is concomitant with callose wall dissolution. The foot layer is thick, and the endexine is thin. The foot layer and the endexine are both continuous. The intine is initially formed in the vacuolated microspore stage. Hollow Ubisch bodies are observed on the inner surface of the tapetum in free microspore stage. Pollen grains are tricolporate and 2-celled at the time of shedding. The numerous anthers of a single flower are at different development stages in both protandrous and protogynous individuals.     

白菜核雄性不育花药超微结构的研究

对白菜核雄性不育两用系的可育与不育花药进行了超微结构的比较观察。结果显示不育花药的造孢细胞核仁靠边分布:包裹小孢子母细胞的胼胝质厚薄不均匀,不完整等早期异常现象。减数分裂后,四分体细胞中常有多个细胞核。从四分体释放出的小孢子外壁的孢粉素物质不均匀沉积．呈不连续的单层异常结构。最后小孢子通过细胞质收缩方式败育。在可育花药中,绒毡层细胞在小孢子发育后期已显示出退化迹象,同时在细胞中开始积累脂类物质。但在同时期的不育花药中, 绒毡层细胞没有显示出退化的迹象,也不合成脂类物质。从时间上看,败育花药中小孢子母细胞及小孢子的异常在先,绒毡层细胞的异常在后。本研究揭示了白菜核雄性不育花药的超微结构特征, 对我们以前的光学显微镜观察结果予以补充和修正。     

白菜核雄性不育花药超微结构的研究

对白菜核雄性不育两用系的可育与不育花药进行了超微结构的比较观察。结果显示不育花药的造孢细胞核仁靠边分布;包裹小孢子母细胞的胼胝质厚薄不均匀,不完整等早期异常现象。减数分裂后．四分体细胞中常有多个细胞核。从四分体释放出的小孢子外壁的孢粉素物质不均匀沉积,呈不连续的单层异常结构。最后小孢子通过细胞质收缩方式败育。在可育花药中．绒毡层细胞在小孢子发育后期已显示出退化迹象,同时在细胞中开始积累脂类物质。但在同时期的不育花药中．绒毡层细胞没有显示出退化的迹象,也不合成脂类物质。从时间上看,败育花药中小孢子母细胞及小孢子的异常在先,绒毡层细胞的异常在后。本研究揭示了白菜核雄性不育花药的超微结构特征．对我们以前的光学显微镜观察结果予以补充和修正。     

Pollen and anther development in Nelumbo (Nelumbonaceae)

The Nelumbonaceae are a small family of aquatic angiosperms comprising Nelumbo nucifera and Nelumbo lutea. Historically, the genus has been considered to be closely related to Nymphaeales, however new systematic work has allied Nelumbo with lower eudicots, particularly Platanus. In recent years, studies of pollen development have contributed greatly to the understanding of phylogenetic relationships, but little has been known about these events in Nelumbo. In this paper, pollen and anther development are morphologically described for the first time in N. lutea. A comprehensive ontogenetic sequence is documented, including the sporogenous tissue, microspore mother cell, tetrad, free spore, and mature pollen grain stages. The deposition of a microspore mother cell coat and callose wall, the co-occurrence of both tetrahedral and tetragonal tetrads, the formation of a primexine in tetrads, and primexine persistence into the late free spore stage are shown. The majority of exine development occurs during the free spore stage with the deposition of a tectate-columellate ectexine, a lamellate endexine, and an unusual granular layer below and intermixed with the endexine lamellae. A two-layered intine forms rapidly during the earliest mature pollen stage. Major events of anther development documented include the degradation of a secretory-type tapetum during the free spore stage and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The majority of mature pollen grains are tricolpate, however less common monosulcate and diaperturate grains also develop. Co-occurring aperture types in Nelumbo have been suggested to be an important transition in angiosperm aperture number. However, aperture variability in Nelumbo may be correlated with the lateness of aperture ontogeny in the genus, which occurs in the early free spore stage. This character, as well as other details of pollen and anther ontogeny in Nelumbo, are compared to those of Nymphaeales and Platanus in an effort to provide additional insight into systematic and phylogenetic relationships. Although Nelumbo is similar to both groups in several characters, the ontogenetic sequence of the genus is different in many ways.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加, 同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少,伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后,小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡,体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大波泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

山麦冬大小孢子发生及雌雄配子体发育研究

采用石蜡切片技术对百合科植物山麦冬大小孢子发生及雌雄配子体发育进行了观察研究。结果表明:(1)山麦冬花药具有4个花粉囊,花药壁的发育方式为基本型,花药壁完全分化时由表皮、药室内壁、中层及绒毡层组成。(2)绒毡层发育类型为分泌型,到四分体孢子彼此分离形成单细胞花粉阶段,绒毡层细胞开始解体退化,花粉成熟时绒毡层细胞完全消失;花粉母细胞减数分裂为连续型,四分体为左右对称形排列,成熟花粉为3-细胞花粉,单萌发沟。(3)子房3室,每室2枚胚珠,胚珠倒生型,双珠被,薄珠心,雌性孢原细胞不经过平周分裂而直接发育而成大孢子母细胞。(4)减数分裂后四分体大孢子呈线型或T型排列,合点端大孢子分化为功能大孢子,胚囊发育为蓼型;花粉母细胞减数分裂过程中,二分体、四分体细胞外方被胼胝质壁所包被,小孢子形成后胼胝质壁逐渐消失。该研究结果丰富了百合科植物生殖生物学研究的内容,也为探讨百合科植物的系统学研究提供了参考。     

Pollen and anther ontogeny in Cabomba caroliniana (Cabombaceae, Nymphaeales)

Cabomba is a small water lily genus that is native to the New World. Studies of pollen development and associated changes in the anther yield valuable characters for considering the evolution of reproductive biology in seed plants. Here we characterized the complete ontogenetic sequence for pollen in Cabomba caroliniana. Anthers at the microspore mother cell, tetrad, free microspore, and mature pollen grain stages were studied using scanning electron, transmission electron, and light microscopy. Tetragonal and decussate tetrads both occur in C. caroliniana, indicating successive microsporogenesis. The exine is tectate-columellate, and the infratectal columellae are the first exine elements to form, followed by a continuous tectum and a thin foot layer. A lamellate endexine initiates in the early free microspore stage, but becomes compressed in mature grains. Tectal microchannels and sculptural rods also initiate during the early free microspore stage, and significant pollenkitt deposition follows, supporting the hypothesis that these elements function in entomophily. The tapetum is morphologically amoeboid, with migratory tapetal cells directly contacting developing free microspores within the anther locule. Results from this study illustrate the importance of including ontogenetic data in analyzing pollen characters and in developing evolutionary and ecological hypotheses. The new palynological data also emphasize the character plasticity that occurs in basal angiosperms.     

含笑花药发育的超微结构研究

对含笑花药发育中的超微结构变化进行观察,结果显示:(1)花粉发育中有三次液泡变化过程——第一次是小孢子母细胞在形成时内部出现了液泡,这可能与胼胝质壁的形成有关;第二次是在小孢子母细胞减数分裂之前,细胞内壁纤维素降解区域形成液泡,它的功能可能是消化原有的纤维素细胞壁;第三次是在小孢子液泡化时期,形成的大液泡将细胞核挤到边缘,产生极性。(2)含笑花粉在小孢子早期形成花粉外壁外层,花粉外壁内层在小孢子晚期形成,而花粉内壁是在二胞花粉早期形成;花粉成熟时,表面上沉积了绒毡层细胞的降解物而形成了花粉覆盖物。研究认为,含笑花粉原外壁的形成可能与母细胞胼胝质壁有关,而由绒毡层细胞提供的孢粉素物质按一定结构建成了花粉覆盖物。     

Microsporogenesis and pollen development in Leymus chinensis with emphasis on dynamic changes in callose deposition

Leymus chinensis is an economically and ecologically important grass that exhibits low seed production. To better understand the causes of its low sexual reproductivity, the microsporogenesis and pollen development of this species were investigated, with emphasis on dynamic changes in callose deposition. A variety of histochemical stains were employed, including Heidenhain's hematoxylin, decolorized aniline blue, DAPI, and acetocarmine, along with a temporary mount method. Microsporogenesis and pollen development generally took place from June 12 to 26. The meiosis of microspore mother cells (MMCs) was of the successive type and the tetrad was isobilateral in shape. Mature pollen grains comprised two sperms and a vegetative nucleus. Callose initially appeared in the center of the anther locule at the premeiotic phase, and then gradually and unevenly deposited around the MMC before the commencement of meiosis. At the onset of meiosis, the accumulation of callose enclosing the MMC peaked, accompanied by the disappearance of callose in the center of the locule. At the dyad and tetrad stages, the dyads and tetrads were surrounded by callose wall and the microspores in the tetrads were isolated by a crossed cell plate composed of callose. Microspores just released from tetrads were still enclosed in callose wall, and then callose gradually disappeared in the pollen wall. Ultimately, callose almost completely disappeared from the walls of mature pollen grains. In the large numbers of sections observed, most of the cases of meiosis of the MMCs, pollen development, and callose dynamics were normal, with only a few abnormities observed. The results suggest that microsporogenesis, male gametogenesis, and callose dynamics during these processes are generally normal in this species, and that the callose wall plays an important role in the production of functional pollen grains. The small numbers of abnormities of these processes that occurred likely do not adversely affect the production of viable pollen grains. Therefore, microsporogenesis and pollen development may not be factors in the low seed production of L. chinensis.     

Functional analysis of a beta-1,3-glucanase gene (Tag1) with anther-specific RNA and protein accumulation using antisense RNA inhibition

A critical stage in pollen development is the dissolution of tetrads into free microspores. Tetrads are surrounded by a wall composed primarily of beta-1,3-glucan. At the completion of meiosis, tetrads are released into the anther locule after hydrolysis of the callose by a beta-1,3-glucanase complex. The cDNA corresponding to a beta-1,3-glucanase cloned from tobacco (Tag 1) represents a gene that is highly similar to other beta-1,3-glucanases and is expressed exclusively in anthers from the tetrad to free microspore stage of pollen development. Tag 1 protein was overexpressed in E. coli, accumulating in insoluble inclusion bodies. Polyclonal antibodies against Tag 1 recombinant protein identify a single 33 kD protein accumulating only in anthers at tetrad and free microspore stages where beta-1,3-glucanase activity is present. Transgenic plants expressing Tag 1 antisense RNA were produced. Although Tag 1 RNA and protein levels were greatly reduced, tetrad dissolution and pollen development were normal. These data indicate that under the conditions these tobacco plants were grown, wild type levels of Tag 1 protein are not necessary for male fertility.     

湖北黄精大小孢子发生及雌雄配子体发育

【目的】研究湖北黄精花部形态结构特征和大小孢子发生及雌雄配子体发育过程,以丰富黄精属植物的生殖生物学理论,为进一步开展湖北黄精的品种选育提供依据。【方法】以不同发育时期的湖北黄精花芽为试验材料,用显微观察法观察花部形态结构特征,石蜡切片技术对单花雌雄蕊进行切片观察。【结果】湖北黄精的花被为白色或淡黄绿色,花被筒近喉部稍缢缩;具6枚雄蕊,花丝下端与花被合生,花药开裂方式为纵裂;雌蕊子房上位,3心皮,花柱与子房等长。湖北黄精花药壁由4层细胞组成,成熟的绒毡层具多核,绒毡层发育类型为分泌型;小孢子母细胞减数分裂为连续型,四分体呈左右对称型排列,成熟花粉粒为2-细胞型;存在小孢子发育不同步的现象。雌蕊胚珠具双珠被、厚珠心;四分体呈直线型排列,胚囊发育类型为蓼型;存在双胚囊胚珠现象。在雄蕊的花药壁和雌蕊的子房壁都观察到有束状草酸钙针晶。【结论】湖北黄精雌雄蕊具有较原始的发育特征,虽然在发育过程中都存在异常现象,但雄蕊最终能形成正常的雄配子体,雌蕊低频率的双胚囊现象对总体受精结果影响很小。湖北黄精杂交育种可以选择花药开裂前一时期的花粉,花药壁和子房壁观察到的束状草酸钙针晶无法作为湖北黄精物种鉴定的...     

A contribution to the embryology of Desmodium motorium (Houtt.) Merr. (= D. gyrans (L.f.) DC.) (Fabaceae)

Abstract

The anthers are tetrasporangiate. The anther wall comprises epidermis, fibrous endothecium, middle layer and tapetal layer. The tapetum is of the Glandular type and its cells remain uninucleate. Meiosis in pollen mother cells is normal and simultaneous cytokinesis leads to the formation of tetrahedral and decussate microspore tetrads. The pollen grains are shed at 2-celled stage. The ovule is campylotropous, bitegmic and crassinucellate. Meiosis in megaspore mother cell results in the formation of linear or occasionally T-shaped megaspore tetrad. The chalazal megaspore develops into Monosporic Polygonum type of embryo sac. Endosperm development is of the Nuclear type.     

蜡梅小孢子发生和花粉形成的研究

通过对蜡梅小孢子发生和花粉形成的研究,结果表明：蜡梅幼小花药中的多列孢原细胞经造孢细胞发良为小孢子母细胞。减数分裂为同时型。四分体呈四面体型排列,同时观察了小孢子在发育过程中液泡的动态变化。成熟花粉为２－细胞型。花药壁的发育为双子叶型。花药壁由５－６层细胞组成,腺质绒毡层。花粉具有异型性现象。     

Cytochemistry of pollen development in Brachypodium distachyon

Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.