Ultrastructure of Male Gametophyte in Wheat II. Formation and Development of Sperm Cell

Ultrastructural events in wheat sperm cell development were examined from the division of generative cell stage to the maturation of sperm cell in pollen grains. The results are smnmarized as follows: 1. The generative cell in forming microspore by mitosis goes through a series of changes including tile displacement and transformation. It finally becomes a spindle-shaped cell getting ready for another mitosis. The generative cell at this stage is naked. it is only surrounded by both membranes of its own and vegetative cell Most part of the generative cell is occupied by the conspicuous elliptical nucleus with highly condensed chromatin. With the exception of ribosomes, the organelles in the thin layer of generativc cell cytoplasm are obviously fewer and smaller than those in the vegetative cytoplasm. The mierotubules may also be seen in the cytoplasm of spindle-shaped generative cell parallel to the long axis of the cell. There is no amyloplast in generative cell. 2. When the generative cell has moved to the position close to the vegetative nucleus again, it begins to divide. The formation of sperm cells as the result of mitosis of generative cell, and the development of sperm cell involves the following main changes. The shape of the sperm cell tranforms from spherical to elliptical, finally it forms an elongated cell with a tail-like structure. At the sametime, the distribution of cytoplasm gradually concentrated at one end of the sperm cell to form the cytoplasmic extension, so that the so called "tail" of the sperm cell is formed. There are more organelles, especially the mitochondria, assembling in this part. The sperm cell just formed after mitosis is naked and the enclosed plasma membrane is discontinuous. The sperm cell membrane is enclosed by vegetative cell membrane, and the double membranes may be completed at a later stage. It is considered that the period which follows is very short, the deposition of wall material, the callose, occurs to fill up continuously the space between two membranes, but soon after this period the cell wall becomes discontinuous and the wall material is obviously decreased. The significance of the position of the generative cell before its mitosis and the morphological changes during the development of the sperm cell are discussed in this paper.     

Variation of Generative Cell and Vegetative Nucleus in Developing Male Gametophyte of Clivia nobilis etc. Cultured in Vitro II. Cytochemical Observations on Adenosine Triphosphatase Distribution

A cytochemical technique was used to determine whether gemerative cell and yegetative nucleus at various stages of the developing male gametophyte of Clivia nobilis and Amaryllis vittata have ATPase activity. All the studies were carried out in Wachstein-Meisel's medium (1957). The pH optimum for this reaction was pH 7.2. Comparing with the results obtained from the normal development %f male gametophytes, under the same measured conditions, the abortive pollen, the boiled and dried male gametophytes, in which protoplasmic streaming had stopped for more than 24 hours, showed negative reaction. The results obtained from the male gametophyies with normal development are briefly summarized as follows: 1. The presence of ATPase in the cultured male gametophyte: By cytochemical procedures for localizing ATPase activity: ATP-dependent reaction product in living samples or in the samples dehydrated by 80% aleohol in a short time as the pretreatment, is found to be confined to: (a) the growth region at the tip of the pollen tube and the dense region of protoplasmic stream, (b) generative cell (sperms) and (c) vegetative nucleus. Therefore, the Mg2+ and ATPase-dependent cytochemical reaction, and the specific localization of reaction product, have strongly suggested that the appearance of lead phosphate precipitate was due to the ATPase activity in the male gametophyte. 2. The range of ATPase relative activity at various stages of development of male gametophyte: the ATPase activity in generative cell at various stages of its development changes in the range from the 1st degree (light-brown) to the 5th degree (black), but in vegetative nucleus it changes only from the 2nd degree (brown) to the 3rd (dark-brown). 3. The relation between ATPase activity and cell action: from the results, it can be seen that the higher the ability of cell movement and cell activation is, the greater is its ATPase activity. So the relative ATPase activity is in close relation to the mechanical movement of generative cell and vegetative nucleus and their physiological state. 4. The vegetative nucleus is a physiologically active organelle, due to its own ATPase system. Many experimental facts revealed that the. vegetative nucleus might play an important role in mitosis of the generative cell and in formation of sperms.     

Microsporogenesis,Megasporogenesis and the Formation of Male and Female Gametophyte in Coix lacryma-jobi L.

The development of the anther wall follows the monocotyledonous type. During meiotic stages of prophase I, some cytoplasmic channels are observed on the walls between meiotic cells. which divide synchronously. Cytokinesis in the microspore mother cell is of the successive type and gives rise to iscbilateral tetrad. The cell wall between the generative cell and the vegetative cell in early stage shows PAS positive reaction. The mature pollen grain is of 3-celled type. The development of the female gametophyte follows the polygonum-type; the antipodal cells proliferate to form a multicellular tissue mass. Many starch grains are present in the central cell. The nucleus of the mature egg cell is located at the micropylar end; a great deal of starch grains are in the cytoplasm surrounding the nucleus, while vacuoles of various size distribute throughout its cytoplasm but are more and larger at the chalaza,1 end. The nucleus of the synergid cell is located at the micropytar end where a filiform apparatus is formed and many small vacuoles are present at the chalazal part.     

焕镛木小孢子发生及雄配子体发育研究

运用常规石蜡切片法和细胞学压片法,对焕镛木[Woonyoungia septentrionalis (Dandy) Law]小孢子发生、雄配子体发育以及花粉萌发进行观察分析.结果显示:焕镛木花单性,雄花的雄蕊多数、离生,每一花药具4个花粉囊,花药壁5～7层.腺质绒毡层具1～2层细胞;小孢子形成时胞质分裂方式为修饰性同时型;四分体排列方式以四面体型为主,并有交叉型和左右对称型,偶见T型和直线型;成熟花粉粒为二细胞型花粉粒.小孢子发生和雄配子体发育过程中存在变形及败育现象.花粉萌发率为79.89%～80.62%.结合焕镛木花粉粒数目和萌发率的分析认为,雄配子体发育中的败育不是影响焕镛木濒危的主要因素.     

江浙獐牙菜的小孢子发生和雄配子体形成及其系统学意义

采用常规石蜡切片技术,对江浙獐牙菜(Swertia hickinii Burk)花药壁形成、小孢子发生和雄配子体形成过程进行了研究。结果表明:(1)花药四室,花药壁由表皮、药室内壁、中层和绒毡层组成,药壁发育为双子叶型。绒毡层异型起源,腺质型。花粉成熟时药室内壁径向加长并纤维状加厚,表皮宿存。(2)小孢子母细胞在减数分裂过程中胞质分裂为同时型;小孢子四分体排列方式主要为四面体型,也有左右对称型和"T"型等其他类型;成熟花粉为3-细胞型,具三萌发沟。另外,对獐牙菜属的雄性胚胎学特征进行了全面总结,并与龙胆属、蔓龙胆属及双蝴蝶属进行比较归纳出其共性。研究认为,花药表皮宿存或退化,是獐牙菜属与双蝴蝶属的重要区别之一。     

甜椒胞质雄性不育雄配子发育的解剖学和超微结构研究

运用石蜡切片和电子显微镜超薄切片方法观察了甜椒(Capsicum annuum L．)细胞质雄性不育系8A和保持系8B雄配子发育过程。结果表明：不育系和保持系都能正常进行减数分裂,绒毡层细胞无明显差异,形成了正常的四分孢子。在四分体单核居中期后,不育系的绒毡层细胞异常膨大并伸进药室,挤压花粉粒,同时绒毡层细胞提前降解,不育系单核晚期花粉粒开始崩出内含物。致使不育系的雄配子在双核花粉粒形成之前就完全裂解,不能发育成正常的花粉粒。此外,超薄切片还观察到不育系花粉粒在单核早期绒毡层细胞线粒体空泡化,这种变化表明雄性不育的遗传缺陷包括在花药发育早期发生的线粒体结构变化。     

Recent Advances in Microtechnic. I. Methods of Studying the Development of the Male Gametophyte in Angiosperms

Among methods used for a study of nuclear details in the development of pollen grains, the following were found to be very satisfactory: (1) warming the entire grains in aceto-carmine and then clearing with chloral hydrate; (2) making smear preparations stained with crystal-violet-iodine or iron alum hematoxylin. For paraffin sections, a counterstain with dilute alcoholic erythrosin is often very useful after the usual iron hematoxylin technic.

A method of making cultures of pollen tubes on slides coated with thin films of sugar agar is described in detail. The tubes can be fixed by immersing the slide in formol-acetic-alcohol and then stained by any desired schedule. Iron alum hematoxylin was found to be the most satisfactory, but the Feulgen reaction is very valuable in such cases where the nuclei are obscured by the density of the pollen tube cytoplasm. Living pollen tubes can be kept under observation by dissolving a small quantity of neutral red or other vital stain in the sugar agar before it is spread on the slide.

For studying stages in fertilization or gametogenesis, styles should be fixed and sectioned only after a preliminary study with iodine-chloral-hydrate or safranin-anilin-blue or aceto-carmine. Once the extent to which pollen tubes grow in a given time in the stylar tissues has been determined, it is possible to fix material with some knowledge of what it is going to show.

Some other methods, that have not been tried by the authors but appear to be valuable, are also briefly described.     

甜瓜花粉母细胞减数分裂及雄配子体的细胞学研究

以薄皮和厚皮类型甜瓜为试材,采用改良的染色体制片方法,系统观察了甜瓜花粉母细胞的减数分裂及雄配子体发育的过程,结果表明:(1)甜瓜细胞核减数分裂的同步性较高,细胞质是同时型分裂,在细胞核分裂的过程中,核仁在前期Ⅰ到中期Ⅰ逐渐消失,在前期Ⅱ再次出现,随后消失,染色体在前期Ⅰ到中期Ⅰ逐渐收缩,变得清晰,至末期Ⅰ变得模糊,在前期Ⅱ再次清晰;(2)2种类型甜瓜终变期的染色体构型都以环状二价体为主;(3)在后期Ⅱ,观察到染色体的垂直和平行2种分离方式;(4)在前期Ⅰ和前期Ⅱ,伽师瓜"形成了多个较小的核仁,呈现一定的特殊性;(5)雄配子体发育经历了单核期和双核期,最后形成了成熟的花粉粒.研究表明,薄皮和厚皮类型甜瓜减数分裂的染色体行为基本一致,没有明显差异;伽师瓜"的核仁数量表现特殊可能与其长期的生态适应性有关.     

Electrorotation of Isolated Generative and Vegetative Cells, and of Intact Pollen Grains of Lilium longiflorum

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m⁻¹. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques. Received: 9 June 1997/Revised: 4 August 1997     

雌、雄配子体间及雌配子体成员细胞间的通讯

被子植物的双受精是一个复杂而精密的调控过程.成功的受精依赖于配子体的正确发育以及雌配子体与雄配子体间的相互识别.研究表明,雌配子体自身成员细胞间存在广泛的胞间通讯.这种通讯不仅影响不同细胞的发育进程,也决定细胞的发育命运,从而保证雌配子体的正常发育.此外,雌配子体与雄配子体间存在胞间通讯,这种胞间通讯是雌配子体与雄配子体间相互识别的分子基础,精确调控了雄配子体准确进入珠孔、在雌配子体内适时停止伸长、尖端破裂并在特定位置释放精细胞等过程.本文概述了这些方面的最新进展,梳理胞间通讯的途径与信号,并展望了未来雌、雄配子体间及雌配子体成员细胞间通讯的研究方向与可能的应用前景.     

小盐芥小孢子发生和雄配子体发育研究

在显微水平上研究了小盐芥的小孢子发生及雄配子体发育过程,以及不同阶段与花蕾外部形态的相关性.本实验报道的小孢子发生及雄配子体发育的研究结果表明:雄蕊为四强雄蕊,每个花药具4个花粉囊.小孢子母细胞减数分裂属同时型,小孢子在四分体中的排列方式属四面体型.成熟花粉粒属3-细胞型,有3个萌发沟.花粉囊壁发育属双子叶型,由4层细胞构成——表皮、药室内壁、中层和绒毡层.绒毡层为腺质绒毡层.植株花蕾肉眼可见时,雄性孢原细胞开始分化.花蕾露白即蕾长1.1~1.7 mm时,形成成熟的雄配子体,即3-细胞花粉粒.     

蒙古莸小孢子发生和雄配子体发育的研究

运用常规石蜡切片技术对蒙古莸小孢子发生和雄配子体发育进行了观察.结果表明:(1)花药4室,花药壁由4层细胞组成,由外向内分别为表皮、药室内壁、1层中层和绒毡层,花药壁发育方式为双子叶型.(2)花药壁表皮具多细胞腺体,药室内壁、药隔部分细胞发育后期均发生纤维性加厚.(3)绒毡层细胞有两种来源,外周部分来源于初生壁细胞,近药隔部分来源于药隔细胞.腺质绒毡层,发育后期为二核.(4)小孢子母细胞减数分裂过程胞质分裂为同时型,四分体多数为四面体型,偶有左右对称型.(5)成熟花粉为2细胞型,具3个萌发沟.     

濒危植物羊角槭小孢子发生与雄配子体发育研究

利用常规石蜡切片法对羊角槭的小孢子发生及配子体发育进行了研究,结果表明：羊角槭花分两性花与雄花,两性花花药不散粉,雄花散粉;花药具四个花粉囊,其发育类型为基本型;药室内壁呈纤维状加厚;绒毡层为腺质型;孢质分裂为同时型;小孢子四分体呈四面体形排列;成熟花粉粒为二细胞型。羊角槭花药发育过程中出现严重败育现象,表现为四分体胼胝质提早溶解、单核小孢子彼此粘连、花粉液泡化等,形成干瘪、内陷的花粉,一定程度上影响花粉质量;另初步测得羊角槭的花粉生活力在36.98%~60.54%之间,平均生活力为45.97%。本文将为羊角槭花药发育、小孢子发生和雄配子体发育过程及出现的异常现象提供解剖学资料。     

蓝猪耳小孢子发生和雄配子体发育

利用常规石蜡切片和超薄切片技术研究蓝猪耳(Torenia fournien)小孢子发生和雄配子体发育过程.蓝猪耳雄蕊4枚,花药具4个花粉囊.小孢子母细胞经减数分裂成四分体,其排列方式为四面体形或左右对称形.成熟花粉属2细胞型,具3个萌发孔.花药壁发育为双子叶型,腺质绒毡层.小孢子母细胞在四分体时期频繁出现细胞质降解的异常现象,其它发育阶段均正常;小孢子母细胞不正常的减数分裂可能导致花粉败育,这可能是蓝猪耳结实率低的原因之一.     

防风大、小孢子发生与雌、雄配子体发育的研究

利用常规石蜡制片法研究了防风大、小孢子发生及其雌、雄配子体的发育过程。主要结果是：(1)小孢子母细胞减数分裂过程中的胞质分裂为同时型,小孢子四分体为四面体形;(2)成熟的花粉粒为三细胞型,具3个孔沟;(3)花药壁发育类型为双子叶型。花药壁由4层结构组成：最外层为表皮,其内分别为药室内壁、1层中层、绒毡层,绒毡层为分泌型;(4)防风的子房为2室,每室1胚珠,单珠被,薄珠心,倒生型胚珠。大孢子母细胞经减数分裂形成线形排列的4个大孢子,合点端大孢子具功能;(5)大孢子发生过程中,具有多个孢原细胞及多个大孢子母细胞的现象,但通常只有一个大孢子母细胞能继续发育;(6)胚囊发育属于蓼型;(7)防风的花为极端的雌雄蕊异熟,雄蕊的发育早于雌蕊的发育。     

柠条锦鸡儿小孢子发生和雄配子体发育

利用常规石蜡切片技术对柠条锦鸡儿小孢子发生及雄配子体发育的过程进行了观察,为柠条锦鸡儿生殖生物学提供基础资料。结果表明:(1)柠条锦鸡儿雄蕊花药4室,花药壁完全分化时,由外到内依次是表皮、药室内壁、中层和绒毡层,花药壁发育为基本型;表皮细胞1层,发育过程中始终存在;药室内壁在花药成熟时形成带状纤维层加厚;幼小花药壁的中层1～2层细胞,在花药发育成熟时退化消失;绒毡层1层细胞,腺质绒毡层,花药成熟时消失。(2)小孢子母细胞减数分裂过程中的胞质分裂为同时型,产生四面体型和左右对称型小孢子。(3)成熟花粉粒为二细胞型,扫描电镜下观察其成熟花粉粒为圆球形,外壁近光滑。(4)花粉母细胞分裂后形成的四分体小孢子中出现多核仁现象,核仁数在2～6个范围变化,推测这可能和末期Ⅱ核仁融合的不彻底有关。研究发现,柠条锦鸡儿小孢子发生和雄配子发育过程没有发现异常现象。     

毛钩藤的小孢子发生和雄配子体发育

在光学显微镜和透射电镜下观察了毛钩藤(Uncaria hirsuta Havil.)的小孢子发生和雄配子体发育过程.结果表明,毛钩藤花两性,具5枚雄蕊,花药4室,花药壁由表皮、药室内壁、中层和绒毡层组成,花药开裂时,药室内壁高度纤维化带状加厚.花药壁的发育方式属于双子叶型,小孢子母细胞减数分裂的胞质分裂为同时型.小孢子在四分体时期开始沉积花粉外壁,小孢子大液泡化时期开始沉积花粉内壁.成熟花粉为2-细胞型.毛钩藤的花粉发育特征和茜草科植物基本一致.毛钩藤绒毡层属于分泌型,双重起源,分别起源于次生周缘层和药隔细胞.小孢子发育早期绒毡层开始降解并分泌形成大量乌氏体,花药开裂时绒毡层完全消失,剩下少量乌氏体.小孢子早期内壁加厚突出形成,小孢子细胞核分裂以后内壁加厚开始脱落,花药开裂时,只剩下少量的内壁加厚突出.初步推测,内壁加厚突出与乌氏体共同作用为雄配子体的发育提供营养物质.     

狼毒大戟的小孢子发生与雄配子体发育

采用石蜡制片技术研究了狼毒大戟（Euphorbia fischeriana）的小孢子发生及雄配子体发育过程,结果表明：狼毒大戟的花药为四分孢子囊,花药壁为五层,发育为基本型;变形绒毡层,细胞为双核;小孢子母细胞减数分裂中的胞质分裂为同时型;四分体排列通常为四面体型,少数左右对称型;成熟花粉为三细胞型,椭球形,具三沟孔。研究结果为狼毒大戟的生物学研究及应用开发积累基础资料。