栽培甜菜卵细胞、合子及二细胞原胚的超微结构

为丰富被子植物生殖生物学资料, 并为甜菜相关研究提供参考, 应用透射电镜技术研究栽培甜菜(Beta vulgaris)卵细胞、合子和二细胞原胚的超微结构特征。结果如下：在成熟卵细胞中多聚核糖体数量不多, 且细胞代谢活性较弱; 初期合子内, 核仁大量合成核糖体前体物质, 胞质中多聚核糖体数目众多, 细胞代谢活性较强; 休眠期合子的核仁变小, 胞质中核糖体数量急剧减少, 仅有少量多聚核糖体, 细胞代谢活性较弱; 合子分裂前期和二细胞原胚期, 核仁显著, 胞质中核糖体的密度增加, 出现大量多聚核糖体, 细胞代谢活性较强。根据上述结果可以得出, 栽培甜菜从卵细胞成熟→合子初期→合子休眠期→合子分裂前期→二细胞原胚的超微结构变化中多聚核糖体的变化最为显著, 表现为“少→多→少→多”的数量变化过程, 反映出细胞代谢状态也经历了“弱→强→弱→强”的变化过程, 这种变化趋势与配子体世代向孢子体世代转变有关。     

甜菜无融合生殖单体附加系M14成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14（Betavulgaris,2n=18＋1）为实验材料,利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明：M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态：（1）极性正常的卵细胞,细胞核位于合点端,细胞质含有大量核糖体、线粒体、内质网等细胞器;（2）细胞核位于细胞中央;（3）细胞核位于珠孔端,且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近;未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显,细胞器的种类与数量多,呈现旺盛代谢活动特征,成为二倍体孢子无融合生殖过程中,卵细胞与次生核自发分裂的细胞学标志。     

篦子三尖杉的胚胎学研究及其系统位置的探讨

篦子三尖杉Cephalotaxus oliveri的成熟花粉包含两个细胞,花粉管在精原细胞产生后 经历了一年的休眠期; 精原细胞分裂产生两个体积不等的精子; 雌配子体游离核经12次分裂后形成细胞壁; 窄长的颈卵器单个顶生; 卵细胞中含有大量类核仁状体; 受精时,雌雄核在分裂中期完全融合; 原胚游离核在16核时产生细胞壁; 原胚具冠细胞; 无裂生多胚现象; 成熟胚 二枚子叶。总之,蓖子三尖杉与本属其它种相比,存在明显的差异。如精原细胞分裂前细胞中心细 胞质浓缩成星状放射区域; 苗端早在胚成熟前分化形成明显的突起,以及成熟胚的子叶极为发达等等。因此,作者赞同将蓖子三尖杉在本属中另立一组的观点。     

烟草合子与二胞原胚在离体培养中的发育

用酶解-研磨法分离烟草与大叶烟草受精后的胚囊,继之以显微解剖分离合子与二胞原胚。将3—5个合子或二胞原胚置于微室的琼脂糖小滴中,以预先培养3—4天、分裂1—2次的烟草、大叶烟草或黄花烟草叶肉原生质体饲养。培养基为KM8p与其它附加成分。在25℃与黑暗下静置培养。培养3—4天,约60%合子完成第一次分裂。多数行不等分裂产生大小两个细胞。12天后形成少数原胚或多细胞团。二胞原胚培养亦分裂为多细胞原胚。研究了合子分离方法、合子发育时期、饲养细胞种类与培养天数等因素对合子离体发育的影响。     

甜菜无融合生殖单体附加系M14 成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14(Beta vulgaris, 2n=18+1)为实验材料, 利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明: M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态: (1)极性正常的卵细胞, 细胞核位于合点端, 细胞质含有大量核糖体、线粒体、内质网等细胞器; (2)细胞核位于细胞中央; (3)细胞核位于珠孔端, 且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近; 未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显, 细胞器的种类与数量多, 呈现旺盛代谢活动特征, 成为二倍体孢子无融合生殖过程中, 卵细胞与次生核自发分裂的细胞学标志。     

莴苣卵细胞、合子与原胚细胞中钙的分布

用焦锑酸盐沉淀法对莴苣开花前后的卵细胞、合子与原胚细胞中的钙颗粒分布变化进行了观察。结果表明,开花前三天,刚形成的卵细胞内钙颗粒很少,开花前二天的卵细胞内钙颗粒开始增多,开花前一天的卵细胞形成了大液泡,建立了极性,细胞内的钙颗粒又减少。开花后、受精前的卵细胞的钙颗粒主要聚集在细胞核中。受精后合子中的钙颗粒又明显增多,在核质中分布一些较大的钙颗粒,在珠孔端大液泡中聚集了较多的絮状钙。二胞原胚中的钙颗粒又开始减少,多胞原胚细胞中的钙进一步减少,但原胚表面分布一层丰富的钙颗粒。探讨了钙在卵细胞分化成熟、受精以及原胚发育初期中的作用。     

棉花小孢子发生过程中细胞质的超微结构变化：着重“细胞质改组”问题

棉花(Gossypium hirsutum L.)小孢子发生过程中,细胞质超微结构发生显著而有规律的变化。这些变化主要涉及细胞质中核糖体、质体和线粒体。减数分裂前期Ⅰ,细胞质中核糖体密度逐渐降低,质体和线粒体结构变得不明显。粗线期至双线期,细胞质中核糖体密度降至极低水平,同时质体和线粒体呈衰退结构状态。中期Ⅰ,细胞质中核糖体恢复致密,质体和线粒体也恢复了正常的形态和结构。来自细胞核的类核仁进入细胞质并扩散。这是恢复中期Ⅰ细胞质中核糖体密度的主要原因。内质网在核糖体数量变化中显示出有重要作用。这些细胞质超微结构的变化可认为与世代转变有关。     

云南油杉受精过程中新细胞质及蛋白泡的动态观察

云南油杉(Keteleeria evelyniana Mast)在受精前,精核与卵核周围的细胞质鞘不明显。受精后,合子核周围出现细密的新细胞质。应用孚尔根核染色法,可以较清晰地将新细胞质染出,呈现较弱的正反应,而合子的核质及受精前的精核与卵核染色极弱。卵细胞质及其中的蛋白泡均为负反应。原胚形成后,除上层外,其余几层细胞质内开始积累淀粉粒。此时胚原细胞核的孚尔根染色深度有所增加。幼胚形成后,在顶端的胚原细胞群中核的孚尔根染色反应已恢复正常。在原胚及幼胚胚原细胞质中也呈现很弱的正反应。在电镜下,胚原层细胞质及新细胞质中均含有核样电子致密小体或称作染色质小体,而原胚莲座层细胞质及四周套细胞质中的线粒体则不含这种核样小体。因此,大蛋白泡在卵核形成的早期数量不多,当合子形成时含量最高,而随着游离核的分裂进程,蛋白泡以及原卵质均逐渐地解体,在原胚形成后全部消解。     

番茄受精作用及其间隔期的研究

利用常规石蜡切片法研究了番茄受精作用的全过程,具体研究结果为:(1)授粉后2 h,花粉粒在柱头上萌发;约2~4 h,花粉管长入柱头,且末端膨大;约8 h后,生殖细胞进入分裂期;并于约两小时后,分裂为两个精细胞。(2)约14 h,花粉管进入子房腔;约18~24 h,花粉管进入胚囊,破坏一个助细胞,并在其珠孔端释放两个精子;随后被释放的精子移到卵细胞与次生核附近。(3)授粉后约30 h精核进入卵细胞;约34 h,精核与卵核融合,并在卵核内出现分散的雄性染色质,进而出现雄性核仁;44~50 h,雌、雄性核仁融合,形成合子;合子的休眠期为10 h左右。60 h之后,合子分裂形成二细胞原胚。(4)约26 h,另一个精子的精核与次生核核膜相贴伏,随后与之融合;约30~34 h,次生核内出现分散的雄性染色质,随之出现雄性核仁;约38~42 h,雌、雄性核仁融合,形成初生胚乳核。约44 h后,初生胚乳核进行有丝分裂,形成两个胚乳细胞。番茄胚乳发育属于细胞型。初生胚乳核无休眠期。(5)精子与次生核的融合比与卵核的融合快。(6)番茄的受精作用属于有丝分裂前配子融合类型。     

韭菜胚囊发育与胚胎发生

韭菜胚囊发育为葱型,胚胎发生属柳叶菜型。成熟胚囊中,三个反足细胞形态上常类似卵器,其中二个呈助细胞状,一个呈卵细胞状。卵状反足细胞可分裂成多细胞原胚,但随着胚乳的发育而退化。在未受精胚囊中,卵细胞和卵状反足细胞均可分裂,它们的发生过程与合子胚相似,但因无胚乳哺育,均不能继续发育。论证了反足细胞胚的性质,初步探讨了胚乳与反足细胞无配子生殖的关系。     

CAPSELLA EMBRYOGENESIS: THE EGG,ZYGOTE, AND YOUNG EMBRYO

The ultrastructure and composition of the egg, zygote, and young embryo of Capsella bursa-pastoris were examined. The egg is a highly polarized cell; one-half to one-third of the micropylar end is filled with a large vacuole while the chalazal end contains the nucleus and much of the cytoplasm of the cell. The wall which surrounds the cell is incomplete at the chalazal end. Ribosomes fill the cytoplasm and show little or no aggregation into polysomes. The structure of the nucleolus suggests that ribosomes are not being produced. Following fertilization and the formation of the zygote, the cell decreases slightly in volume as the large central vacuole becomes smaller. The zygote soon increases in size as the small chalazal vacuoles present before fertilization begin to enlarge. The dictyosomes become active and a continuous wall forms around the zygote. Aggregation of the ribosomes begins and numerous polysomes are formed. Before division of the zygote all plasmodesmata between the zygote and the surrounding cells are lost. The first division of the zygote is unequal as a result of its marked polarity. A large basal cell and a small terminal cell are produced. The basal cell appears to contain more protein, RNA, carbohydrate, and cell organelles than the terminal cell. Ribosomal aggregation is even more pronounced at this stage. Starch accumulates in the plastids. Numerous plasmodesmata are present between the terminal and basal cells but there are no connections between the endosperm or other cells. The basal cell divides next to give rise to a three-celled linear embryo consisting of the basal cell, the suspensor cell, and the terminal cell. The terminal cell stains more intensely for protein and RNA as a result of increased numbers of ribosomes. Starch in all the cells is about equal and reaches a maximum in the embryo at this stage.     

Orientation of microtubules suggests a role in mRNA transportation in fertilized eggs of Chinese pine (Pinus tabulaeformis)

Summary. Polysomes become associated with microtubules (MTs) in egg cells of Chinese pine upon fertilization, providing direct evidence for MT-based intracellular mRNA and polysome localization. We have investigated by immunoelectron microscopy the orientation and spatial distribution of MTs and their association with polysomes in the fertilized egg cells. There is a perinuclear accumulation of MTs and polysomes in the zygote soon after fertilization. At this time, some of the MTs are perpendicular to the nuclear envelope and directly connected to the outer membrane or nuclear-pore complexes (NPC) at one end, and the other ends reach to the outer tier or cortical MTs that are parallel to the long axis of the zygote. The polysomes in the perinuclear region show the same spatial and temporal pattern as the MTs. Immunolocalization of the mRNA-binding protein hnRNP indicates that the mRNAs are loaded onto the nucleus-associated MTs immediately after their export from the nuclear-pore complexes. The polysomes and mRNAs are then transported from these MTs to the outer tier and/or cortical MTs, where they further localize to the polar region of the cell. Correspondence (present address): Fengli Guo, Stowers Institute for Medical Research, 1000 E 50th Street, Kansas City, MO 64110, U.S.A.     

ORCHID EMBRYOLOGY: MEGAGAMETOPHYTE OF EPIDENDRUM SCUTELLA FOLLOWING FERTILIZATION

The megagametophyte of Epidendrum scutella, an orchid, was examined with the electron microscope after the entrance and discharge of the pollen tube. The pollen tube enters the embryo sac by growing through the filiform apparatus of a synergid and discharges through a terminal pore into the degenerating cytoplasm of the synergid. The synergid nucleus appears pushed to one side by the discharge of the pollen tube. What is believed to be the remains of the vegetative nucleus has been found in the degenerate synergid, but no trace of the sperm cytoplasm has been seen. The zygote is approximately the same size as the egg. The ribosomes become grouped into polysomes. Both the egg and the zygote apparently completely lack dictyosomes. The polar nuclei partially fuse before fertilization, but fusion of the sperm nucleus with the polar nuclei does not occur and no endosperm is produced. Polysome formation occurs in the central cell and large amounts of tubular, smooth ER are seen. The antipodals remain following fertilization, undergoing ultrastructural changes similar to the central cell.     

FINE STRUCTURAL DEVELOPMENT OF THE MEGAGAMETOPHYTE OF ZEA MAYS FOLLOWING FERTILIZATION

The changes in cytoplasmic fine structure during the interval between pollination and zygotic division in the megagametophyte of Zea mays are reported. The rate of membrane synthesis appears to be low in the mature unfertilized megagametophyte. This megagametophyte is suggested to be in a state of relative metabolic inhibition. The rate of membrane synthesis is high in the zygote and free nuclear endosperm. After fertilization the swelling of the mitochondria in all cytoplasms is interpreted to reflect a presumed increase in metabolic rate relative to growth. In the zygote and endosperm, endoplasmic reticulum (ER) production is associated with the nuclear envelope. In all of the material the ribosomes not associated with the ER are helical polysomes which increase in length after fertilization. On the basis of permanganate staining, the ribonucleoprotein of the endosperm is suggested to be different from that of the other cytoplasms. The specific plastid form of each cell type does not change during the first few hours after fertilization. Dictyosomes are most numerous and active in the antipodals. They are least numerous in the egg and zygote. The smallest dictyosome vesicles are in the synergids. Dictyosome number and vesicle size increase in the egg and central cell after fertilization. Similarities among the megagametophytes of several angiosperms are enumerated.     

Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Changes of Plastids Mitochondria and Their DNA in the Egg Cell Before and After Fertilization in Pharbitis

The ultrastructure and cytoplasmic DNA in the egg cell and zygote of Pharbitis purpurea, (L.) Voyght and P. limbata Lindl. which were studied with electron microscopy and DNA epifiuorescence microscopy. The egg cell before fertilization was highly vacuolated with only a few cytoplasmic plastids and mitochondria. Plastids were spherical and/or rod- shaped containing 1 ～ 2 large starch grains. Most of the mitochondria were cup and/or circular. The cytoplasm in the zygote was much more abundant than that in the egg cell. The number of plastids and their electronic density were greatly increased, in most of which containing osmiophilic bodies. The mitochondria were rich and spherical-shaped in the zygote. Two types of cytoplasmic DNA nucleoids were detected in the egg cell, the more abundant one being big and circle-shaped and the other dot-shaped. Only dot-shaped nucleoids were present in the zygote. The content of nucleoids in the zygote was much less than that in the egg cell. Authors propose that some cytoplasmic DNA may degenerate after fertilization. The ultrastructural characteristics of the egg cell and the reduction of cytoplasmic DNA in the zygote may related to the mechanisms of plastid unipaternal inheritance in Pharbitis.     

水稻胚囊超微结构的研究

水稻(Oryza sativa L.)胚囊成熟时,卵细胞的合点端无细胞壁,核居细胞中部,细胞器集中在核周围,液泡分散于细胞周边区域。助细胞珠孔端有丝状器,合点端无壁,核位于细胞中部贴壁处,细胞器主要分布在珠孔端,液泡主要分布在合点端。开花前不久,一个助细胞退化。中央细胞为大液泡所占,两个极核靠近卵器而部分融合,细胞器集中在极核周围和靠近卵器处,与珠心相接的胚囊壁上有发达的内突。反足细胞多个形成群体,其增殖主要依靠无丝分裂与壁的自由生长,反足细胞含丰富活跃的细胞器,与珠心相接的壁上有发达的内突。开花后6小时双受精已完成,合子和两个助细胞合点端均形成完整壁。合子中开始形成多聚核糖体、液泡减小。退化助细胞含花粉管释放的物质,其合点端迴抱合子。极核已分裂成数个胚乳游离核,中央细胞中细胞器呈活化状态。反足细胞仍在继续增殖。讨论了卵细胞的极性、助细胞的退化、卵器与中央细胞间界壁的变化、反足细胞的分裂特点等问题。     

Differences in the initiation of the zygotic and parthenogenetic pathway in the Salmon lines of wheat: ultrastructural studies

 The ultrastructure of the egg apparatus of the sexual (aestivum)-Salmon line (aS) and the isogenic but alloplasmic (kotschyi)-Salmon line (kS) of the Salmon system of wheat was studied by transmission electron microscopy 3 days before and during anthesis. Additionally, the zygotic stage of aS, 17 h after pollination, was included. Metabolic activity of egg cells from the sexual line aS was low 3 days before anthesis and increased dramatically after pollination and fertilization. This timing of increased activity was evident because of changes occurring in the egg cell nucleus and nucleolus, polysomes, endoplasmic reticulum and Golgi apparatus, and the completion of the cell wall around the zygote. In contrast to the sexual line, the egg cell of the parthenogenetic line showed high activity 3 days before anthesis. The metabolic and ultrastructural characters observed in the nucleus and cytoplasm of the kS line 3 days before and during anthesis corresponded with those of the isogenic sexual line aS during anthesis and 17 h after pollination, respectively. High metabolic activity observed in the persistent synergid of kS may be connected with the occurrence of additional embryos in seeds (twins) of this line. Received: 25 November 1997 / Revision accepted: 16 April 1998