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。     

大葱卵器及受精后助细胞的超微结构

章丘大葱（Ａllium fistulosum L.cv.Zhangqiu)的卵器由１个卵细胞及２个助细胞组成,观察到不少卵器没有卵细胞,只有２个助细胞。卵细胞的核及大部分细胞质位于细胞的合点端,１个大液泡占据了细胞其他部位。卵细胞含有很多的核糖体及多聚核糖体、嵴明显的线粒体、粗面内质网、高尔基体具小泡,卵细胞似是一个活跃的细胞。细胞外被细胞壁,其合点端及侧方与助细胞共同壁不连续,助细胞有一较大的核,位于细胞膨大的部位,众多的小液泡遍布细胞中。核糖体及聚合核糖体、线粒体,粗面内质网及风心圆环状粗面内质丰富,高尔基体及小泡常见,反映了其活跃的代谢作用。助细胞合点端及侧方与卵细胞、中央细胞的共同壁不连续,与卵细胞共同壁含胞间连丝,壁不连续处,有不状多层膜结构伸入卵细胞质,显示助细胞可能对卵细胞提供营养,伟粉后,一个助细胞退化,宿存助细胞至随胚胚期尚存在,它经历了一个缓慢的退化过程,出现质壁分离,细胞质变稀,液泡扩大,细胞器逐渐减少,在椭形胚期,宿存助细胞核内的染色质及核仁消失,有细胞质侵入核内,因宿存助细胞壁变厚,细胞质出现现脂滴,宿存助细胞可能仍有合成功能,宿存助细胞壁出现若干无壁部位,细胞内的营养物质可能通过无壁部位向胚乳转运,供游离核胚乳及胚乳细胞化初期的发育。     

Observations on Fertilization in Sugar Beet

The whole process of double fertilization in sugar beet has been observed, the main results are as follows: About 2 hours after pollination, the pollen grains germinate, the sperms in the pollen tube are long-oval. 15 hours after pollination, the pollen tube destroys a synergid and releases two sperms on one side or at the chalazal end of the egg cell. The sperms are spherical each having a cytoplasmic sheath. 17 hours after pollination, one sperm enters the egg cell, and the sperm nucleus fuses with the egg nucleus rapidly. 21 hours after pollination, the zygote is formed. In the meantime, the primary endosperm nucleus has divided into two free endosperm nuclei. 25 hours after pollination, the zygote begins to divide, forming a two-celled proembryo. The dormancy stage of the zygote is about 4 hours. In the meantime the endosperm is at the stage of four free nuclei. 17 hours after pollination, the sperm nucleus comes into contact and fuses with the secondary nucleus. The sperm nucleus fuses with the secondary nucleus, faster than the sperm with the egg. he first division of the primary endosperm nucleus is earlier than that of the zygote, it takes place about 20 hours after pollination, the dormancy stage of the primary endosperm is about 2 hours. The endosperm is free nuclear. The fertilization of sugar beet belongs to premitotic type of syngamy. From the stage of zygote to the two-celled proembryo, it can be seen that addition- al sperms enter the embryo sac, but polyspermy has not been observed yet.     

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.     

Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote

Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical-basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical-basal axis for its first cleavage.Key words: asymmetric division, egg cell, fertilization, gamete fusion, rice, sperm cell, two-celled proembryo, zygote     

Localization of arabinogalactan proteins in egg cells, zygotes, and two-celled proembryos and effects of beta-D-glucosyl Yariv reagent on egg cell fertilization and zygote division in Nicotiana tabacum L

Arabinogalactan proteins (AGPs) have been implicated in a variety of plant development processes including sexual plant reproduction. As a crucial developmental event, plant sexual reproduction generally occurs inside an ovule embedded in an ovary. The inaccessibility of the egg cells, zygotes, and embryos has hindered our understanding of the importance of AGPs in the early events involving fertilization, zygotic division, and early embryogenesis. In this study, the well-established in vitro zygote and ovary culture systems, together with immunofluorescence and immunogold labelling techniques, were employed to investigate the role of AGPs in the early events of sexual reproduction in Nicotiana tabacum. Dramatic changes in AGP content during ovule development were evidenced by western blotting. Subcellular localization revealed that AGPs are localized in the plasma membrane, cell wall, and cytoplasm of pre- and post-fertilized egg cells, and cytoplasm and vacuoles of two-celled proembryos. Abundant AGPs were detected in unfertilized egg cells; however, the level of AGPs substantially decreased in fertilized egg cells. Polar distribution of AGPs in elongated zygotes was observed. The early two-celled proembryos just from zygote division displayed accumulation of AGPs at a low level, while in the elongated two-celled proembryos at the late stage, the AGP content clearly increased. Provision of betaGlcY, a synthetic phenylglycoside that specifically binds AGPs, to the in vitro cultures of isolated zygote and fertilized ovaries increased abnormal symmetrical division of zygotes. In the culture of pollinated but unfertilized ovaries, addition of betaGlcY resulted in arrest of fertilization of the egg cells, but had no effect on fertilization of the central cells. The possible roles of AGPs in fertilization, zygotic division, and proembryo development are discussed.     

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     

The zygote and proembryo of alfalfa: quantitative,three-dimensional analysis and implications for biparental plastid inheritance

Summary Genetic studies have demonstrated biparental inheritance of plastids in alfalfa. The ratio of paternal to maternal plastids in the progeny varies according to the genotypes of the parents, which can be classified as strong or weak transmitters of plastids. Previous cytological investigations of generative cells and male gametes have provided no consistent explanation for plastid inheritance patterns among genotypes. However, plastids in the mature egg cells of a strong female genotype (6–4) were found to be more numerous and larger than in mature eggs of a weak female genotype (CUF-B), and the plastids in 6–4 eggs are positioned equally around the nucleus. In CUF-B, the majority of plastids are positioned below (toward the micropyle) the mid level of the nucleus, which is the future division plane of the zygote. Since only the apical portion of the zygote produces the embryo proper, plastids in the basal portion were predicted to become included in the suspensor cells and not be inherited. In the present study, we examined zygotes and a two-celled proembryo from a cross between CUF-B and a strong male genotype (301), a cross that results in over 90% of the progeny possessing paternal plastids only. Our results indicate that the distribution of plastids observed in the CUF-B egg cell is maintained through the first division of the zygote. Further, paternal plastids are similarly distributed; however, within the apical portion of the zygote and in the apical cell of the two-celled proembryo, the number of paternal plastids is typically much greater than the number of maternal plastids. These findings suggest that maternal and paternal plastid distribution within the zygote is a significant factor determining the inheritance of maternal and paternal plastids in alfalfa.