Developmental synchronization of male and female gametophytes in Ginkgo biloba and its neck mother cell division prior to fertilization

This study investigated male and female gametophytes in Ginkgo biloba, while a droplet of fluid was present in the fertilization chamber and found that the central cell, the generative cell and the neck mother cell divided simultaneously prior to fertilization. In male gametophytes, the generative cell divided to yield two sperm cells. Concomitantly, the two neck mother cells of the archegonium increased in size then divided asymmetrically resulting in two big cover cells and two small base cells. Each cell had a fixed end in direct contact with an adjacent jacket cell and a free end overlapping its counterpart. This unique arrangement could allow for their free ends to swing into the fertilization chamber as a result of the force from the interior of the archegonium where a polar periclinal division had occurred to produce a canal cell and an egg. The subsequent withdrawal of the content of the archegonium may facilitate the entry of sperm into the archegonium. The neck apparatus closed after the fertilization occurred. The concurrence of the above divisions and the delicate structure of neck apparatus suggest that the gametophytes undergo a synchronization process to become receptive at the time of fertilization. However, the formation of neck cells and the opening time of neck apparatus of the archegonia within the same ovule were slightly different, which could lead to the formation of zygotes at a temporally distinct interval. The earlier formed zygote may progress as the only mature embryo in the ovule.     

Antheridia and Archegonia of the Apogamous Fern Pteris cretica

The external appearance of gametophytes and gametangia of Pteriscretica closely resembled those of sexually reproducing ferns.Antheridia were fully functional but archegonia were not. Despitethe formation of egg, ventral and neck canal cells, the flask-shapedarchegonia failed to open and Pteris spermatozoids failed toexhibit a chemotactic response to them. Cryo SEM revealed thatthe topmost cells of mature archegonia had collapsed, and thisis thought to account for the loss of archegonial function. Pteris cretica, fern, apogamy, antheridia, archegonia, cryo SEM     

Fertilization and rejection of spermatozoids by egg cells in artificially pollinated ovules ofEncephalartos (Zamiaceae)

The structure and behaviour of free female, male and proembryonal nuclei ofEncephalartos villosus Lem. were studied during a light-microscopical investigation of serially sectioned archegonia in successfully pollinated ovules. Before spermatozoids were released from the pollen tubes into the archegonial chamber, the ventral canal nucleus had disintegrated in the neck region of the egg cell among minute, amoeboid bodies with PAS-positive granules. In archegonia containing multiple spermatozoids, the egg nucleus was unobtrusive and syngamy followed by proembryo formation regularly resulted. The egg cell usually reacted violently in archegonia penetrated by a single spermatozoid. These reactions were regarded as rejection phenomena and considered as indicators that the egg cell can differentiate between compatible and incompatible male gametes.     

Structure and function of the neck cell during fertilization in Ginkgo biloba L.

Key message

Neck cells in Ginkgo biloba contribute to archegonial opening through morphological changes and might be involved in the production of fertilization liquid to attract spermatozoids toward archegonia.

Abstract

Neck cells are an essential part of the archegonium in archegoniate gymnosperms, but their function in the sexual reproductive process remains unclear, particularly in zoidogamous gymnosperms. To clarify the structural characteristics of neck cells and their role in fertilization, we examined the neck cells of Ginkgo biloba L. by means of scanning electron microscopy and transmission electron microscopy. The two curved inner neck cells, which are covered imbricately by the two turgid outer neck cells, were pushed to two sides during fertilization, which indicated that morphological changes in these cells contribute to archegonial opening. The neck cells contained many secretory organelles with some material accumulated outside the cell wall, thus the neck cells might be involved in the production of fertilization liquid to attract spermatozoids toward the archegonium. In addition, the surrounding surface cells of the female gametophyte also cooperate to produce the liquid. Taken together, these results indicate that the neck cells provide an effective mechanism by which zoidogamous gymnosperms achieve reproductive success through altering the morphology and cellular physiology of the neck cells.     

Comparative morphology of the gametophytes of the genus Equisetum, subgenus Equisetum

When grown under a variety of axenic conditions gametophytes of E. arvense, E. telmateia, E. sylvaticum, E. palustre , and E. fluviatile are all morphologically distinct. Lamellae from archegonial branches provide several diagnostic features but lamellae associated with antheridia are much less distinct. The archegonia of all five species have two neck canal cells and three tiers of neck cells and are of limited use taxonomically. Although antheridium development is basically similar in all species, during the metamorphosis of the spermatocytes specific differences arise in the shape of the cavity, degree of emergence and the number of opercular cells. A taxonomy of the gametophytes based on antheridium morphology corresponds very closely to the classification of the subgenus Equisetum into three sections based on the coning behaviour of the sporophytes.     

海金沙配子体发育及卵发生的显微观察

用光镜观察海金沙(Lygodium japonicum)配子体发育和卵发生。海金沙孢子为四面体形,具三裂缝,孢子萌发方式为密穗蕨型(Anemia-type);配子体的发育形态多样,通常丝状体长至3~5个细胞时通过顶细胞纵分裂发育为片状体,进而发育为心形原叶体,在心形原叶体上可产生精子器和颈卵器。但在培养过程中也可产生10个细胞以上的丝状体,这种丝状体发育成的片状体和原叶体形态通常不规则,只产生精子器,不产生颈卵器。原叶体发育是铁线蕨型(Adiantum-type),性器官是薄囊蕨型(Leptosporangiate-type)。切片观察海金沙颈卵器产生于生长点下方表面细胞,经两次分裂形成了顶细胞、初生细胞和基细胞。其中初生细胞再经两次不等分裂产生卵细胞、腹沟细胞和颈沟细胞,此时三个细胞紧密相连,随发育,颈沟细胞和和腹沟细胞退化,卵周围形成了分离腔,光镜观察显示成熟卵细胞上无典型卵膜形成,未观察到受精孔的结构。     

极危孑遗物种东方水韭雌配子体及胚胎的解剖学观察

以人工培养的国家一级保护植物东方水韭(Isoetes orientalis)为材料,采用切片技术对雌配子体和胚胎的发育进程进行解剖学观察研究,探讨其有性生殖过程及濒危机制。结果表明:(1)东方水韭大孢子3~5d萌发,成熟雌配子体呈球形,无假根,三裂缝处发育出多个颈卵器,成熟颈卵器只有颈壁细胞与颈沟细胞,无腹沟细胞。(2)多数雌配子体只发育出一个胚胎,偶见多胚共存现象;胚胎发育时期,第一叶原基相比第二、三叶原基发育迅速。(3)颈卵器部分组织常出现分化紊乱,导致雌配子体败育。该研究结果支持"根叶理论",并讨论了腹沟细胞的退化以及双胚共存机制,认为东方水韭雌配子体常停留在游离核阶段、颈卵器形态或位置不规则、卵细胞排列紊乱等可能是其败育的原因。     

乌蕨配子体发育及卵发生的显微观察

采用光学显微镜对乌蕨的配子体发育及卵发生的过程进行了研究,以阐明蕨类植物颈卵器发育特征,为揭示蕨类植物有性生殖机制以及鳞始蕨科的演化提供依据。结果表明:(1)乌蕨孢子黄褐色,具单裂缝,表面平滑或呈疣状纹饰;孢子接种12d萌发,萌发类型为书带蕨型,原叶体发育类型为铁线蕨型。(2)半薄切片观察表明,乌蕨颈卵器产生于原叶体生长点下方的表面细胞,即颈卵器原始细胞,该细胞经过两次分裂形成纵向3层细胞,最上层细胞发育为颈卵器的颈部壁细胞,中间层细胞即初生细胞再经过两次不等分裂产生颈沟细胞、腹沟细胞和卵细胞,此三细胞最初紧密贴合,随着颈卵器的发育,卵细胞与腹沟细胞间从两侧向中间产生分离腔,且腹沟细胞与颈沟细胞开始退化;分离腔逐渐向中间扩大,直至出现孔状结构,即受精孔;颈卵器发育后期,在卵细胞上表面形成染色较深的卵膜,颈沟细胞与腹沟细胞退化成絮状物。     

阔鳞瘤蕨颈卵器形成与卵发生的初步研究

运用光学显微镜与透射电镜对阔鳞瘤蕨（Phymatosorus hainanensis(Noot.) S.G.Lu）颈卵器形成和卵发生进行了研究。阔鳞瘤蕨颈卵器产生于雌配子体生长点下方分枝毛状体内侧。切片观察表明颈卵器起源于配子体表面的原始细胞,该细胞经两次不等分裂形成3个细胞,上下两个细胞分别发育为颈卵器的颈部与底部壁细胞,中间的细胞为初生细胞,含有较丰富的细胞器。初生细胞进行两次不等分裂产生颈沟细胞、腹沟细胞与卵细胞。成熟颈卵器内颈沟细胞和腹沟细胞退化,卵细胞上表面产生受精孔。本研究阐述了阔鳞瘤蕨颈卵器形成和卵发生的细胞学过程,对阐明蕨类植物雌性生殖器官的发育特征有一定的科学意义。     

野雉尾金粉蕨配子体发育及其系统学意义

野雉尾金粉蕨为中国蕨科金粉蕨属植物,而金粉蕨属的系统位置一直存在争议。该研究用原生境腐殖土和改良克诺普氏(Knop’s)营养液对野雉尾金粉蕨的孢子进行培养,培养条件为温度25℃、光照强度2 500 lx、光照12 h/d,在光学显微镜下观察记录其孢子萌发和配子体发育过程。结果表明:野雉尾金粉蕨的孢子为黄褐色,四面体型,三裂缝,赤道面观为扇形,具周壁,外壁表面具网状纹饰。孢子培养7 d后开始萌发,孢子萌发类型为书带蕨型(Vittaria-type)。孢子萌发后,配子体原始细胞经多次横向分裂形成3~9个细胞的丝状体,丝状体细胞呈圆筒形,壁薄,侧壁向外鼓起,含有颗粒较大且数量较多的叶绿体。15 d左右发育为片状体,片状体多为匙状。25 d左右形成幼原叶体,幼原叶体不对称,配子体发育类型为水蕨型(Ceratopteris-type)。在原叶体发育过程中分枝丝状体非常发达,配子体呈丛状生长,整个发育过程没有毛状体产生。野雉尾金粉蕨的假根为单细胞管状,偶有分支,内无叶绿体。45 d左右精子器开始出现,精子器顶面观近圆形,侧面观为近椭圆形或短柱状。精子器成熟时,盖细胞裂开,精子逸出。60 d左右颈卵器出现,颈卵器比较大,基部略大于顶部,侧面观呈烟囱状,顶面观为铜钱状,颈部由四列细胞构成。90 d左右发育出肉眼可见的幼孢子体。从研究结果看,其配子体发育的特征与凤尾蕨科(Pteridaceae)凤尾蕨属(Pteris L.)相似,支持金粉蕨属归于凤尾蕨科的观点。该研究结果为野雉尾金粉蕨系统学研究提供了配子体发育方面的证据。     

Preprophase band loses its function as a cytokinetic apparatus in mitosis of neck canal mother cell

Summary. Preprophase bands in the neck canal mother cell and the central cell of the archegonium of the fern Dryopteris crassirhizoma are observed with immunofluorescence microscopy. No phragmoplast is found during mitosis of the neck canal mother cell; however, the phragmoplast develops very well in the central cell. The neck canal mother cell undergoes karyokinesis but not cytokinesis and finally produces only one binucleate neck canal cell. However, the central cell undergoes cytokinesis and produces an egg cell and a ventral canal cell. These observations suggest that the preprophase band in the neck canal mother cell loses its function as a cytokinetic apparatus and becomes an evolutionary vestige in the development of the archegonium. Received January 22, 2002; accepted April 26, 2002; published online October 31, 2001 RID="*" ID="*" Correspondence and reprints: College of Life Sciences, Peking University, Beijing 100871, Peoples' Republic of China. E-mail: rlyou@pku.edu.cn     

峨眉凤丫蕨配子体发育及卵发生的研究

用显微观察及透射电镜技术对峨眉凤丫蕨的配子体发育及卵发生过程进行了观察研究,以探讨其卵发生细胞学机制及蕨类植物演化关系。结果表明:(1)峨眉凤丫蕨孢子接种7～9d萌发,经丝状体和片状体阶段发育为心形原叶体,成熟原叶体雌雄同株,在原叶体基部产生精子器,在原叶体生长点下方产生颈卵器。(2)卵发生研究表明,峨眉凤丫蕨颈卵器产生于生长点下方的表面细胞,该细胞经2次分裂形成3层细胞,中间者为初生细胞,它经2次不等分裂产生卵细胞、腹沟细胞和颈沟细胞;新产生的卵与腹沟细胞间连接紧密,有发达的胞间连丝,随着发育,卵细胞与腹沟细胞之间产生分离腔,而腹沟细胞与卵细胞始终通过孔区相连;发育中期,卵核形成大量核外突;发育后期,在卵细胞外侧形成卵膜,孔区演变为受精孔,核外突数量减少。     

水蕨颈卵器的形成与发育

主要运用电子透射显微镜技术对水蕨（Ceratopteris thalictroides（L．）Brongn）颈卵器形成与发育进行了研究。结果表明：水蕨颈卵器是由原叶体分生组织内颈卵器原始细胞形成的。该原始细胞经2次分裂形成3层细胞,上下两层发育成颈卵器颈部与底部的壁细胞,中层为初生细胞。初生细胞是颈卵器内雌配子发生的第一个细胞,该细胞经2次不等分裂形成1个卵细胞,1个腹沟细胞、1个双核的颈沟细胞。本研究首次阐明了水蕨颈卵器内细胞的发育顺序和特征。     

Formation of archegonium chamber is associated with nucellar-cell programmed cell death in Ginkgo biloba

Summary. The archegonium chamber in Ginkgo biloba L. is a pathway for spermatozoids swimming towards the archegonium for fertilization. The objective of this study was to elucidate the mechanism of archegonium chamber formation. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and DNA ladder demonstrated that the nucellar cell death, coordinated with the archegonium chamber formation, was a process of programmed cell death. Cytochemical localization of Ca²⁺ in these nucellar cells was determined by means of in situ precipitation with potassium pyroantimonate and electron microscopic visualization, in order to study the relation between Ca²⁺ and programmed cell death. The results showed an early uptake of the mitochondrial calcium particles in the nucellar cells undergoing programmed cell death. Together with other dynamic changes in Ca²⁺ subcellular distribution, this indicates that Ca²⁺ may play a role in the regulation of mitochondria-mediated programmed events in the nucellar cells. Correspondence and reprints: School of Pharmaceutical Sciences, Peking University, Beijing 100083, People’s Republic of China.     

红松配子体的发育过程

红松是我国主要用材树种,材质优良。我国主要分布在长白山和小兴安岭,随着森林工业的发展,大面积的原始森林迅速减少,人工更新日趋重要。在生产中采用种子育苗,因此对红松种子的发育规律应全面掌握,以便为种子园建立、良种选育采取有效措施提供理论依据。根据国内外有关报导,红松雌雄配子的形成、受精作用已有一定的研究^[14、15],但这些研究只侧重在小孢子母细胞的减数分裂和受精作用。故对雌雄配子体的发育过程仍需深入研究,尤其是授粉后的雄配子体发育和精子进入颈卵器的过程,均未见详细报导。     

GAMETOPHYTE OF LYCOPODIUM DIGITATUM

Gametophytes of Lycopodium digitatum A. Braun are subterranean, mycorrhizal structures. Externally, they are conical and divided into three principal regions: tapering base, ring meristem, and gametangial cap. Internally, the endobiont is restricted to specialized tissues in the base. Gametangia typical of those described for L. complanatum are found along the cap margin near the ring meristem. Comparative analysis of these and other related gametophytes leads to the conclusion that Bruchmann's Type I (L. clavatum) and Type II (L. complanatum) gametophytes cannot be consistently separated on the basis of 1) mycorrhizal tissues, 2) form of gametophyte, and 3) number of neck canal cells in the archegonium, as he had proposed. Consequently, it is suggested on the basis of ring meristem and radial symmetry, mycorrhizal tissue specialization, and lack of sterile paraphyses or lobes that Type I and Type II gametophytes be merged as a single type. The now broadened clavatumcomplanatum type is restricted to subgenus Lycopodium (sensu Wilce) where it is the only known type.     

水蕨颈卵器的形成与发育

主要运用电子透射显微镜技术对水蕨(Ceratopteris thalictroides(L.)Brongn)颈卵器形成与发育进行了研究。结果表明:水蕨颈卵器是由原叶体分生组织内颈卵器原始细胞形成的。该原始细胞经2次分裂形成3层细胞,上下两层发育成颈卵器颈部与底部的壁细胞,中层为初生细胞。初生细胞是颈卵器内雌配子发生的第一个细胞,该细胞经2次不等分裂形成1个卵细胞,1个腹沟细胞、1个双核的颈沟细胞。本研究首次阐明了水蕨颈卵器内细胞的发育顺序和特征。     

云贵水韭雌配子体发育及其濒危机制的研究

为了解云贵水韭(Isoetes yunguiensis)的生殖过程和濒危原因,采用人工培养获得其雌配子体,用半薄切片法观察雌配子体发育过程。结果表明,云贵水韭的大孢子萌发率约为26%;雌配子体异养,终生都在大孢子细胞壁内发育;颈卵器发育只有2枚颈沟细胞而无腹沟细胞。雌配子体发育停止在游离核时期、颈壁细胞数量偏少或排列紊乱、颈沟细胞异常、卵细胞凋亡等可能是导致生殖濒危的原因。同时,游离核是水韭生殖生物学研究和系统学研究的重要环节之一;缺失腹沟细胞可能是水韭类简化性进化的特征之一。     

Secretions from the female gametophyte and their role in spermatozoid induction in Cycas revoluta

In cycads, spermatozoids are released from pollen tubes and swim in fluid toward the archegonia. The source of this fluid was examined using Cycas revoluta Thunb. ovules placed in culture. Dissected female gametophytes just before fertilization produced copious fluid on their upper surface. The fluid first appeared around the archegonial chamber and then on the inside of the archegonial chamber. When this fluid was applied to dry turgid pollen tubes, they discharged spermatozoids 12 h later. The archegonial neck appeared as two semi-spherical swellings, whereas the four neck cells later became visible and they separated in a schizogenous manner. Many globose particles appear on the top of the archegonial neck cells when the fluid is present. The contents of pollen tubes, spermatozoids and surrounding liquid intermingle with the secreted fluid. The female gametophyte differs in ultrastructure during the stages before and after fluid secretion, the latter showing changes suggestive of fluid secretion from the female gametophyte.     

肾蕨配子体发育的研究

利用光学显微镜观察肾蕨(Nephrolepis auriculata)孢子的萌发及配子体发育过程。结果表明:成熟的孢子棕黄色,不透明,极面观圆球形,赤道面观豆形,单裂缝,表面光滑。孢子萌发类型为书带蕨型,播种后15 d左右萌发,形成5-12个细胞长的丝状体。原叶体发育为三叉蕨型。30 d左右发育为片状体,45 d左右形成幼原叶体,幼原叶体不对称,成熟原叶体心脏形对称。原叶体边缘及背腹面都具毛状体,毛状体由单细胞构成。60 d左右精子器开始出现,精子器近圆球形,由3个细胞构成。70 d左右颈卵器出现,成熟颈卵器颈部由4列细胞构成,3-5层细胞高。原叶体受精后1个月内可看到幼胚生成。