银杏雌雄配子体发育及胚胎形成的研究进展

银杏（Ginkgo biloba）是现存最古老的裸子植物之一, 其生殖过程表现出许多原始特征和独特性状, 长期以来备受国内外专家的关注。经过近100年的研究取得了显著成果： （1） 银杏雄配子体发育周期长, 经历了从平周分裂到斜背式分裂,并最终垂周分裂形成带有鞭毛的游动精子; （2） 银杏雌配子体发育经历较长的游离核期和细胞化期, 分化形成颈卵器母细胞并经平周分裂、垂周分裂和斜向分裂形成成熟的颈卵器（包括有4个颈细胞和1个卵细胞）; （3） 推测其精细胞中的液泡状结构为受精过程中的遗传物质载体; （4） 原胚的形成经历了游离核期和细胞化期。该文针对国内外最新银杏生殖生物学方面的研究进展, 对银杏雌雄配子体发育、受精过程以及胚胎形成等方面进行较为系统全面的分析和总结, 为进一步的银杏生殖生物学研究提供有价值的参考资料。     

银杏雌性生殖器官发育过程的显微观察

对银杏(Ginkgo biloba)雌性生殖器官的发育过程进行了连续显微观察。结果表明: 功能大孢子经过大约1个月的分裂形成约5 000个游离核后开始细胞化。授粉后约45天近珠孔端两侧各产生1个颈卵器母细胞。授粉后约50天, 颈卵器母细胞 平周分裂形成初生颈细胞和中央细胞。授粉后约55天, 初生颈细胞垂周分裂形成2个扁平状次生颈细胞, 之后次生颈细胞体积逐渐增大并突入颈卵器腔。授粉后约130天, 2个次生颈细胞斜向分裂形成4个颈细胞, 中央细胞不均等分裂形成腹沟细胞和卵细胞。套细胞起源于颈卵器母细胞的周围细胞, 授粉后70天至受精作用发生前, 套细胞内不断积累营养物质, 且套细胞与中央细胞间的细胞壁以及套细胞之间角隅处的细胞壁均出现明显增厚现象。在受精及胚胎早期发育过程中, 套细胞内营养物质逐渐消失, 细胞逐渐解体。授粉后55天, 2个颈卵器之间的一些细胞向上突起形成帐篷柱, 之后帐篷柱体积逐渐增加, 并突入颈卵器腔。自授粉后120天至受精前帐篷柱细胞内开始积累大量营养物质, 随后这些营养物质在受精过程中被逐渐消耗。到了原胚游离核后期, 帐篷柱的顶端细胞发生变形并解体。     

银杏雌性生殖器官发育过程的显微观察

对银杏(Ginkgo biloba)雌性生殖器官的发育过程进行了连续显微观察.结果表明:功能大孢子经过大约1个月的分裂形成约5000个游离核后开始细胞化.授粉后约45天近珠孔端两侧各产生1个颈卵器母细胞.授粉后约50天.颈卵器母细胞平周分裂形成初生颈细胞和中央细胞.授粉后约55天,初生颈细胞垂周分裂形成2个扁平状次生颈细胞,之后次生颈细胞体积逐渐增大并突入颈卵器腔.授粉后约130天,2个次生颈细胞斜向分裂形成4个颈细胞,中央细胞不均等分裂形成腹沟细胞和卵细胞.套细胞起源于颈卵器母细胞的周围细胞,授粉后70天至受精作用发生前,套细胞内不断积累营养物质,且套细胞与中央细胞间的细胞壁以及套细胞之间角隅处的细胞壁均出现明显增厚现象.在受精及胚胎早期发育过程中,套细胞内营养物质逐渐消失,细胞逐渐解体.授粉后55天,2个颈卵器之间的一些细胞向上突起形成帐篷柱,之后帐篷柱体积逐渐增加,并突入颈卵器腔.自授粉后120天至受精前帐篷柱细胞内开始积累大量营养物质,随后这些营养物质在受精过程中被逐渐消耗.到了原胚游离核后期,帐篷柱的顶端细胞发生变形并解体.     

银杏套细胞发育的解剖学研究

银杏套细胞起源于近雄配子体表达的颈卵器母细胞周围细胞,中央细胞形成时,套细胞呈明显的一层,围绕颈卵器紧密排列,随着颈卵器的发育,套细胞体积逐渐地大,细胞质变浓厚,细胞质中脂滴增多,套细胞和中央细胞的接触壁开始出现局部加厚,在颈卵器发育的泡沫化阶段,套细胞和中央细胞的接触壁不均匀加厚较为显著,在较薄的区域可见胞间连丝,受精前,套细胞中液泡增多,脂滴迅速减少,造淀粉体增大,套细胞与卵细胞的接触壁的不均匀加厚非常明显,精核进入颈卵器以后,卵细胞与套细胞的接触壁和卵细胞的质膜之间形成一个薄厚不均的间隔层,受精卵分裂时,受精卵细胞与套细胞接触壁的凹陷处可见许多小泡和内质网,游离核期时,套细胞内出现大量小液泡,细胞内含物迅速消失,套细胞外形变长,胚胎长出颈卵器后,套细胞逐渐解体消失。     

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。     

福建柏的配子体发育

福建柏花粉在4月中传粉。5月初,不育细胞、管细胞和精原细胞已经形成。6月底,精原细胞开始分裂,产生2个在大小和形态上都相似的雄配子。这两个精子都有授精的功能。福建柏起作用的大孢子,经过12次连续有丝分裂后,产生4096个(实际计算为3733—4224个)游离核,而后在核之间形成雌配子体胞壁。颈卵器数目为6—16个,多数为9—12个。复合颈卵器被2—3层套细胞所包围。颈细胞1—2层,共4个细胞。6月底,中央细胞分裂形成腹沟核和卵核。受精作用在卵细胞的中部进行。从配子体发育来看,福建柏属与圆柏属有许多共同之处。     

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。     

樟子松受精作用和原胚的选择

樟子松发育成熟的雄配子体中的精子6月15日左右在颈卵器中上部与卵细胞结合,进行受精作用,其后,受精卵进行游离核分裂,形成8个子核时,开始形成细胞壁。它们再分裂1次,形成16个细胞的原胚。接着胚柄细胞层迅速生长、伸长,把下面的原胚送出颈卵器基部的细胞壁,进入胚乳中的溶蚀腔。原胚吸收溶蚀腔中的营养,生长发育。初期,胚的数目往往很多,但常常只有1个发育成熟。     

阔叶鳞盖蕨配子体发育及卵发生的研究

采用显微镜和透射电镜对阔叶鳞盖蕨(Microlepia platyphylla)的配子体发育和卵发生过程进行了观察,以阐明其卵发生的细胞学机制,探讨其演化地位。阔叶鳞盖蕨孢子褐色,四面体形,具三裂缝,接种5~10d后孢子萌发,经丝状体和片状体阶段发育为心形原叶体,原叶体发育是铁线蕨型,通常为雌雄异株,精子器产生于不规则配子体的表面,颈卵器产生于心形原叶体生长点的下方,性器官是薄囊蕨型。卵发生研究表明,阔叶鳞盖蕨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3个细胞通过胞间连丝紧密相连,随发育,腹沟细胞与卵细胞间形成了分离腔,但在孔区处始终通过胞间连丝相连,成熟卵细胞上形成了卵膜和受精孔,卵核表面产生了核外突,通过比较表明阔叶鳞盖蕨卵发生与蕨（Pteridium aquilinum）卵发生相似。     

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。     

HETEROCHRONY AND DEVELOPMENTAL INNOVATION: EVOLUTION OF FEMALE GAMETOPHYTE ONTOGENY IN GNETUM,A HIGHLY APOMORPHIC SEED PLANT

Seed plant female gametophytes are focal points for the evolutionary modification of development. From a structural perspective, the most divergent female gametophytes among all seed plants are found in Gnetum, a clade within Gnetales. Coenocytic organization at sexual maturity, absence of defined egg cells (free nuclei are fertilized), lack of centripetal cellularization, and postfertilization development of embryo-nourishing tissues are features of the female gametophytes of Gnetum unparalleled among seed plants. Although the female gametophyte of Gnetum retains the three basic phases of somatic development common to female gametophytes of plesiomorphic seed plants (free nuclear development, cellularization, cellular growth), the timing of fertilization has been accelerated relative to the rate of somatic development. As a consequence, the female gametophyte of Gnetum matures sexually (is fertilized) at a juvenile (compared with the ancestral somatic ontogeny) and free nuclear stage of somatic development, thereby precluding differentiation of egg cells. Unlike progenetic animals, where truncation of somatic ontogeny evolves in tandem with acceleration in the timing of sexual maturation, the female gametophyte of Gnetum completes the entire ancestral somatic ontogeny after precocious sexual maturation. This results in the evolution of postfertilization development of embryo-nourishing female gametophyte tissues, a phenomenon unique among seed plants. Nonheterochronic developmental innovations have also played important roles in the evolution of the female gametophyte of Gnetum. Centripetal cellularization, which is always associated with the phase change from coenocytic to cellular organization among plesiomorphic seed plant female gametophytes, is lacking in Gnetum. Instead, during early phases of development, apomorphic free nuclear organization is coupled with a highly anomalous pattern of cellularization. Stage-specific innovations during early development in the female gametophyte of Gnetum do not affect plesiomorphic aspects of later phases of development. Thus, a complex array of heterochronic and nonheterochronic developmental innovations have played critical roles in the ontogenetic evolution of the highly apomorphic female gametophyte of Gnetum.     

Microtubules in early development of the megagametophyte of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Food storage tissue in the seeds of gymnosperms is female gametophyte (megagametophyte) that develops before fertilization, whereas, in seeds of angiosperms, food is stored as endosperm initiated by double fertilization. The megagametophyte is haploid, and endosperm is usually triploid, at least initially. Despite differences in origin, ploidy level, and developmental trigger, the early events of female gametophyte development in ginkgo are very similar to nuclear endosperm development in the seeds of angiosperms. In both, development begins as a single cell that undergoes multiple mitoses without cytokinesis, to produce a large syncytium. This study provided evidence that microtubule involvement in organization of the syncytium into nuclear cytoplasmic domains (NCDs) via nuclear-based radial microtubule systems is a critical developmental feature in the ginkgo megagametophyte, as it is in endosperm. Once the initial anticlinal walls have been deposited at the boundaries of NCDs, cellularization proceeds by the process of alveolation. Continued unidirectional growth of the alveolar walls is an outstanding example of polar cytokinesis. Ginkgo megagametophyte development appears to occur uniformly throughout the entire chamber, whereas nuclear type endosperm usually exhibits distinct developmental domains. These observations suggest that there is a fundamental pathway for the development and cellularization of syncytia in seed development.     

Development of endosperm in Arabidopsis thaliana

 The process of endosperm development in Arabidopsis was studied using immunohistochemistry of tubulin/microtubules coupled with light and confocal laser scanning microscopy. Arabidopsis undergoes the nuclear type of development in which the primary endosperm nucleus resulting from double fertilization divides repeatedly without cytokinesis resulting in a syncytium lining the central cell. Development occurs as waves originating in the micropylar chamber and moving through the central chamber toward the chalazal tip. Prior to cellularization, the syncytium is organized into nuclear cytoplasmic domains (NCDs) defined by nuclear-based radial systems of microtubules. The NCDs become polarized in axes perpendicular to the central cell wall, and anticlinal walls deposited among adjacent NCDs compartmentalize the syncytium into open-ended alveoli overtopped by a crown of syncytial cytoplasm. Continued centripetal growth of the anticlinal walls is guided by adventitious phragmoplasts that form at interfaces of microtubules emanating from adjacent interphase nuclei. Polarity of the elongating alveoli is reflected in a subsequent wave of periclinal divisions that cuts off a peripheral layer of cells and displaces the alveoli centripetally into the central vacuole. This pattern of development via alveolation appears to be highly conserved; it is characteristic of nuclear endosperm development in angiosperms and is similar to ancient patterns of gametophyte development in gymnosperms. Received: 21 September 1998 / Revision accepted: 17 November 1998     

The peroxin loss-of-function mutation abstinence by mutual consent disrupts male-female gametophyte recognition

In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.     

Development of the endosperm in rice (Oryza sativa L.): Cellularization

The syncytial endosperm of rice undergoes cellularization according to a regular morphogenetic plan. At 3 days after pollination (dap) mitosis in the peripheral synctium ceases. Radial systems of microtubules emanating from interphase nuclei define nuclear-cytoplasmic domains (NCDs) which develop axes perpendicular, to the embryo sac wall. Free-growing anticlinal walls between adjacent NCDs compart-mentalize the cytoplasm into open-ended alveoli which are overtopped by syncytial cytoplasm adjacent to the central vacuole. At 4 dap, mitosis resumes as a wave originating adjacent to the vascular bundle. The spindles are oriented parallel to the alveolar walls and cell plates formed in association with interzonal phragmoplasts result in periclinal walls that cut off a peripheral layer of cells and an inner layer of alveoli displaced toward the center. Polarized growth of the newly formed alveoli and elongation of the anticlinal walls occurs during interphase. The next wave of cell division in the alveoli proceeds as the first and a second cylinder of cells is cut off inside the peripheral layer. The periods of polarized growth/anticlinal wall elongation alternating with periclinal cell division are repeated 3–4 times until the grain is filled by 5 dap.     

A developmental and evolutionary analysis of embryology in Platanus (platanaceae), abasal eudicot

The Platanaceae are an early derived eudicot lineage and therefore occupy a key position for understanding reproductive character diversification associated with the early evolutionary radiation of flowering plants. We conducted an embryological study of Platanus racemosa in order to provide critical data on defining angiosperm reproductive characters for this important group. Female gametophyte development is monosporic. Embryogenesis occurs in a series of stages including zygote elongation and division, development of a linear proembryo, formation of the embryo proper, histogenesis, organogenesis, and growth. Endosperm development is a complex process that includes four distinct phases: free nuclear proliferation, cellularization of the chalazal zone, centripetal cellularization of the micropylar zone, and cellular differentiation and growth. Only the outer endosperm layer persists at seed maturity. Our findings differ significantly from previously published reports for Platanus, in which endosperm development was described as ab initio cellular. A comparison of endosperm development in Platanus with several closely and distantly related free nuclear taxa reveals considerable developmental variability, consistent with a hypothesis of multiple origins of free nuclear endosperm in angiosperms. Our analysis indicates that much remains to be learned about embryology in basal angiosperms. Additional developmental and comparative studies will likely reveal critical insights into the early evolution of flowering plants.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma