Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indicaljaponica Hybrids in Rice

Embryo sac abortion is one of the major masons for sterility in indicaljaponica hybrids In rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indicaljaponica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagamatogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucallus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.     

Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indica/japonica Hybrids in Rice

Embryo sac abortion is one of the major reasons for sterility in indica/japonica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indica/japonica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.     

A New Method for Embryo Sac Isolation and In Situ Fusion of Egg and Synergid Protoplasts in Nicotiana tabacurn

A new method combining enzymatic maceration with osmotic shock was developed for isolation of living embryo sac and its protoplasts in Nicotiana tabacum L. The principle of this method was that the ovules submitted to enzymatic treatment and osmotic shock could release embryo sacs along with some internal ovular cells through either the funicle cut end or the micropyle. Factors affecting embryo sac isolation were investigated, including concentration of mannitol as a shock osmoticum and in enzymesolution ,duration of enzymatic maceration,and duration of osmotic shock. As a result a procedure was established: Ovules at mature embryo sac stage were macerated for 2. S h in 1 %–1.5% cellulase R-10 and 0. 5% macerozyme R-10 (or 1% Pectinase,Serva) dissolved in 13% mannitol solution using microshaker,followed by osmotic shock for 15–30 min with enzyme free 8% mannitol solution and gentle agitation using a pipette. Using a capillary,50–70 embryo sacs could be collected manually in one hour. The embryo sacs thus isolated could be kept viable from which protoplasts of egg cell and other componcnt cells could be further isolated. An additional interesting phenomenon was that osmotic shock often caused in situ fusion the protoplasts of egg cell and synergids. The rate of fusion ranging 9%—71.9% could be controlled by modification of the procedure. This phenomenon merits further attention both from basic and practical point of view. The present method gives the advantages of faciliting isolation and promoting good harvest of viable embryo sacs/female protoplasts within a relative short time.     

利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察

应用改进的整体染色透明激光扫描共聚焦显微术(WCLSM),对同源四倍体水稻PDER-2B-4x胚囊的形成与发育过程进行观察。发现其胚囊的形成发育过程与二倍体的一致,可以清楚地划分为8个发育时期,即孢原细胞形成期、大孢子母细胞形成期、大孢子母细胞减数分裂期、功能大孢子形成期、单核胚囊形成期、胚囊有丝分裂期、八核胚囊发育期和成熟胚囊期。除正常发育的过程外,大孢子发育的各个过程均出现一些异常现象,包括:细胞退化、核位置异常、核数目异常和细胞分化异常等。这些异常可能最终导致多种结构异常成熟胚囊的形成。     

Study on the Structure and Genetic Polymorphism of Embryo Sac in Polyembryonic Rice Strain AP Ⅳ

By using the technology of GMA half-section the comparative studies on the structure of embryo sac between polyembryonic rice strain AP IV and haploembryonic rice cuhivar “IR36” showed: In AP IV, except 7.8% of ovaries in which the embryo sac degenerated, there were only 27.1% of ovaries in which the structure of embryo sac was similar to that of "IR36' which was of the Polygonum type; 65.1 % of ovaries were different from “IR36”, in which some changes happened in the embryo sac: Of most two kinds of variant embryo sac, referred as poly-egg apparatus embryo sac (53.3%) and double set of embryo sac (6.3%) were found. In the poly-egg apparatus embryo sac, three egg-apparatuses were the majority which could be further divided into three types, namely, 5-2-1 type, 6-2-0 type and 5-3-0 type, based on the whole structure of embryo sac. The double set of embryo sac characterized with a large embryo sac inlaid with a small embryo sac, and with the eggs far from the micropyle part. Moreover, the other variants were also found. According to the analysis, genetic polymorphism of the structure of embryo sac was found in single-panicle in AP Ⅳ.     

鹤顶兰胚囊发育过程中Ca~（2＋）分布的超微细胞化学定位（英文）

用焦锑酸盐沉淀法对鹤顶兰（Phaius tankervilliae）胚囊发育过程中的Ca2＋状态进行超微细胞化学定位。观察结果发现：功能大孢子时期,珠孔端的胚囊壁上开始出现小颗粒的Ca2＋沉淀,但功能大孢子细胞内未见明显的Ca2＋标记;四核胚囊时期胚囊壁上的Ca2＋沉淀明显增多,液泡膜上有Ca2＋沉淀出现,珠孔处的Ca2＋沉淀颗粒较大;成熟胚囊时期,胚囊壁上的Ca2＋沉淀进一步增多,且胚囊内Ca2＋分布明显增多,且极性明显,珠孔端助细胞、卵细胞比合点端反足细胞有更多的Ca2＋沉淀。鹤顶兰成熟胚囊内Ca2＋积累的来源有：（1）在胚囊成熟前主要由珠被细胞、珠细胞通过胞间连丝向胚囊运输;（2）以沉淀有大量Ca2＋的小泡形式跨过胚囊壁进入胚囊。     

Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.     

大叶杨配囊及胚珠的形成和发育

本文应用细胞化学方法研究了大叶杨胚珠、胚囊的形成和发育过程中核酸、蛋白质及不溶性多糖的分布和消长。大孢子母细胞、大孢子四分体及功能大孢子中含较少不溶性多糖,但却含丰富的RNA和蛋白质。功能大孢子经分裂发育成八核的蓼型胚囊。四核胚囊开始积累细胞质多糖,成熟胚囊中除反足细胞外充满淀粉粒。反足细胞形成后不久即退化。助细胞具多糖性质的丝状器,受精前两个助细胞退化。卵细胞核对Feulgen反应呈负反应。二极核受精前由胚囊中部移向卵器,与卵器接触后融合形成次生核。发育早期的胚珠为厚珠心,双珠被。晚期,内珠被退化,故成熟胚珠为单珠被。四核胚囊时期,珠孔端珠心组织退化,胚囊伸向珠孔形成胚囊喙。合点端珠心组织含丰富的蛋白质和核酸,这一性质与绒毡层性质相似,可能涉及胚囊的营养运输。胚囊的营养来源于子房和胎座细胞内贮存的淀粉粒。     

水蔗草胚珠附器的研究

本文对水蔗草的胚珠附器进行研究,结果表明：在功能大孢子时期,珠孔端的1～3个珠心细胞开始特化,发育成胚珠附器;胚珠附器发生时,有些胚珠同时出现无孢子生殖原始细 胞;有性生殖和无孢子生殖的胚囊中均有胚珠附器存在;但在无孢子生殖的胚囊中,胚珠附器一般很大,长约是宽的1～３倍;而有性生殖胚囊的胚珠附器的长约是宽的1～２倍;和有性生殖胚囊相比,无孢子生殖胚囊的胚珠附器更加发达;存在发达的胚珠附器是水蔗草无孢子生殖胚囊的特点之一。     

水蔗草胚珠附器的研究

本文对水蔗草的胚珠附器进行研究,结果表明：在功能大孢子时期,珠孔端的1—3个珠心细胞开始特化,发育成胚珠附器;胚珠附器发生时,有些胚珠同时出现无孢子生殖原始细胞;有性生殖和无孢子生殖的胚囊中均有胚珠附器存在;但在无孢子生殖的胚囊中,胚珠附器一般很大,长约是宽的1—3倍;而有性生殖胚囊的胚珠附器的长约是宽的1—2倍;和有性生殖胚囊相比,无孢子生殖胚囊的胚珠附器更加发达;存在发达的胚珠附器是水蔗草无孢子生殖胚囊的特点之一。     

Embryo Sac Development in Almond [Prunus dulcis (Mill.) D. A. Webb] as Affected by Cross-, Self- and Non-pollination

Embryo sac development in ‘Nonpareil’ almond wasstudied following cross-, self- and non-pollination under fieldand greenhouse conditions. The embryo sac, which develops accordingto the Polygonum type, does not begin to differentiate untilanthesis in contrast to other Prunus spp. where a well-developedembryo sac is present at the time of flower opening. The developingmegagametophyte appears to be isolated from surrounding nucellartissue by the deposition of a ring of callose, which, as indicatedby aniline blue-induced fluorescence in the walls of nucellarcells, encloses the embryo sac during its elongation. Developmentand growth of the embryo sac following the different pollinationtreatments indicated that embryo sac development was stimulatedby the presence of compatible pollen tubes in the style andfinal elongation growth of the embryo sac was promoted by cross-pollination.Irregularities in megagametophyte development, including delayeddifferentiation of the megaspore mother cell, embryo sac abortionand lack of polar nuclei fusion and embryo sac elongation, werefrequently noted in ovules of self- and non-pollinated flowers. Almond, callose, embryo sac, megagametophyte, pollination, Prunus dulcis (Mill.) D. A. Webb.     

Isolation and preservation of the living embryo sac ofCrinum asiaticum L. var.japonicum baker

The enzymatic maceration method was used to isolate an intact embryo sac ofCrinum asiaticum and its component cells. Best results were obtained when using enzyme solutions that contained pectinase hemicellulase, cellulase and pectolyase. Aseptic ovules were incubated in the enzyme solution for 1.5 hr at 25 C. This allowed the isolation of embryo sacs to yield up to 20% of the amount present. An isolated embryo sac usually consists of an egg cell, synergids, antipodals and a central cell. Some embryo sacs can be digested as gametophytic protoplast. The size, shape and position of the isolated embryo sac seemingly possessed similarities with those of the fixed embryo sac in the ovary. An isolated embryo sac can be in a living state when the result of the fluorochromatic reaction (FCR) and protoplasmic streaming is positive. When cultured in proper media, 68% of the isolated gametophytic protoplasts were observed to have sustained their positive FCR for more than 1 month.     

水稻胚囊壁的形成与发育观察

通过透射电镜对水稻(Oryza sativa L.)功能大孢子形成开始至胚囊成熟期间胚囊壁的形成与发育进行观察,结果表明:水稻胚囊壁是在原有功能大孢子壁的基础上,通过与其周围退化珠心细胞留下的壁相叠合,使壁加厚。功能大孢子近合点端壁存在胞间连丝,其中个别胞间连丝可保留到八核胚囊。胚囊壁上内突最早于四核胚囊近珠孔端发生。八核胚囊形成后,内突的发育在胚囊不同的细胞中表现不同,其中以中央细胞最具特点,表现为先在中央细胞与珠心相接的近珠孔端和近合点端两个区域的胚囊壁上形成,以后近珠孔端胚囊壁上的内突大量增加,而近合点端的却增加不明显,中部胚囊壁上的内突出现的时间相对较晚。到胚囊成熟时,近珠孔端胚囊壁上内突的分布密度最大,中部次之,近合点端的最小,三个区域上内突的形态各异。反足细胞与珠心相接的胚囊壁上内突的形成时间较早,但以后的发育却相对缓慢,数量增加不明显。2个助细胞交界处胚囊壁上的丝状器在胚囊未明显膨大时已形成。卵细胞除在与助细胞交界处的壁外,其它部位不形成明显的内突结构。     

Fertilization of Different Types of Embryo Sacs and Its Embryo Formation in Polyembryonic Rice Strain APⅣ

The ferflization and its embryo fonnation of different types of embryo sacs were studied by using the technology of GMA half-section in the APIV strain of polyembryonic rice ( Oryza sativa L. ). In rare cases,all three egg cells in the embryo sac of 5-2-1 type could fertilize and develop into three embryos. But in most cases only one or two egg cells fertilized and developed into one or two embryos in the respective type of embryo sac. The frequency of poly-egg fertilization in total all was very low in the embryo sac of 6-2-0 type and 5-3-0 type. These results indicated that the polyembryos in APIV originated mainly from overall fertilization and develotment of the embryo sac with poly-egg apparatus. This was observed,for example, in 5-2-1 type in which three embryos were fertilized and developed from three egg cells and two embryos from two egg cells. The fertlization process of double set of embryo sac was most complicated, all often abnormal. Only in few ovaries the egg cells in both large and small embeyo sac could fertilize simultaneously. The fertilization and development of egg cells in the large embryo sac might be the main cause of the formation of the so called "mid-seated embryo" (the embryo far from the micropyle end).     

多胚水稻品系APⅣ不同类型胶囊的受精及其胚胎形成

通过GMA半薄切片技术对APⅣ不同类型水稻(Oryza sativa L.)胚囊的受精及其胚胎发育的研究表明,APⅣ中5-2-l型胚囊的3个卵细胞在少数情况下都可受精并发育形成3个胚;但多数情况只有 1个或2个卵细胞受精发育成1个胚或2个胚。6-2-0型和5-3-0型胚囊多个卵受精频率都很低。由此证明APIV多胚是来自如5-2-1型胚囊的多卵卵器胚囊多个卵细胞都受精的结果,其中3胚来自3个卵细胞受精发育,2胚来自2个卵细胞受精发育。双套结构胚囊受精最为复杂,多数情况是受精不正常,只有少数子房大、小胚囊中的卵细胞都能正常受精。大胚囊中的卵细胞受精发育可能是形成所谓中位胚(远离珠孔端胚)的主要原因。     

广东高州普通野生稻生殖特性的研究Ⅱ.胚囊育性、胚囊发育、胚胎发生和胚乳发育

利用整体染色激光扫描共聚焦显微镜术（WCLSM）,对采自广东省高州市6个地点共141个编号的高州普通野生稻的成熟胚囊育性和胚囊形成发育特点等进行研究。结果表明,供试的绝大多数高州普通野生稻材料成熟胚囊均存在不同程度的育性异常现象,包括雌性生殖单位退化、极核位置异常、极核数目异常、胚囊退化等。这些异常结构的胚囊由于没有正常的卵细胞,不能正常受精,影响子粒结实。141个编号平均异常胚囊频率为11．11％,最高异常率为67．86％。高州普通野生稻胚囊发育过程与正常栽培稻一致,属寥型。对一些结实率偏低材料的研究,发现在胚囊发育过程的不同时期存在一些异常现象,包括功能大孢子退化,二至八核胚囊发育异常等。对柱头上的花粉量调查,发现观察的69个编号中,多数编号柱头上花粉量偏少。研究表明,花粉量偏少影响受精是导致结实率偏低的最主要原因之一。本文对导致结实率偏低的综合因素进行了讨论。     

Iatercellular Migration of Protoplasm and Its Relation to the Development of Embryo Sac in Nucellus of Wheat

During the development of the ovule before pollination, deterioration of successive layers of nucellar tissue, beginning from the nnermost, constantly takes place and consistently forms a zone of disorganization surrounding the periphery of the enlarging embryo sac. Nucellar tissue deteriorates much more profusely near the antipodal end of the sac. "Nuclear extrusion" taken as an indication of intercellular movement of the protoplasm which has undergone partial disassembly, can be seen among the nucellar tissues and between the nucellus and the embryo sac. The intruding nuclear fragments, some of which assume the form of nucleolus, can be found in the antipodal cells. The results interpreted according to our previous hypothesis, are as follows. The nucellar cell by means of intercellular movement of its own protoplasm in the state of partial disassembly, furnishes the embryo sac with composite units of various polymers and organelles. Consequently, the antipodal cells proliferate and flourish The interrelationship between nucellus and embryo sac has been discussed from the viewpoint of supply and utilization of nourishment, which is necessary for the rapid development of the embryo sac.     

Isolation of Viable Embryo Sacs and Their Protoplasts of Nicotiana tabacum

Isolation of fixed and fresh embryo sacs has been reported. However,the isolation of protoplasts of embryo sac elements is reported here for the first time.The protoplasts of egg cell, synergids, central cell and antipodal cells have been isolated with the retaining of their viability. Though this is a preliminary work, it indicatesthe potentiality of isolation of naked female gametes of angiosperms, which may beused in genetic manipulation and plant biotechnology. Nicotiana tabacum was grown in the greenhouse of the Department of Biology,Peking University. From opened and unpollinated flowers, the ovaries were removedand sterilized with 70% alcohol. The ovules were dissected out from those ovaries andfollowed by incubation (4–8 hrs. 28℃) in anenzyme solution containing 2% driselase, 0.65 M mannitol and 0.25% potassium dextran sulfate. Ovules from 3 4 ovariescould be incubated with 1 ml of enzyme solution in a 3 cm petri dish. All these manipulations and the following procedures were carried out under sterile conditions. Afterincubation, ovules were washed 3 times with a washing solution of 0.65 M mannitol.The isolated embryo, sacs and their protoplasts were obtained by gently squashing digested ovules in a small volume of washing solution on a slide. When the fresh ovules were incubated 3–3.5 hrs in the enzyme solution, the embryosacs may be successfully isolated in an intact manner, either for mature or immatureembryo sacs. The isolated embryo sac looked plump, viable and very distinct in itsstructure. If the isolated embryo sacs were incubated in 0.01% fluorescein diacetate(FDA) used as a test for the viability of the embryo sac, and observed under fluorescein microscope, the cytoplasm of all embryo sac elements, including egg cell, synergids,central cell and antipodal cells, showed strong fluorescence. It is proved that these iso-lated embryo sacs are still viable. When the incubation of ovules was prolonged as to 8 hrs in certain cases, theboundary wall of the embryo sac may be partially digested and the protoplasts of embryo sac elements came out from micropylar or chalazal end after squashing. The difference of the protoplasts derived from different embryo sac elements could be recognized by their relative size and other characteristics. The egg protoplast is smallerthan that of the synergid. However, the protoplasts of antipodal cells were. obviouslysmaller than that of egg. But the central cell protoplast was the largest among theseprotoplasts and possessed two polar nuclei and a very large central vacuole. All theseisolated protoplasts of embryo sac elements were also proved viable with FDA method. The importance of isolated protoplasts of embryo sac elements is discussed withrespect to genetic manipulations.     

Development and structure of the female gametophyte in <Emphasis Type="Italic">Austrobaileya scandens</Emphasis> (Austrobaileyaceae)

Austrobaileyales, comprising the four families Austrobaileyaceae, Trimeniaceae, Schisandraceae, and Illiciaceae, are included in the basal angiosperms along with Amborellaceae and Nymphaeaceae. Here, we present the first developmental study of the female gametophyte in Austrobaileya scandens, the only species of Austrobaileyaceae, which are sister to the rest of the Austrobaileyales. Austrobaileya scandens has a four-celled/four-nucleate embryo sac as in the derived families of the order, e.g., Illiciaceae and Schisandraceae. It is monosporic, with the chalazal megaspore of a tetrad developing into the embryo sac composed of an egg cell, two synergids, and one polar nucleus. This mode of embryo sac formation was first reported in Schisandra over 40 years ago and should now be established as the Schisandra type. Its occurrence in A. scandens shows that the Schisandra-type embryo sac is likely common to the whole Austrobaileyales as well as to Nymphaeaceae. Amborellaceae were recently reported to have an eight-celled/nine-nucleate embryo sac, clarifying that none of the basal angiosperms has the seven-celled/eight-nucleate Polygonum-type embryo sac found in the majority of angiosperms, and that the Polygonum-type embryo sac represents a derived character state in angiosperms.