Abnormal macrosporogenesis in five alfalfa (Medicago sativa) mutants producing 4n pollen

Summary An analysis of micro- and macrosporogenesis in five diploid alfalfa mutants was carried out using a stain-clearing technique. All plants produced tetranucleated microspores and jumbo pollen due to the complete failure of the postmeiotic cytokinesis as well as bi- and trinucleated macrospores. The latter was due to the absence of cytokinesis after the first and second meiotic division of macrosporogenesis. Only one out of the five clones analyzed formed tetranucleated macrospores as a consequence of the total lack of cytokinesis after both meiotic divisions. The fusion of nuclei within binucleated macrospores resulted in 2n macrospores of the SDR type, recognizable on the basis of nucleolus dimension, confirming the ability of jumbo pollen (jp) mutants to produce 2n eggs at a high frequency. Nuclear fusion was also observed within tri- and tetranucleated macrospores. Although having the same genetic background, the five clones showed significant variability in the expression of abnormal cytokinesis during macrosporogenesis.Research supported by National Research Council of Italy, special project RAISA, sub-project no. 2, paper no. 527     

Nuclear DNA amount during sporogenesis and gametogenesis in sexual and aposporous buffelgrass

Nuclear DNA amounts (C values) were measured in Feulgen-stained sections of anthers and ovules of sexual plant B-2s (genotype aaaa) and aposporous cultivar Higgins (genotype AAaa) of buffelgrass (Pennisetum ciliare). The mass of the unreplicated nuclear genome of a gamete equals 1C DNA. In both lines, pollen mother cell nuclei were 4C before leptotene; anther wall, dyad, 1-nucleate pollen, and generative cell nuclei were 2C; microspore tetrad, enlarging microspore, and sperm nuclei were 1C. The tapetum persisted as uninucleate cells with 4C DNA. Archespores (2-4C) of both lines initiated meiosis to form megaspore tetrad nuclei with 1-2C DNA. In B-2s, chalazal megaspores (2-4C) formed reduced 8-nucleate Polygonum type embryo sacs, and sacs at 2- and 4-nucleate stages showed distributions with peaks near C1 and C2, corresponding to G1 and G2 cell cycle phases; this is characteristic of active mitosis. Nuclei of 8-nucleate sacs and of eggs and polars were 1C, indicating chromosomes were not duplicated before fertilization. Antipodal nuclei had levels from 1 to 36C, possibly due to polyteny or endopolyploidy. In Higgins, aposporous initials and 2-nucleate embryo sacs showed bimodal distributions of 2n nuclei with peaks at 2C and 4C DNA. Nuclei of newly formed 4-nucleate Panicum type aposporous sacs and of polars were 2C; aposporous eggs stained too faintly for reliable measurement.Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA     

Cytoembryological Studies on Polyembryonic Line SB-1 of Oryza sativa: Polyembryong and Its Origin

Anatomical observations, genetic and cytoembryological studies were conducted to determine the cytological factors in relation to the production of diploid polyembryony within one embryo sac occurring in polyembryonic line SB-1 of O. sativa L. The results showed that the diploid polyembryony in SB-1 was not originated from the fertilization of synergids, but presumably from the megagametophytes with supernumerary egg cells. Potentially polyembryonic megagametophytes at maturity contained either a conventional 3-celled egg apparatus (92.60% of megagametophytes) or the supernumerary eggs forming either a 4-celled (5.2 %) or 5-celled (2.2 %) egg apparatus. In megagametophyte with one additional supernumerary egg cells were almost situated side by side in adjacent to the synergids and recessed from the micropyle, whereas in megagametophyte with two additional supernumerary eggs, one egg cell occupied the conventional possition and the other two lay above the synergids. The arrangement of embryos in polyembryonic caryopsis at various developmental stages was concordant to that of egg cells in megagametophytes with supernumerary eggs. Karyotype analysis showed that most of the twins and triplets were of 2n--2n and 2n-2n-2n respectively. Polyembryony was observed in Fo and F1 caryopsis of cross SB-1 × Malaihong (Malaihong is of non-polyembryony) and in Fl caryopsis of its reciprocal but not in Fo caryopsis. This implies that polyembryony occurring in SB-1 is of a dominant character of embryo sac. Anatomical observations proved that each seedling from twins or triplets was completely independent with no vascular bundle connected with each other. The causes of polyembryony within one embryo sac, the entry of supernumerary sperms and the origin of supernumerary egg cells are discussed.     

四倍体双穗雀稗兼性无孢子生殖的研究

研究了四倍体双穗雀稗(Paspalum distichum L)无孢子生殖胚囊、胚胎发育以及假受精特点。当其大孢子母细胞发育至四分体阶段时,大多数情况下会发生四分体退化,同时有多个特化珠心细胞发育为1—3个无孢子生殖胚囊的现象。成熟无孢子生殖胚囊一般3核,包括1个卵细胞和2个极核。卵细胞在抽穗前就能自发分裂形成原胚团,而极核则在抽穗和传粉后参与假受精形成胚乳。当胚珠内存在多个无孢子生殖胚囊时,只是靠近珠孔端的1个无孢子生殖胚囊内的极核与精核结合,而其它的并不参与。种子成熟后出现很低频率的二胚苗。此外,还能观察到少量的有性生殖胚囊的发育以及有性生殖胚囊和无孢子生殖胚囊在同一胚珠中的发育现象,因此判断该类群为兼性无孢子生殖体。     

Cytological evidences of SDR-FDR mixture in the formation of 2n eggs in a potato diploid clone

Summary Macrosporogenesis and microsporogenesis were investigated in a diploid S. tuberosum x S. chacoense potato hybrid, characterized by more than 50% 2n egg formation. Fifty-five percent of dyad formation of 2n macrospores is ascribed to two meiotic abnormalities: omission of the second meiotic division, occurring at a frequency of 38%, and irregular spindle axis orientation at metaphase I at a frequency of 16%: These abnormalities give origin to a mixture of 2n eggs, composed of mostly second division restitution (SDR) and a small portion of first division restitution (FDR). Microsporogenesis showed rare dyads of 2n microspores depending on parallel spindles observed in anaphase II.Contribution no. 53 from the Center of Vegetable Breeding, CNR, Portici, Italy     

Abnormal Behavior of Nuclei and Microtubule (MT) Organizational Changes During Embryo Sac Development in the Poly-Egg Mutant,APⅣ of Rice

APⅣ is a rice mutant that develops poly-egg apparatus in its embryo sac. All the eggs that make up the poly-egg apparatus can be fertilized respectively resulting in the development of polyembryony. The routes taken in the development of polyembryony appear to fall mainly into three variant polygonum pattern types, designated as 5-2-1 , 5-3-0 and 6-2-0 types. Out of the embryo sacs of APⅣ studied about 50% exhibited variant polygonum type with associated abnormal nuclear behavior and microtubule organizational changes. Some of the major abnormal features shown by the three variant polygonum types were described and they included the following: For the 5-2-1 type At the beginning of the four-nucleate embryo sac development, one pair of nuclei became located to the micropylar end and the other pair to the chalazal end. As embryo sac further developed, long connecting microtubule (MT) bundles that existed between the two nuclei in the chalazal end play a role in the movement and positioning of that nucleus. As a result of the activities of these MT, one of the nuclei in the chalazal end moved to the micropylar end resulting in the micropylar end having three nuclei and the chalazal end only one. For the 5-3-0 type In the two-nucleate embryo sac of the 5-3-0 type, one nucleus remained at the micro-pylar end, while the other one became located near the central region. In the four-nucleate embryo sac, the pair of nuclei aligned in parallel to the micropylar-chalazal axis often having one of its nuclei relocated to the micropylar end as a result of associated MT activities. For the 6-2-0 type All the nuclei in the megaspore, two- and four-nucleate embryo sacs became located to the micropylar end. At the early stages of the eight-nucleate embryo sac development, the two nuclei in the central region of the embryo sac (originally at the micropylar end) became polar nuclei. All the other nuclei remained at the micropylar end were surrounded by reticulate MT. The relationship between abnormal behavior of nuclei and MT organi-zation in the development of rice embryo sac was discussed.     

In Vitro Embryogenesis in Unfertilized Embryo Sacs of Oryza sativa L.

Haploid rice plantlets were induced from cultured ovaries in our previously reported experiment. The present paper is an embryological study on this subject. Young flowers of two japonica cultivars were excised and cultured just in the same manner as before. Liquid medium used for float culture was N6+3% sucrose+0.125 ppm MCPA. The inoculated materials were checked to be at late uninuclcate pollen stage which corresponded mainly to the uuinuelcate embryo sac stage, but as well as some 2- or 4-nucleate embryo sacs. Samples were fixed at 23 days intervals in acetomethanol (1:3), stained in toto with diluted Ehrlich's hematoxylin and sectioned by paraffin method for microscopical observation. 4 days after inoculation most of the embryo sacs developed up to 8-nucleate stage with polarized differentiation of the egg apparatus, central cell and antipodals. From 7th day on, proembryos of various sizes and shapes appeared in the micropylar region of some embryo sacs; some consisted of meristcmatic ceils, others were highly vacuo]ated. One-celled as well as linear multicellular suspensors atypical of in vivo zygote proembryos were observed, ttowever, it was uncertain whether the proembryos originated from the egg cell, the synergids, or the differentiating egg apparatus as a whole. Another peculiar event occured during culture was the formation of endosperm-like free nuclei from the unfertilized polar nuclei in some embryo sacs. Sometimes the free nuclei were numerous and showed a tendency of cell formation in localized areas. 12–15 days after inoculation, the proembryos developed into mieroseopieal ealli with globular or pearlike shape, wbieh continued enlarging to visible size with naked eyes at about 18–24 th day. Further growth eventually led the ealli protruding out the ovary wall beyond 32–35 the day. These observations indicate that the embryo sue, similarly as the pollen, can be induced to embryogenesis in vitro. This may open a new way to study the mechanism controlling gametophytie and sporophytie developmental pathways of embryo sac and provide means for large-scale production of "embryo sac plants" in future.     

On the Fertilization of Oryza sativa L. × Sorghum vulgare Pets.

1.The pollen germination of Sorghum vulgate appeared normal on the stigma of the Oryza sativa, but the pollen tubes grew slowly in the style. Some of the pollen tubes may become enlarged in their tips or sometimes bursting, while others have continued to grow and entered the embryo sacs. 2. The growth rate of the pollen tubes varied widely. A few pollen tubes were observed in the embryo sacs of the materials 2 hours after pollination, but most of them entered the embryo sacs much later. 3. The zygote associated with a paucity of endosperm nuclei was observed in the materials 1 day after pollination. The double fertilization and 8–12-celled proembryo associated with a number of the free nuclei of the endosperm appeared with a rather high frequency (10.3%) in the materials 3 days after pollination. Some of them are normal in appearance and others may show more or less abnormalities. 4. No division figure was found except in one single case in which mitoses have occurred in both the proembryo and the endosperm. It is most likely that in such case the proembryo and the endosperm if left intact might develop further. 5. A 80-celled embryo was the biggest one which appeared in the materials 5 days after pollination. In general, no cells were ever formed in the endosperm, except in one instance among the 7 days materials the endosperm became cellular in micropylar end. In all other cases the endosperm either ceased to develop early or disorganized. The disorganized endosperm materials are considered to be utilized by the embryo. 6. In certain instances the free nuclei of the endosperm were not distributed at random. They were not equal in size and might fuse into giant nuclelei. 7. The most striking feature is that in the embryo sacs, in which double fertilization or proembryo and endosperm have occurred, a dark stained pollen tube was commonly present. This fact leads us to the conviction that in general only if a healthy pollen tube entered the embryo sac, double fertilization can take place and further development can proceed. 8. In certain cases the protoplasm of the embryo cells appeared scanty. It is apparently that the normal metabolism of the embryo was disturbed owing to the lack of nutrient, and the death of the embryo ensued. 9. No differentiated embryo was observed and no mature seeds were produced. The materials fixed 12 days after pollination showed a variety of abnormalities and collapses. The authors believe that the failure of seed production of rice X kaoliang was primarily due to the fact that the pollen tubes in the style grew too slowly to reach the embryo sacs in time. The consequence is that the double fertilization took place only in a late stage while the male and female gametes may have already become unhealthy. In addition, in this late stage the stored starch in the maternal tissues having gradually disappeared, the nutrient supply to the embryo sac was therefore limited and the young embryo and endosperm were finally in starvation.     

Localization of fibrillarin, 53 kDa protein and Ag-NOR proteins in the nuclei of giant antipodal cells of the wheat Triticum aestivum

Distribution of nucleolar argentophylic proteins, fibrillarin and 53 kDa protein, in highly polyploid nuclei of antipodal cells of Triticum aestivum L. was studied at different stages of the embryo sac development. The main results are as follows. 1. Ag-NOR proteins and fibrillarin form clusters are distributed in the giant nucleoli, whereas 53 kDa protein is mainly localized on the nucleolar periphery. Ag-NOR proteins and fibrillarin are accumulated as globular nucleolar-like particles--micronucleoli. 2. Dynamics of Ag-NOR proteins, fibrillarin and 53 kDa protein depends on the proliferative activity of endosperm cells. In embryo sacs with non-dividing endosperm cells at interphase stages, Ag-NOR proteins and fibrillarin were observed only within nucleoli and micronucleoli. In embryo sacs with dividing endosperm cells, fibrillarin and 53 kDa protein formed heterogeneous globular bodies varying in size. Simultaneously, some argentophylic material was observed in giant chromosomes. This may be due, presumably, to a partial or complete disappearance of the nucleoli of antipods and transition of some nucleolar components into the peripheral material of giant polytene chromosomes. We suggest that giant nuclei of antipodal cells may undergo cyclic transformation similar to those in the nuclei of dividing cells.     

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。     

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。     

Anatomy of Watermelon Embryo Sacs Following Pollination, Non-pollination or Parthenocarpic Induction of Fruit Development

There is little information on the fate of embryo sacs in plantovules if pollination is prevented. In this study embryo sacsfrom watermelon were observed over a 13 day period followingflowering with (a) normal pollination, (b) non-pollination and(c) induction of parthenocarpic fruit development with naphthaleneacetic acid. Following pollination, and prior to fertilizationapproximately 2 days later, the embryo sacs completed developmentand consisted of two synergids with prominent filiform apparatus,an egg cell, a central cell with two polar nuclei and threeantipodal cells. Sperm nuclei were observed within the embryosac at 2 days and by 4 days the endosperm was proliferating.In the non-pollination treatment the embryo sac was still intactafter 4 days although the antipodal nuclei were becoming hardto distinguish. By 7 days only the two synergids and the eggcell were still well defined, the polar nuclei appeared in somepreparations to be fused, and the antipodals had degenerated.By 10 days the embryo sac was a structure-less watery mass.In parthenocarpic fruit the fate of the embryo sac was similarto that in non-pollinated fruit except that final breakdownwas delayed past 10 days. Maturity of the majority of embryo sacs in an ovary appearedto be contemporaneous with penetration of the pollen tube, andon the basis of the anatomical results it seems possible thatembryo sacs could be fertilized up to 2 days beyond the normaltime. Citrullus lanatus, watermelon, embryo sac, anatomy, pollination, parthenocarpy     

The Development of Gynoecium after Anthesis and Fertilization in Eleutherococcus senticosus (Araliaceae)

The development status of gynoecia in Eleutherococcus senticosus flowers is different from that in ordinary plants. Female gametophytes of E. senticosus have not become mature until the 6th day after anthesis. On the 6th day, 82.25% of embryo sacs in female plants, and 67.25％ of those in hermaphroditic plants become mature, while the rest are sterile, immature or degenerated with no fertilized embryo sacs observed. At the same time, all embryo sacs degenerated and flowers withered in male plants. On the 7th day, a few embryo sacs in female and hermaphroditic plants start being fertilized. Accompanying the differentiation of embryo sacs, styles of female and hermaphroditic flowers start to expand and their nectaries become mature gradually. After the 4th or 6th day of anthesis, stigmatic papillae become conspicious and stigmata become white and open. In the meantime, the stigmata become receptive and the nectaries get active or reactive. By the 9th or 10th day, 40～65 ％ of embryo sacs in female plants and 25～41% of those in hermaphroditic plants have been fertilized. The whole process of fertilization in E. senticosus was observed. About 2 or 3 days after pollination, the two sperm nuclei start to fuse with the egg and the secondary nucleus. The fertilization of E. senticosus belongs to the premitotic type of syngamy. The essential process of the fusion of male and female nuclei during syngamy may be generalized as follows: (1) the contacting of male nucleus with the female one; (2) the fusion of nuclear membranes between the male and female nuclei; (3) the despiralization of male spireme and the appearance of male nucleolus inside the fertilized female nucleus; (4) the dispersion of male chromatin and the mergence with the female chromatin, which is the sign of completion of the fusion of the two nuclei. In addition, degeneration types of mature embryo sacs were observed. And typical polyspermy and a series of cases in which extra sperms enter the em-bryo sac are recorded.     

Mosaic haploid-diploid embryos and polyspermy in the tellinid bivalve Macoma balthica.

We investigated meiosis, fertilization, and early development in eggs of the tellinid bivalve Macoma balthica (L.), which has external fertilization. Meiosis is standard but polyspermy is found to be very common. In all eight crosses examined, mosaic embryos consisting of a mixture of diploid (2n = 38) and haploid cells occur at a frequency ranging from 2.7 to 29.1%. The earliest mosaic found is in the two-cell stage. We propose that an androgenic haploid cell lineage can originate from one supernumerary sperm that decondenses into a functional haploid nucleus, starts mitosis, and is incorporated in the developing embryo.     

Nuclear size and DNA content of the embryo and endosperm during their initial stages of development in Glycine max (Fabaceae)

A technique was developed for isolating embryo sacs from ovules of soybean and for separating embryo from endosperm. Image analysis and cytophotometry were used to determine the relative mass of DNA and size of nuclei of endosperm and embryo cells. Analyses were done at the globular through late heart-shaped embryo stages to correlate ploidy level or nuclear size, and differentiation in these tissues. Mean size of embryo nuclei was fairly constant through all stages studied. Ploidy condition of the embryo was stable, 95%–99% of the nuclei were distributed in a bipolar pattern by relative mass at 2C and 4C. Few embryo nuclei (3%) had ploidy levels above 4C at the late heart-shaped embryo stage. Variability in size of endosperm nuclei seemed correlated with the morphological state of these nuclei (free-nuclear vs. cellular). Most endosperm cells did not show significant polyploidy with 84%–92% of nuclei in the expected 3C–6C range, but some nuclei with elevated ploidy levels were noted during endosperm cellularization. Endosperm senescence was correlated with nuclear DNA loss over time. Polyploidy seems to have no direct role in the early differentiation of the soybean embryo and endosperm, but these stable conditions may be necessary for the early establishment of the embryo.     

甜菜单体附加系M14无融合生殖的细胞胚胎学研究

利用常规研究方法,对甜菜单体附加系M 14品系(B eta vu lg aris L.,VV 1C、2n=18 1)的生殖方式进行细胞学与胚胎学研究.结果表明:(1)甜菜单体附加系M 14的4代细胞学检查表明:染色体组分别为VV 1C、2n=18 1;VV 0、2n=18 0;VV 2C、2n=18 2;VVV 0、2n=27 0;VVV 1C、2n=27 1;VVV 2C、2n=27 2等,其中VV 1C、2n=18 1的植株传递率平均为96.7%,表现为稳定传递,具有二倍体孢子无融合生殖特性;其余各种分离植株的传递率总计为3.25%,有性生殖发生率较低.(2)胚胎学研究表明,二倍体孢子无融合生殖的胚珠中,珠孔处看不到花粉管,胚囊没有发生受精作用.2个助细胞提前退化,半数卵细胞的极性与正常卵细胞相反;卵与次生核不经受精而自发分裂,卵细胞自发分裂产生无性胚,次生核自发分裂产生核型胚乳,而且次生核自发分裂早于卵细胞分裂;有性生殖胚珠中,珠孔处可见多条花粉管,胚囊里见到精卵融合的图像.表明甜菜单体附加系M 14是以二倍体孢子无融合生殖为主要繁殖方式,有性生殖为次要敏殖方式的兼性无融合生殖体.     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsis binata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料.龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%.胚囊发育属大黍型.多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%.胚珠内没有有性胚囊的发育.胚的发生有两种类型:(1)早发生胚(74%),开花前1～2 d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2～3 d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚.存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%.胚乳由极核不经受精自发分裂产生.     

The indeterminate gametophyte1 gene of maize encodes a LOB domain protein required for embryo Sac and leaf development

Angiosperm embryo sac development begins with a phase of free nuclear division followed by cellularization and differentiation of cell types. The indeterminate gametophyte1 (ig1) gene of maize (Zea mays) restricts the proliferative phase of female gametophyte development. ig1 mutant female gametophytes have a prolonged phase of free nuclear divisions leading to a variety of embryo sac abnormalities, including extra egg cells, extra polar nuclei, and extra synergids. Positional cloning of ig1 was performed based on the genome sequence of the orthologous region in rice. ig1 encodes a LATERAL ORGAN BOUNDARIES domain protein with high similarity to ASYMMETRIC LEAVES2 of Arabidopsis thaliana. A second mutant allele of ig1 was identified in a noncomplementation screen using active Mutator transposable element lines. Homozygous ig1 mutants have abnormal leaf morphology as well as abnormal embryo sac development. Affected leaves have disrupted abaxial-adaxial polarity and fail to repress the expression of meristem-specific knotted-like homeobox (knox) genes in leaf primordia, causing a proliferative, stem cell identity to persist in these cells. Despite the superficial similarity of ig1-O leaves and embryo sacs, ectopic knox gene expression cannot be detected in ig1-O embryo sacs.     

Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection

 Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac. The results show that the mature embryo sac was located deep inside the hemitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of flowers 1 day after anthesis, and treated with an enzyme solution for digesting cell walls on a gyratory shaker. After 12 h of enzyme treatment, these ovules were dissected using a glass needle under an inverted microscope to release the embryo sacs. The embryo sacs, characterized by their specific size, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetate staining. Fluorescent staining with 4,6-diamidino-2-phenylindole revealed the nuclei of the egg apparatus in the isolated embryo sacs. The procedure for isolating embryo sacs established in this study will offer a new approach to further in vitro studies on fertilization in Dianthus. Received: 20 January 1999 / Revision received: 12 July 1999 / Accepted: 17 August 1999     

Fertilization in maize indeterminate gametophyte1 mutant

Summary. Mature embryo sacs of the maize mutant indeterminate gametophyte1 displayed different cellular patterns compared to those of the wild type. About 40% of the ig1 embryo sacs contained three or more synergids and two or more egg cells at the micropylar end. During fertilization in embryo sacs with two synergids, both of them frequently degenerated and were penetrated by two pollen tubes. 75% of the embryo sacs containing three or more synergid cells were penetrated by two or more pollen tubes, although most of them had only one degenerated synergid. Multiple fusions between the sperm cells and eggs frequently occurred in the same embryo sac, which subsequently generated multiple embryos. There were two or more central cells in about 33% of ig1 embryo sacs. The largest central cell was usually adjacent to the egg apparatus and contained two unfused polar nuclei, while those extra central cells located at the chalazal end usually had a single nucleus. Fertilization occurred only between the male gamete and the largest binucleate central cell. The extra central cells eventually degenerated after fertilization.Present address: GI Basic Research Center, Mayo Clinic, Rochester, Minnesota, U.S.A.Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, Peoples Republic of China.