Microscopical Observations on the Embryoid Formation in Cultured Unfertilized Ovules of Helianthus annuus L.

A recent advance in plant experimental embryology is the induction of haploid plants via in vitro culture of unfertilized ovules. Using float culture method on liquid media, we have raised haploid as well as diploid embryoids in sunflower cultivars by ovule culture. The present investigation was aimed to know the origin and developmental processes of these embryoids. Young flowers 1–4 days before anthesis were dissected and ovules were inoculated on N6 medium supplemented with 0.5–2 ppm MCPA and 6% sucrose. During culture period, samples were collected at intervals, fixed, stained and sectioned by paraffin method. Fifty one gynogenie embryoids of various sizes were observed among Ca. 2000 ovules. They were located at the micropylar end of the embryo sacs and proved to be originated from the unfertilized egg cells. At the early stages, they bore a strong resemblance to the zygotic proembryos in vivo, but after a considerable enlargement, they grew into globular, ovoid or elongated big bodies without polarized organ differentiation. Chromosome counts on some mitotic figures in these embyoids revealed their haploid nature. Embryoids were also produced from the endothelial tissue, which proliferated markedly after inoculation, especially at the chalazal parts, resulting in massive multilayered irregular folds and then degenerated. In some eases, cell divisions at one or several places led to embryoid or callus formation. The problems of how to regulate the growth of in vitro ovules in order to promote the gynogenic embryoids and inhibit the somatic embryoids or calli are left for future research.     

Further Embryological Studies on the in Vitro Apogamy in Oryza saliva L.

In the cultured young ovaries of rice, the processes of megagametophyte develop- ment could be switched to the formation of various abnormally organized embryo sacs and then to the initiation of synergid apogamy. The main pathway leading to apogamy was found to go via a linearly oriented 4-nucleate embryo sac to the formation of a li- nearly oriented egg apparatus, from which it was usually the chalazal synergid giving rise to an apogamous proembryo, and the micropylar synergid degenerated. The proembryo thus produced was located at the base of a vacuolated egg cell (Plate Ⅰ, 1–7). The second pathway went through a nonlinearly oriented 4-nucleate embryo sac to the formation of an egg apparatus in which the two synergids were located at one side of the egg and oriented longitudinally. In this case it was often the chalazal synergid that could be triggered to apogamy, resulting in a hook-shaped proembryo embracing the egg cel1 from one side (Plate Ⅰ, 8–11). When ovaries with nearly matured embryo sac were cultured, in a few cases where apogamy was induced, the proembryos observed were all situated at one side of the egg and were hook-shaped (Plate Ⅰ, 12). All these pathways are summarized in a diagram (Fig. 23). Some interesting changes were observed in the synergid and the egg cell of the cultured ovaries by PAS reaction and mercuric bromphenol blue staining. The egg cells, in contrast to in vivo condition, often contained abundant starch grains,. The synergids and synergid proembryos were rich of cytoplasmic protein (Plate Ⅱ, 13, 14). We supposed that the egg may supply some nutrients as well as stimulants to the developing synergid in the course of apogamy. The distribution of starch and protein in apogamous embryoids during subsequent development was also described in this paper (Plate, 15–22).     

Regulation of In Vitro Parthenogenesis and Somatic Proliferation in Sunflower by Several Factors

Culture of unfertilized ovules in sunflower (Helianthus annuus L.) could be induced to form parthenogenetic embryoids, endothelial embryoids and integumentary calli. Various factors exerted regulatory influences on the production of these structures. The key factors were exogenous hormone, sucrose concentration and cold-pretreatment. In hormone-free condition, parthenogenesis was promoted without any occurence of somatic embryoids or calli. Addition of exogenous hormone MCPA induced proliferation of sporophytic tissue and meanwhile, reduced the induction frequency of parthenogenesis. Relatively high sucrose level favored the Froduction of parthenogenetic embryoids. Reducing sucrose concentration favored the integumentary calli. The optimal sucrose concentration for endothelial embryoids was intermediate between them. Cold-pretreatment also played an effective role in promoting parthenogenesis and inhibiting integumentary calli. By combining these three factors, we were able to. work out a culture technique which significantly enhanced the inductionfrequency and growth. of parthenogenetic embryoids and completely eliminated somatic embryoids and calli.     

An electron microscope study on in vitro parthenogenesis in sunflower

Summary Electron microscope studies have been conducted on the parthenogenesis induced by in vitro culture of unfertilized ovules of sunflower (Helianthus annuus). In comparison with the state of the egg prior to inoculation, some eggs 5 days after culture show striking ultrastructural changes, which include, among others, nuclear migration, an increase in the number and activity of the organelles, a loss of polarity and wall formation at the chalazal end of the cell. Most of these changes are similar to those that occur normally in the zygote, indicating that parthenogenic development has been triggered in these eggs. Such eggs have been termed activated and are presumed to be capable of undergoing parthenogenesis. The parthenogenic proembryos which result share some features in common with zygotic proembryos. In addition, some parthenogenic proembryos exhibit unique properties not found in zygotic proembryos. These include embryos that consist of two parts differing markedly in density, an inversion of polarity, the frequent occurrence of autophagic vacuoles, the thickening of cell walls, a centripetal growth mode of wall formation, the appearance of an incomplete cell wall, free nuclear division, amitosis and degeneration. We believe that these ultrastructural peculiarities are the effects of in vitro culture.     

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。     

The cytological changes of tobacco zygote and proembryo cells induced by beta-glucosyl Yariv reagent suggest the involvement of arabinogalactan proteins in cell division and cell plate formation

ABSTRACT: BACKGROUND: In dicotyledonous plant, the first asymmetric zygotic division and subsequent several cell divisions are crucial for proembryo pattern formation and later embryo development.. Arabinogalactan proteins (AGPs) are a family of extensively glycosylated cell surface proteins that are thought to have important roles in various aspects of plant growth and development, including embryogenesis. Previous results from our laboratory show that AGPs are concerned with tobacco egg cell fertilization and zygotic division. However, how AGPs interact with other factors involved in zygotic division and proembryo development remains unknown. RESULTS: In this study, we used the tobacco in vitro zygote culture system and series of meticulous cell biology techniques to investigate the roles of AGPs in zygote and proembryo cell division. For the first time, we examined tobacco proembryo division patterns detailed to every cell division. The bright-field images and statistical results both revealed that with the addition of an exogenous AGPs inhibitor, beta-glucosyl Yariv (beta-GlcY) reagent, the frequency of aberrant division increased remarkably in cultured tobacco zygotes and proembryos, and the cell plate specific locations of AGPs were greatly reduced after beta-GlcY treatment. In addition, the accumulations of new cell wall materials were also significantly affected by treating with beta-GlcY. Detection of cellulose components by Calcofluor white stain showed that strong fluorescence was located in the newly formed wall of daughter cells after the zygotic division of in vivo samples and the control samples from in vitro culture without beta-GlcY treatment; while there was only weak fluorescence in the newly formed cell walls with beta-GlcY treatment. Immunocytochemistry examination with JIM5 and JIM7 respectively against the low- and high-esterified pectins displayed that these two pectins located in opposite positions of zygotes and proembryos in vivo and the polarity was not affected by beta-GlcY. Furthermore, FM4-64 staining revealed that endosomes were distributed in the cell plates of proembryos, and the localization pattern was also affected by beta-GlcY treatment. These results were further confirmed by subsequent observation with transmission electron microscopy. Moreover, the changes to proembryo cell-organelles induced by beta-GlcY reagent were also observed using fluorescent dye staining technique. CONCLUSIONS: These results imply that AGPs may not only relate to cell plate position decision, but also to the location of new cell wall components. Correlated with other factors, AGPs further influence the zygotic division and proembryo pattern establishment in tobacco.     

五唇兰合子与原胚的分离

分离合子和原胚可以为植物受精和胚胎发生提供很好的研究材料, 因此具有重要意义。用酶解-振荡法分离出五唇兰(Doritis pulcherrima)受精后胚囊, 然后以显微解剖获得合子和原胚。酶解液组成为0.7%–1.3%纤维素酶、0.6%–1.0%果胶酶和10%甘露醇, pH值为5.8, 酶解时间0.5–3.0小时。在分离的早期原胚和接近成熟的球形胚中, 珠孔端均有较发达的胚柄吸器。实验获得了对五唇兰胚胎发育的新认识, 合子和原胚的成功分离也为进一步的细胞学和分子生物学研究打下基础。     

Distribution of Gibberellins A7 and A4 in Tobacco Proembryos Using Immunoelectron Microscopy

The ovules of Nicotiana tabacum var. macrophylla 8 days after pollination were fixed successively with 2% EDC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) and a mixture solution of paraformaldehyde and glutaraldehyde, then slightly postfixed in 0.5 % osmium solution. UI- trathin sections of 6-, 9- or 12- celled proembryos embedded in Epon 812 resin were stained in an anti-GA MAb and sheep anti-mouse IgG-colloidal gold (10 nm). This MAb specifically recognizes methyl esters of GA7 and GAn. Therefore, it could be used as a probe to localize GA7 and GAn in cells after EDC fixation. The 12-celled proembryo is composed of a 9-celled embryo and a 3-celled suspensor. Wide distribution of GA7 and GAn was observed in all proembryo cells and most organelles at subeellular level, including walls, plasmodesmata, plasma membrane, cytoplasmic matrix, mitochondria, plastids, vacuoles, endoplasmic reticulum and nuclei. Clusters of gold granules were found in nuclear envelope, nucleomatrix, nucleolus, chromosome, cytoplamic matrix. In a region composed of cytoplasmic matrix, a vacuole and a mitochondrium, such concentrated gold granules were particularly obviously observed. There appeared a gradient distribution of GA7 and Gan from embryo cells decreasingly to suspensor cells. GA7 and GAn could be translocated via intercellular walls, plasmodesmata and vesicles between embryo and suspensor. 1he authors suspect the direction of GA translocation in proembryo may be from suspensor to embryo. To author's knowledge, this is the first report to indicate subcellular and gradient distributions of bioactive gibberellins in plant proembryos.     

Characterization of the natural chemical and osmotic environment of early wheat embryogenesis

Soluble carbohydrates, amino acids, and major inorganic ions were quantified and compared with the total osmolarity of the ovule. Most of the analyzed components increased rapidly in concentration, reaching a peak at 1 day post anthesis (DPA) and decreasing quickly 1 day later. Within this time period, the osmolarity decreased from 1.49 to 1.18 MPa. The osmolarity then increased to 1.39 MPa at 4 DPA, and yet the overall concentration of analyzed molecules decreased. At 1 DPA, inorganic ions were observed at levels that could produce a high osmolarity (1.11 MPa), followed by the carbohydrates (0.37 MPa) and amino acids (0.07 MPa). Maltose was detected in ovules only from 0 to 2 DPA. Inositol was abundant in the ovules on the day of anthesis, but decreased to non-detectable levels after 4 DPA. This suggested that ovules allow the rapid entry of solutes into the syncytial endosperm after fertilization, but are osmotically buffered through the increase and decrease of partly identified substances. Specific gravity tests on the liquid squeezed from young endosperm sukpported this new hypothesis. Osmolarity in ovules seems only indirectly related to morphogenetic regulation mechanisms, and it may play a lesser role than the fluctuations of certain specific substances. The observed chemical changes offer insights into the rapidly varying nutritional needs of proembryos. Thus, in this research yielded a useful complex amino acid formula, derived from knowledge of the natural environment of the proembryo, and also an improved proembryo and ovule culture medium.     

Young microspore-derived maize embryos show two domains with defined features also present in zygotic embryogenesis

In this work we report the existence of two domains in early microspore maize proembryos displaying similar features of zygotic embryogenesis. The large or so-called endosperm-like domain exhibits specific features: coenocytic organisation, synchronous mitosis, vacuolated cytoplasm, starch granules, incomplete walls containing callose and differential tubuline organisation. The small or embryo-like domain displays small polygonal uninucleate cells with typical organisation of proliferating cells. The structural organisation and the subcellular localization of specific cytoplasmic and cell wall components (starch, tubuline and callose) in both proembryo domains have been determined by using specific cytochemical and immunocytochemical methods. Four morphological types of proembryos containing both domains have been characterised. Taking into account their relative size, the high asynchrony of the culture and the homologies between structural features of endosperm-like domain and zygotic endosperm development, they could represent different stages in microspore embryogenesis development. The ZmAE1 and ZmAE3 genes expressed in the Embryo Surrounding Region of the endosperm during zygotic embryogenesis (Magnard et al., 2000), were revealed to be expressed in early microspore proembryos by in situ hybridization at light and electron microscopy levels. This data supports the existence of an endosperm-like function during early microspore embryogenesis and provides new insights into the onset of microspore embryogenesis in maize, and its parallelism with zygotic embryogenesis.     

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.     

石刁柏胚乳愈伤组织内胚胎发生的组织细胞学观察

石刁柏(Asparagus officinalis L.)的幼嫩胚乳接种于MS+NAA 5ppm+6-BA 1ppm和MS+NAA 1ppm+6-BA 0.5ppm的培养基上诱导产生愈伤组织。将愈伤组织转移到MS+6-BA 1ppm+NAA 0.5ppm的分化培养基上,培养30天后即可形成胚状体,组织细胞学观察表明:胚状体起源于愈伤组织内或近表层的单个胚性细胞。在胚状体发生的早期阶段,观察到与柳叶菜型和藜型胚胎发育大致相似的细胞分裂方式,从而出现了T型或线型的四个细胞的原胚。在多细胞原胚及球形胚期具有单列或不规则排列的多细胞胚柄。石刁柏胚乳愈伤组织中的胚胎发生是不同步的,在同一块愈伤组织的切片中可以观察到不同发育阶段的胚状体。在外形上还可以观察到一个子叶、两个子叶或四个子叶等多种不同形态的胚状体。一部份胚状体能发育成完整小植株。     

Studies in Garcinia, dioecious tropical forest trees: agamospermy

RICHARDS, A. J., 1990. Studies in Garcinia , dioecious tropical forest trees: agamospermy . In Garcinia , agamospermy is known or suspected for ten species. Most are facultative agamosperms, with males occurring, but males are probably lacking in G. mangostana . In G. mangostana and G. hombroniana , adventitious embryony occurs autonomously, and haploid parthenogenesis may also occur rarely. In G. parvifolia , it is reported previously that gametophytic agamospermy occurs, and adventitious embryos are suppressed. Autonomous endospermy is found in G. mangostana and G. parvifolia . In G. hombroniana , endosperm probably only develops after PEN fertilization. In G. hombroniana , some proembryos are formed precociously, and further proembryo formation is inhibited by sexual embryos. Asexual proembryos tend to occur in large ovules in small ovaries, and sexual embryos tend to occur in small ovules in large ovaries. It is considered that facultative agamospermy renders the genus Garcinia particularly suitable for the development of new types of fruit. Agamospermy in a dioecious genus, adventitious embryony in the absence of fertilization and/or pseudogamy, and the co-occurrence of gametophytic and sporophytic agamospermies in the same genus are unusual phenomena, which are discussed in the context of the lifestyle of a tropical forest tree. For G. hombroniana, n = 24, and for G. cowa, n = 26 are reported.     

Ultrastructural Observations on Parthenogenesis and Antipodal Apogamy of Allium tuberosum Roxb

Alliurn tuberosum Roxb is a species characterized by spontaneous parthenogenesis and antipodal apogamy. This paper deals with the ultrastructural changes during these processes. Before pollination, the mature egg cell contained abundant mitochondria with well developed cristae, spherical or ellipsoidal plastids and some polyribosomes, which suggested a relatively high metabolic activity. After fertilization, in zygotes the mitochondria changed to irregular shape and their cristae degenerated, the plastids elongated or became cup-shaped, the polyribosomes decreased and the free ribosomes increased in number. Some unfertilized egg ceils, two days after anthesis, showed similar ultrastructural changes as those taking place in zygote, which. seemed to be a sign of triggering to parthenogensis. In Allium tuberosum Roxb, the three antipodai cells bore a close resemblance to the egg apparatus: Among them two antipodal cells were similar to the synergids with a filiform apparatus-like structure and plentiful organelles at their chalazal end. The other was an egg-like antipodal cell which could undergo similar ultrastructural changes as those happened in zygote, leading to divide into apogamic proembryos two days after anthesis. The problems of parthenogenesis and antipodal apogamy in Allium tuberosum Roxb is diicussed in view of ultrastructural features of the egg and egg-like antipodal cell.     

Parthenogenetic egg cells of wheat: cellular and molecular studies

The 'Salmon' system of wheat comprises three isogenic alloplasmic lines with either zygotic (aS) or autonomous, fertilisation-independent (cS kS) embryo development. While the initiation of embryogenesis from the isolated sexual egg cell depends on in vitro fertilisation, the corresponding parthenogenetic egg cell develops into an early embryo without fertilisation. This demonstrates that parthenogenesis is an inherent feature of the isolated egg cell. Based on this observation, we have constructed egg-cell-specific cDNA libraries and report first results of a sequencing project aimed at the isolation of putative egg-cell-specific and parthenogenesis-related genes.     

A novel approach to differentiated embryos in the absence of endosperm

Summary In all of the Poaceae tested (Bromus, Festuca, Hordeum, Lolium, Poa, Triticum) the formation of grains without endosperm was induced from unpollinated ovules by treatment with the following synthetic auxins: DIG; 2,4-D; 2,4,5-T; or CPAA. Cytokinins (BAP, ZTN) as well as adenine or gibberellic acid (GA₃) alone were ineffective. In parthenogenetic lines auxin treatment resulted in grains with mature embryos without endosperm. Differences in embryo differentiation were found, which were dependent on the synthetic auxins used, their concentrations, and the developmental stages of the treated spikes or panicles. Thus, the regulation of embryogenesis by the endosperm can be replaced by exogenous auxin application. The developing proembryos of grasses did not need nutritive support from the endosperm.Auxin treatment to give mature embryos without endosperm enables the screening of apomictic species for sexual plants and sexual species for parthenogenetic individuals. It opens ways for inducing haploid parthenogenesis and improves methods for overcoming interspecific crossing barriers.     

蓝猪耳卵细胞和合子的分离

蓝猪耳(Torenia fournieri)胚囊部分裸露出胚珠,在光学显微镜下能清楚观察到卵细胞和助细胞的形态结构.用解剖和酶解-解剖两种方法都能分离出生活卵细胞.用前种方法机械分离出的卵细胞数量较少(5%),但避免了酶对配子识别研究的干扰.在后种方法中加入0.1%纤维素酶和0.1%果胶酶既能使分离更加容易操作,又对卵细胞没有致命伤害,能在短时间内分离出较多的卵细胞(18%).用酶解-解剖方法也可分离出授粉14 h后的合子细胞.     

茴香组织培养中体细胞胚胎发生的组织细胞学研究

将茴香幼茎或叶柄的愈伤组织转入附加6-BA和低浓度2,4-D的MS培养基以后,愈伤组织逐步由松软状转变成为颗粒状的胚性愈伤组织,胚状体起源于胚性愈伤组织中的单个细胞或胚性细胞团。在含NAA和6-BA的培养基中,胚状体发育成熟,并再生小植株。茴香的胚状体主要以单细胞内起源方式发生。首先由胚状体单个原始细胞分裂形成2-细胞原胚,2-细胞原胚以三种方式进行分裂:1.T- 形分裂;2.直线形分裂;3.田字形分裂。不同的分裂方式决定了胚柄的有无。茴香胚状体的发育过程与合子胚基本相同。由原胚发育成为球形胚,依次经过心形胚和鱼雷胚阶段,形成成熟的子叶胚。在胚状体发育的每一个阶段,都有其分生组织的活动中心。球形胚期,两团分生组织位于胚体中部对应的两点;心形胚期,位于两侧和中部;鱼雷胚期,分生组织的分布在子叶形成区域呈倒“U”形,在下胚轴部位呈中空的梭形。到子叶期,分生组织从两片子叶伸向胚根,呈“Y”形分布。两子叶间产生茎生长点,由生长点分化出叶原基。胚状体最终发育成为完整植株。     

韭菜幼小子房培养时胚囊的发育和一些异常现象

对韭菜开花前1天左右的子房进行培养可获得大量的单倍体植株。观察表明单倍体植株起源于未受精的卵细胞和反足细胞。为了探索培养不同发育时期的子房对单倍体原胚发生频率的影响,我们又对大孢子母细胞时期的幼     

Life-history changes that accompany the transition from sexual to parthenogenetic reproduction in Drosophila mercatorum

In spite of the predicted genetic and ecological costs of sex, most natural populations maintain sexual reproduction, even those capable of facultative parthenogenesis. Unfertilized eggs from natural populations of Drosophila mercatorum occasionally develop into viable adults, but obligately parthenogenetic populations are unknown in this species. To evaluate the microevolutionary forces that both favor and constrain the evolution of parthenogenesis in D. mercatorum, we have measured parthenogenetic rates across a natural, sexually reproducing population and characterized the life-history changes that accompany the transition from sexual to parthenogenetic reproduction in laboratory strains. A highly significant difference in parthenogenetic rate was found between two populations in close geographic proximity, with increased rate found with lower population density. Laboratory strains of parthenogenetic females suffered increased mortality and reduced egg viability relative to their virgin counterparts from a sexual strain. Lifetime egg production was similar across all strains, but a shift in peak egg production to an earlier age also occurred. The combination of these life-history traits resulted in a higher net reproductive value for sexual females, but because they also had a longer generation time, intrinsic rate of increase was not as dramatically different from parthenogenetic females. In environments with high early mortality, there may be no fitness disadvantage to parthenogenesis, but the predicted ecological advantage of a twofold increase in intrinsic rate of increase was not realized. These results support the theory of Stalker (1956) that parthenogenesis is favored in environments in which sexual reproduction is difficult or impossible.