Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

Hierarchical regulation of organ differentiation during embryogenesis in rice

In plants, genetic mechanisms leading to shoot and root formation are almost unknown. Because basic body organization of such organisms is established during embryogenesis, induction and isolation of embryonic mutants is a promising approach to the study of plant development. The study of available embryonic mutants of rice indicates the existence of three major developmental processes taking place during embryogenesis before morphogenetic events start: determination of organ differentiation, positional regulation of organs and size regulation of the embryo. The consideration of specific rice mutants supports the existence of two types of mutations in each regulatory process, one affecting the embryo as a whole and the second concerning more restricted embryonal regions. A hierarchical type of control of rice embryogenesis is suggested.     

The asymmetric division of the Arabidopsis zygote: from cell polarity to an embryo axis

During plant embryogenesis, a simple body plan consisting of shoot and root meristem that are connected by the embryo axis is set up by the first few rounds of cell divisions after fertilization. Postembryonically, the elaborate architecture of plants is created from stem cell populations of both meristems. Here, we address how the main axis (apical-basal) of the plant embryo is established from the single-celled zygote and the role that the asymmetric division of the zygote plays in this process. We will mainly draw on examples from the model plant Arabidopsis, for which several key regulators have been identified during the last years.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

Control of root meristem establishment in conifers

The evolution of terrestrial plant life was made possible by the establishment of a root system, which enabled plants to migrate from aquatic to terrestrial habitats. During evolution, root organization has gradually progressed from a very simple to a highly hierarchical architecture. Roots are initiated during embryogenesis and branch afterward through lateral root formation. Additionally, adventitious roots can be formed post‐embryonically from aerial organs. Induction of adventitious roots (ARs) forms the basis of the vegetative propagation via cuttings in horticulture, agriculture and forestry. This method, together with somatic embryogenesis, is routinely used to clonally multiply conifers. In addition to being utilized as propagation techniques, adventitious rooting and somatic embryogenesis have emerged as versatile models to study cellular and molecular mechanisms of embryo formation and organogenesis of coniferous species. Both formation of the embryonic root and the AR primordia require the establishment of auxin gradients within cells that coordinate the developmental response. These processes also share key elements of the genetic regulatory networks that, e.g. are triggering cell fate. This minireview gives an overview of the molecular control mechanisms associated with root development in conifers, from initiation in the embryo to post‐embryonic formation in cuttings.     

In vitro culture of Arabidopsis embryos within their ovules

Embryogenesis of flowering plants establishes a basic body plan with apical-basal, radial and bilateral patterns from the single-celled zygote. Arabidopsis embryogenesis exhibits a nearly invariant cell division pattern and therefore is an ideal system for studies of early plant development. However, plant embryos are difficult to access for experimental manipulation, as they develop deeply inside maternal tissues. Here we present a method for the culture of zygotic Arabidopsis embryos in vitro. The technique omits excision of the embryo by culturing the entire ovule, thus greatly facilitating the time and effort involved. It enables external manipulation of embryo development and culture from the earliest developmental stages up to maturity. Administration of various chemical treatments as well as the use of different molecular markers is demonstrated together with standard techniques for visualizing gene expression and protein localization in in vitro cultivated embryos. The presented set of techniques allows for so far unavailable molecular physiology approaches in the study of early plant development.     

The Putative Factors Involved in the Induction of Embryogenesis in Angiosperms

We quantified various endogenous cytokinins during wheat (Triticum aestivumL.) and dandelion (Taraxacum officinaleWeb.) ovary development. Wheat ovaries were studied at the following developmental stages: the mature embryo sac with eight nuclei (stage 1), the interphasic zygote 12 h and 24 h after fertilization (stage 2), and the onset of zygote division (stage 3). The dandelion ovaries were studied at the stage of the mature embryo sac (stage 1), in the interphase of the parthenogeneticaly developing ovule (stage 2), and during its first division (stage 3). The material was analyzed by the method of competitive solid-phase immunoenzyme assay (ELISA) using peroxidase-labeled anti-rabbit antibodies. The onset of embryogenesis in wheat and dandelion ovules was accompanied by the substantial rearrangement of their hormonal complexes, which preceded the morphogenetic processes leading to seed formation. This implies that the hormonal system of the whole maternal plant is involved in the induction of embryogenesis. The final stages of embryogenesis depend on the hormonal systems in the flower, ovary, and ovule.     

被子植物胚胎发育的分子调控

被子植物的胚胎发育受到精确的遗传调控。从双受精开始到种子成熟, 胚胎发育经历了合子激活、细胞分裂与分化、极性建立、模式形成、器官发生和储藏物质累积等重要过程。过去20年来的分子遗传学研究鉴定了很多调控胚胎发生的基因,为了解胚胎形成的分子机理提供了大量信息。本文对这一领域的主要研究进展进行了简要评述, 重点阐述了植物的早期胚胎发生过程, 对尚未解决的科学问题及未来发展方向进行了综合分析。     

EARLY DEVELOPMENT OF THE BARLEY EMBRYO: FINE STRUCTURE

The ultrastructure of early stages in embryogenesis in barley was examined. Post-fertilization shrinkage does not occur. Plasmodesmata were not observed in cell walls of the zygote and outer cell walls of embryos. There is little evidence of cellular specialization in earliest embryonic stages, and planes of cell division tend to be irregular although a pattern of cell disposition characteristic of some grass embryos can be discerned. The embryo appears polarized after 2–3 division cycles, but no evolving of dorsiventrality occurs during this period. A basal supensor cell “anchors” the embryo during early embryogenesis, but by about five division cycles the embryo loses its attachment to the nucellus and is completely surrounded by endosperm. An increase in number of ribosomes and mitochondria takes place during early embryogenesis, and mitochondrial dimensions are reduced. A shift in vacuole distribution occurs.     

被子植物胚胎发育的分子调控

被子植物的胚胎发育受到精确的遗传调控。从双受精开始到种子成熟,胚胎发育经历了合子激活、细胞分裂与分化、极性建立、模式形成、器官发生和储藏物质累积等重要过程。过去20年来的分子遗传学研究鉴定了很多调控胚胎发生的基因．为了解胚胎形成的分子机理提供了大量信息。本文对这一领域的主要研究进展进行了简要评述,重点阐述了植物的早期胚胎发生过程,对尚未解决的科学问题及未来发展方向进行了综合分析。     

Comparative anatomy of embryogenesis in three species of Podostemaceae and evolution of the loss of embryonic shoot and root meristems

During embryogenesis in angiosperms, the embryonic shoot and root meristems are created at opposite poles of the embryo, establishing a vertical body plan. However, the aquatic eudicot family Podostemaceae exhibits an unusual horizontal body plan, which is attributed to the loss of embryonic shoot and root meristems. To infer the embryogenetic changes responsible for the loss of these meristems, we examined the embryogenesis of three podostemads with different meristem characters, that is, Terniopsis brevis with distinct shoot and root meristems, Zeylanidium lichenoides with reduced shoot and no root meristems, and Hydrobryum japonicum with no shoot and no root meristems. In T. brevis, as in other eudicots, the putative organizing center (OC) and L1 layer (=the epidermal cell layer) arose to generate a distinct shoot meristem initial, and the hypophysis formed the putative quiescent center (QC) of a root meristem. Z. lichenoides had a morphologically unrecognizable shoot meristem, because a distinct L1 layer did not develop, whereas the putative OC precursor arose normally. In H. japonicum, the vertical divisions of the apical cells of eight-cell embryo prevented putative OC initiation. In Z. lichenoides and H. japonicum, the putative QC failed to initiate because the hypophysis repeated longitudinal divisions during early embryogenesis. Based on their phylogenetic relationships, we infer that the conventional embryonic shoot meristem was lost in Podostemaceae via two steps, that is, the loss of a distinct L1 layer and the loss of the OC, whereas the loss of the embryonic root meristem occurred once by misspecification of the hypophysis.     

The ultrastructure of zygotic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of zygotic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Embryogenesis was initially characterized by the presence of a vacuolated egg cell and zygote. The increased presence of Golgi bodies in the zygote suggested it was metabolically more active than the egg cell. The first zygotic division resulted in a densely cytoplasmic apical cell and a highly vacuolated basal cell. The club-shaped proembryo displayed a large amount of endoplasmic reticulum (ER) and ribosomes, very few lipids, and a continuous gradient of vacuoles from the highly vacuolated basal suspensor cells to the densely cytoplasmic apical cells. The embryo had well-defined parts by 8 days after pollination, including shoot and root meristems, coleoptile, scutellum, provascular system, and the first leaf primordium. Large increases in ER, lipids, starch, and vacuoles occurred in the scutellum during the maturation of the embryo, except in the provascular cells. Throughout zygotic embryogenesis, embryo cells were connected by plasmodesmata except where intercellular spaces occurred. Ultrastructural, morphological, and histological observations of zygotic embryogenesis in pearl millet are in agreement with previous reports for other grass species.