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

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

野芹菜体细胞胚胎发生早期变化的细胞学研究

通过光学与电子显微镜观察研究了野芹菜(Angelica polymorpha Maxim.)叶柄外植体胚性细胞的起源与原胚状体的发生。叶柄切段植入 MS 2mg/L2,4-D 0.25 mg/L KT固体培养基后,以DNA合成和细胞分裂为指标,判明了胚状体发生与紧贴维管束的鞘细胞层密切相关;鞘细胞通过有丝分裂形成多层结构的细胞群,它们仍含大液泡及薄层胞质,成片被覆在维管束表面;胚性细胞团即不同步地发生在该多层组织较内层的局部位点上,细胞具大核,胞质稠密,经持续有丝分裂发展成大小不同呈瘤状突起的原胚状体。伴随着鞘细胞的剧烈变化,韧皮部薄壁细胞亦分裂增殖,原有筛管变形衰退,部分新增殖的细胞分化为新筛分子;木质部中,原导管束附近朝向韧皮部一侧的部分薄壁细胞亦进一步分化为孔纹导管。外植体中维管系统的再次分化,显然是与输导功能强化以适应原胚状体发生时对营养物质的大量需求有关。     

Fine Structure of and Embryoid Development from Embryogenic Ovular Callus of 'Shamouti' Orange (Citrus sinensis Osb.)

A morphologically, anatomically and physiologically unique callushas been developed from unfertilized ovules of Shamouti orange.The callus, which is suspected to be of nueellar origin, isnot made up of the normal unorganized parenchymatous tissue,but solely of numerous proembryoids which vary between 0.1 and1.0 mm in diameter. Adventive embryogenesis in this tissue isautonomous and is in fact depressed by the inclusion of growthregulators in the medium, despite having been in culture formore than 2 years. Embryogenesis occurs in single cells on the periphery and withinexisting proembryoids. Cells destined to form new proembryoidsare surrounded by greatly thickened cell walls which lack plasmodesmata.Cell divisions occur within the thickened walls to give riseto globular proembryoids which are freed from encasing thickwalls as these degenerate. Proembryoids may enlarge into spherical pseudobulbils up to4 mm in diameter with an epidermal cell layer but no vascularization.Such structures rarely develop into plantlets but do form furtherproembryoids from surface cells. Alternatively, proembryoidsmay develop into heart-shaped, torpedo, and cotyledonary embryoids,and thence into plantlets with varying degrees of organ fasciation. Since plantlets are derived from single, usually surface cells,this system holds great promise for the production of solidgenetic mutants by irradiation.     

Ectopic divisions in vascular and ground tissues of Arabidopsis thaliana result in distinct leaf venation defects

Leaf venation patterns vary considerably between species and between leaves within a species. A mechanism based on canalization of auxin transport has been suggested as the means by which plastic yet organized venation patterns are generated. This study assessed the plasticity of Arabidopsis thaliana leaf venation in response to ectopic ground or procambial cell divisions and auxin transport inhibition (ATI). Ectopic ground cell divisions resulted in vascular fragments between major veins, whereas ectopic procambial cell divisions resulted in additional, abnormal vessels along major veins, with more severely perturbed lines forming incomplete secondary and higher-order venation. These responses imply limited vascular plasticity in response to unscheduled cell divisions. Surprisingly, a combination of ectopic ground cell divisions and ATI resulted in massive vascular overgrowth. It is hypothesized that the vascular overproduction in auxin transport-inhibited wild-type leaves is limited by simultaneous differentiation of ground cells into mesophyll cells. Ectopic ground cell divisions may negate this effect by providing undifferentiated ground cells that respond to accumulated auxin by differentiation into vascular cells.     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

Division patterns in the thallus ofPelvetia compressa embryos and the effects of gravity

Summary The pattern of divisions in the thallus ofPelvetia compressa embryos was determined with respect to the embryonic growth axis. To detect all possible division planes, embryos were viewed from two vantages which permitted observations of (1) the thallus pole and (2) the longitudinal embryonic profile. Following formation of rhizoid and thallus cells by any asymmetrical division transverse to the embryonic axis that is established prior to any divisions, the thallus cell divided twice along the embryonic axis (axial divisions) in orthogonal planes, and then divided transverse to the growth axis. This division pattern produced an eight-cell thallus with four cells in each of two layers. The spatial relation between gravity and the first axial division was investigated, and gravity was found to have little effect on the alignment of this division. The reproducible pattern of divisions in the thallus indicates spatial control of spindle positioning.Abbreviations ASW artificial seawater - AF after fertilization     

Cleavage in vivo and in culture in the Dasyurid marsupial Antechinus stuartii (macleay)

Cleavage in the brown marsupial mouse, Antechinus stuartii, from the zygote to the unilaminar blastocyst, was observed in vivo and in culture and in sections of embryos. The first three divisions were meridional and passed from the yolk pole to the opposite pole. Deutoplasmolysis, resulting in a distinct yolk mass, occurred during the first two divisions. Prior to the third and fourth divisions, the blastomeres elongated and flattened against the zona pellucida. The fourth division was latitudinal and resulted in two histologically distinct rings of eight blastomeres which were at first rounded and then became flattened against the zona. Further divisions and flattening of the blastomeres resulted in a complete unilaminar blastocyst by the time the blastocyst numbered 22 to 30 cells. Some expansion, causing compression of the zona and mucoid layers, occurred before completion of the blastocyst, but most expansion occurred once the blastocyst was complete. No histological differences could be detected between the blastomeres at this stage. Embryos were successfully cultured from the zygote to the rounded four-cell stage and from the flattened four-cell stage to the completed unilaminar blastocyst of around 32 cells. Total estimated cleavage times were slower in culture than in vivo, but the relative lengths of time for each division were approximately the same.     

Pollen Ultrastructure in Anther Cultures of Nicotiana tabacum: III . THE FIRST SP.OROPHYTIC DIVISION

Ultrastructure of embryogenic tobacco pollen was examined atthe first division of the vegetative cell. Structurally emptyzones formed before division by the degradation of gametophyticcytoplasm were rapidly re-populated with ribosomes, mitochondria,dictybsomes, and organelles interpreted as lipid centres. Thelatter appeared to be active in synthesis, and were associatedwith a vesicular component. Starch accumulation also began atthe first division in plastids carried over from the mothercell. Each daughter cell possessed a typical primary wall confluentwith the inner layers of the intine. Plasmodesmata traversedthe wall between the two cells. The cells were either equalor unequal; equal divisions occurred in both the longitudinaland transverse planes. In many proembryoids, the generativecell showed signs of degeneration. The cell was either attachedto the intine or detached from it. Detached cells sometimesdivided. The data, are discussed in relation to other embryogenicand morphogenic systems induced from normally quiescent or highlydifferentiated cells.     

Quantitative control of end products in the melanocyte lineage of the ascidian embryo.

Terminal amounts of tyrosinase (EC 1.10.3.1) activity and melanin pigment in the giant melanocytes of cleavage-arrestedCiona intestinalis (L.) embryos are regulated independently of cell size and number of nuclei in the cells. Embryos were cleavage-arrested in cytochalasin B at a time before the last two divisions of the melanocyte lineage took place. The resulting two giant melanocytes, one from each of the two bilateral melanocyte lineages, developed tyrosinase and melanin. The cells were about three times larger in volume than the normal larval melanocytes and each contained four nuclei instead of just one. Quantitative measurements of melanin synthesized and tyrosinase activity in embryos with the giant melanocytes revealed amounts identical to those found in normal embryos. This specification of exact quantities differs markedly from the situation in mammalian melanocytes where cell volume and gene dosage influence the extent of melanotic differentiation. Quantitative control of differentiation in ascidian melanocytes appears to be mediated by a cytoplasmic determinant segregated through the melanocyte lineage and inherited by one daughter at each division of the lineages.     

Light microscopical studies of chromosomes in embryos of cod, Gadus morhua L.

The chromosomes in the cells of 1–2-day-old cod embryos were studied using light microscopic techniques. The observations revealed slightly non-synchronous cell divisions, with at least three different mitotic phases present in a 1-day-old embryo. Surprisingly high percentages of abnormal metaphases and anaphases were found in normal-looking embryos. In the 2-day-old embryos, free-lying nuclei were observed in the periblast zone. Chromosome studies of these nuclei revealed a cleavage pattern of the chromosomes which differed from that of the other cells.     

Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.     

Nuclear transfer from teratocarcinoma cells into mouse oocytes and eggs

A spontaneous ovarian teratocarcinoma was isolated from a LT/Sv mouse female and converted into an ascites tumor from which embryonal carcinoma (EC) cells were dissociated. Non-enucleated and enucleated, activated oocytes were fused with EC cells and either cultured in vitro or transferred into ligated oviducts of Swiss/A females. The nucleocytoplasmic hybrids cultured in vitro up to 22 h were examined cytologically at various time intervals. EC nuclei showed morphological remodelling in the foreign cytoplasm. EC chromosomes and female pronuclear chromosomes together formed a common metaphase. The nucleocytoplasmic hybrids developed in vivo were analyzed cytologically between the first and third day after oviduct transfer. The majority of embryos developed abnormally and, in a few instances, they had passed several cleavage divisions and reached, at best, a developmental stage resembling a premature morula. Fertilized, enucleated eggs were fused with EC cells or microinjected with EC nuclei. The resulting nucleocytoplasmic hybrids were either cultured in vitro or in vivo up to the fourth day. Enzyme tests were carried out on the nuclear transplant embryos, using electrophoretic variants of glucose phosphate isomerase (GPI) in order to distinguish between EC nuclei (GPI-A) and recipient eggs (GPI-B). The EC-specific GPI could be detected in about one third of the embryos analyzed and, in several instances, also together with the egg-specific GPI. Most of them were arrested during early cleavage divisions. Some embryos cleaved abnormally or mimicked normal embryogenesis. In a few instances, development resulted in embryos that resembled late preimplantation embryos.     

Symmetrically dividing cell specific division axes alteration observed in proteasome depleted C. elegans embryo

A fertilised Caenorhabditis elegans embryo shows an invariable pattern of cell division and forms a multicellular body where each cell locates to a defined position. Mitotic spindle orientation is determined by several preceding events including the migration of duplicated centrosomes on a nucleus and the rotation of nuclear-centrosome complex. Cell polarity is the dominant force driving nuclear-centrosome rotation and setting the mitotic spindle axis in parallel with the polarity axis during asymmetric cell division. It is reasonable that there is no nuclear-centrosome rotation in symmetrically dividing blastomeres, but the mechanism(s) which suppress rotation in these cells have been proposed because the rotations occur in some polarity defect embryos. Here we show the nuclear-centrosome rotation can be induced by depletion of RPN-2, a regulatory subunit of the proteasome. In these embryos, cell polarity is established normally and both asymmetrically and symmetrically dividing cells are generated through asymmetric cell divisions. The nuclear-centrosome rotations occurred normally in the asymmetrically dividing cell lineage, but also induced in symmetrically dividing daughter cells. Interestingly, we identified RPN-2 as a binding protein of PKC-3, one of critical elements for establishing cell polarity during early asymmetric cell divisions. In addition to asymmetrically dividing cells, PKC-3 is also expressed in symmetrically dividing cells and a role to suppress nuclear-centrosome rotation has been anticipated. Our data suggest that the expression of RPN-2 is involved in the mechanism to suppress nuclear-centrosome rotation in symmetrically dividing cells and it may work in cooperation with PKC-3.     

Histodifferentiation of somatic embryos in cotyledons of pineapple guava (Feijoa sellowiana Berg)

Summary Somatic embryos of pineapple guava (Feijoa sellowiana Berg, Myrtaceae) were induced particularly well from the adaxial face of the cotyledons of zygotic embryos cultured on MS medium containing 1.0 mg/l 2,4-D and 0.3 M sucrose. Somatic embryos were never obtained from globular and heart-shaped zygotic embryos and embryos at the torpedo stage produced somatic embryos at lower frequencies than mature zygotic embryos. At the time of explantation, cotyledonary cells were rich in storage proteins and lipids but no starch was found. After the first 5 days of culture most of the reserves had been mobilized in cotyledons of germinating embryos, but were still present in large amounts in cotyledons undergoing embryogenie induction. In contrast to cotyledons following the normal pattern of development, cells of embryogenically-induced cotyledons accumulated starch, especially those cells not involved in the embryogenie process. Two patterns of somatic embryo differentiation were observed: (1) from single epidermal cells or (2) from groups of meristematic cells near the adaxial surface. Comparative observations on cotyledons from germinating embryos and those undergoing embryogenesis suggest that the meristematic layer arises as the result of successive divisions of cells that, under normal conditions, would form the palisade parenchyma. These were the only mesophyll cells that showed mitotic divisions during the normal development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FAA formalin/acetic acid/ethyl alcohol - PAS periodic acid-Schiff     

A novel regeneration system for a wild passion fruit species (<Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 μM 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog’s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.     

Effect of antimediator preparations on the intercellular relations in early embryos of the sea urchin]

The dated treatment of the early embryos of an irregular (flat) sea urchin Scaphechinus mirabilis by neuropharmacological drugs (anti-neurotransmitters) during one of the first four cleavage divisions results in the impairment of intercellular connections and leads to the formation of twin embryos, dwarf embryos, embryos of the dumb-bell shape etc. In the experiments with some of the drugs under study such developmental abnormalities were not seen or were expressed much more weakly when serotonin or bufotenin (N,N-dimethylserotonin) were added to the medium. A suggestion is put forward that the early embryos possess an intracellular mechanism participating in the interaction between the cells and operating via endogenous monoamines, primarily serotonin.     

The TORMOZ gene encodes a nucleolar protein required for regulated division planes and embryo development in Arabidopsis

Embryogenesis in Arabidopsis thaliana is marked by a predictable sequence of oriented cell divisions, which precede cell fate determination. We show that mutation of the TORMOZ (TOZ) gene yields embryos with aberrant cell division planes and arrested embryos that appear not to have established normal patterning. The defects in toz mutants differ from previously described mutations that affect embryonic cell division patterns. Longitudinal division planes of the proembryo are frequently replaced by transverse divisions and less frequently by oblique divisions, while divisions of the suspensor cells, which divide only transversely, appear generally unaffected. Expression patterns of selected embryo patterning genes are altered in the mutant embryos, implying that the positional cues required for their proper expression are perturbed by the misoriented divisions. The TOZ gene encodes a nucleolar protein containing WD repeats. Putative TOZ orthologs exist in other eukaryotes including Saccharomyces cerevisiae, where the protein is predicted to function in 18S rRNA biogenesis. We find that disruption of the Sp TOZ gene results in cell division defects in Schizosaccharomyces pombe. Previous studies in yeast and animal cells have identified nucleolar proteins that regulate the exit from M phase and cytokinesis, including factors involved in pre-rRNA processing. Our study suggests that in plant cells, nucleolar functions might interact with the processes of regulated cell divisions and influence the selection of longitudinal division planes during embryogenesis.     

Small-scale spatial dynamics in a fluctuating ungulate population

1. The scale at which population dynamics are analysed is important, as results from analyses at different spatial scales can differ and affect interpretation.
2. In this study, detailed census data collected over a 10-year period from a population of Soay sheep ( Ovis aries L.) on the Island of Hirta in the St Kilda archipelago, Scotland, is used, together with cluster analysis, to distinguish a temporally stable spatial substructure.
3. Structured demographic accounting of the variance in population change (SDA) is also used to analyse the dynamics of the whole population treated as (a) one unit; (b) one unit subdivided into three subunits; and (c) three independent units.
4. Differences in survival, recruitment and dispersal rates are demonstrated between divisions of the population, which are probably associated with variation in grazing quality.
5. If these groups were not coupled by dispersal and density-independent entrainment, the population dynamics of the three groups would diverge, however, the dynamics of the three subunits are strongly correlated.     

Patterned cell development in the secondary phloem of dicotyledonous trees: a review and a hypothesis

The secondary phloem of dicotyledonous trees and shrubs is constructed of sieve tube cells (S) and their companion cells, as well as parenchyma (P) and fibre (F) cells. Different species have characteristic sequences of these S, P and F cells within the radial files of their phloem. The sequences are recurrent, and are evidence of rhythmic cell determination and differentiation. A model was devised to account for the sequences found in various dicot tree species. It is based on the pattern of radial displacement of cells through a gradient of morphogen which supports secondary phloem development. According to this model, each tree species shows a particular pattern of post-mitotic cellular displacement along each radial file as a result of a corresponding sequence of periclinal division in the cambial initial and its descendents. The divisions and displacements ensure that at each timestep (equivalent to an interdivisional interval) each cell resides in a specific location within the morphogenic gradient. Cells then emerge from the post-mitotic zone of cell determination, having acquired different final positional values. These values lie above a series of thresholds that permit the respective determination and subsequent differentiation of one or other of the three cell types S, P and F. The recurrent nature of the sequences of the three cell types within each radial cell file, as well as their tangential banding, are a consequence of a shared rhythmic spatio-temporal pattern of periclinal cambial divisions. With a single set of morphogen parameters required for cell determination, and using three positions for cambial cell divisions, all the cellular sequences of secondary phloem illustrated in the literature can be accounted for.This is an invited article.