Generation of bilateral symmetry in Anthozoa: a model

Polyps of Anthozoa usually display bilateral symmetry with respect to their mouth opening, to their pharynx, and in particular to the arrangement of their mesenteries. Mesenteries, which are endodermal folds running from the apical to the basal end of the body, subdivide the gastric cavity into pouches. They form in a bilateral symmetric sequence. In this article I propose that early in polyp development the endoderm subdivides successively into three different types of compartments. A mesentery forms at the border between compartments. Two of the compartments are homologous to those of Scyphozoa. They form by mutual activation of cell states that locally exclude each other. The third compartment leads to siphonoglyph formation and is an evolutionary innovation of the Anthozoa. The mechanism that controls the number and spatial arrangement of the third type of compartment changes the radial symmetry into a bilateral one and occasionally into a different one. The dynamics of its formation indicate an activator-inhibitor mechanism. Computer models are provided that reproduce decision steps in the generation of the mesenteries.     

Development of an in vitro model of Toxoplasma gondii cyst formation

Abstract Toxoplasma gondii tissue cyst reactivation is a major pathogenic mechanism in ocular toxoplasmosis, disease associated with AIDS and organ transplantation. The mechanisms associated with cyst formation and reactivation have not been elucidated. The complexity of studying these issues in animal models has led to the development of in vitro tissue culture strategies for cyst formation. In the present study we have adopted the human embryonic lung fibroblast (HEL) as the host cell and have compared the cyst forming abilities of eight clinical isolates. We describe by transmission electron microscopy and quantitative light microscopy the development of cysts in vitro. The numbers of in vitro cysts increased with time for all isolates. Cyst cultures were stabilised by manipulation of the free parasite load, an observation not previously recorded. Thus, in this paper we describe a viable model for the analysis of the mechanisms of Toxoplasma cyst development.     

Axis formation in half blastoderms of the chick: Stage at separation and the relative positions of fused halves influence axis development

The blastoderm of the avian embryo acts during the early stages of development as an integrative system programmed to form a single embryonic axis. Isolated parts of the blastoderm are known to each form an axis, owing to the system's properties. In the work reported here, the regulative capability of the right and left halves of chick blastoderms to form an embryonic axis was examined systematically at different stages. This revealed a progressive change in the developing blastoderm. After early separation, the axis in each half will form at some distance from the blastoderm's original midline, while with late separation the axis will form next to the original midline and may even lack one row of somites at the medial rim. Since development stops in culture after about 2 days, axis development after early separation ceases before somites are formed, whereas after late separation somites and brain vesicles can develop. In addition, an attempt was made to learn whether the two halves of blastoderm, when shifted along the midline and then reunited in staggered fashion, act as a single or two separate embryonic fields. When reunion of the right and left halves was achieved so that the posterior end of one half was adjoining the posterior area pellucida region of the other half, a single embryonic axis developed. When, on the other hand, the shift was larger so that the posterior end was fused to the central area pellucida of the other half, two separated embryonic axes developed.     

Changes in the microtubular cytoskeleton precede in vitro tuber formation in potato

Summary An in vitro system for tuber formation was used to study early morphological and cytological changes occurring during tuber formation in potatoes, with special emphasis on the orientation of the microtubular cytoskeleton, visualized immunocytochemically. Axillary buds from potato plants were cultured in the presence or absence of gibberellin (GA), resulting in either tuber formation (without GA) or shoot formation (GA added). Tuber formation in the absence of GA was highly synchronous in individual buds, enabling the dissection of various aspects of tuberization. Under both conditions, starch started to accumulate. In the absence of GA, starch levels rapidly increased, concomitantly with tuber formation, whereas it slightly decreased in the presence of GA. Up to 4 days, the cortical MTs in the cells were oriented perpendicular to the longitudinal axis of the developing buds. Under tuber-inducing conditions this orientation changed into a longitudinal one at day 5. This change preceded a change in the direction of cell expansion. In the presence of GA no such reorientation was observed, cells continued to grow longitudinally, and a stoloniferous shoot was formed. The cytoskeletal changes preceded the visible swelling of the buds, observed after day 5, demonstrating that the reorientation of the microtubular cytoskeleton is one of the earliest steps observed so far in tuber formation in potatoes.Abbreviations GA gibberellin - MTs microtubules - PBS phosphate buffered saline - SD short-day     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.     

Oosome formation during in vitro oogenesis inBradysia tritici (syn.Sciara ocellaris)

Summary The development of follicles fromBradysia tritici (syn.Sciara ocellaris) during in vitro culture was studied. When follicles are isolated from 12-h-old females and placed in Robb's R-14 medium, their nurse cells regress with the same kinetics as in vivo and a histologically normal oosome forms at the posterior pole of the oocyte. Protein synthesis during in vitro development was studied by labelling follicles for 15 min and culturing them in vitro until the oosome had formed (28 h after eclosion of the donor). The time-course of protein labelling was defined by studying the incorporation kinetics of³H-amino acids into TCA-precipitable material; 50% of the radioactivity in the follicles was incorporated into TCA-precipitable material in less than 30 min. Autoradiographs of follicles labelled at different stages of oogenesis always showed a labelled oosome even if the labelling period was hours before oosome formation. These results indicate that the synthesis of oosome material starts long before the oosome forms at the end of vitellogenesis. Oosome formation can be inhibited by colchicine (20 g/ml) and is, therefore, likely to be dependent directly or indirectly on microtubule function.     

生长调节剂对马蹄金外植体离体培养的影响

以草坪地被植物马蹄金的无菌苗子叶、叶片和下胚轴作为外植体进行离体培养,探讨了不同植物生长调节剂对其愈伤组织诱导及分化的影响.结果表明,从不同外植体在含有不同生长调节剂的诱导培养基中进行离体培养时,都可诱导出愈伤且出愈率为43.3%～100%,其中诱导培养基(MS 100 mg/L水解酪蛋白 0.5 mg/Lα-NAA 0.2 mg/L ZT)适合于三种外植体的愈伤诱导,出愈率为100%,愈伤生长旺盛.在所有愈伤分化试验中均形成了绿色芽点,其频率为25.0%～95.0%,仅从下胚轴诱导的愈伤分化获得了再生植株,且在分化培养基(MS 100mg/L水解酪蛋白 0.1 mg/Lα-NAA 3.0 mg/L 6-BA)上的最高愈伤分化频率为20.0%.     

Whole-embryo culture of Drosophila : development of embryonic tissues in vitro

Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.     

Plantlet formation from embryonic tissue of chestnut grown in vitro

Development of axillary shoots was induced when isolated embryonic axes of chestnut (Castanea sativa Mill.) were cultured on a defined medium containing 6-benzyl-aminopurine (BAP). The optimal concentrations of BAP were determined for development of axillary shoots from both embryonic axes and subcultured shoots. After shoot multiplication a great number of shoots have been maintained sequentially without significant change in the proliferative rate for one year. Limited rooting has been obtained with excised shoots. Indole-3-butyric acid (IBA) at 2 mg/l was used to induce root primordia. After 8 days of treatment the plantlets were transferred to an auxin-free medium for root development.     

In vitro somatic embryogenesis and plant regeneration in Zantedeschia hybrids

A method for regeneration of plants from tuber explants of a Zantedeschia hybrid via somatic embryogenesis was developed. In vitro cultures were initiated starting from both anthers and tubers. Somatic embryogenesis was only achieved from tuber explants. 6-Benzyladenine (BA) at 0.6 or 2 mg l⁻¹ in combination with 2 mg l⁻¹ α-naphthaleneacetic acid (NAA) yielded the highest number of embryos per explant. The somatic embryos converted into plantlets on Murashige and Skoog basal medium supplemented with vitamins, micro- and macronutrients, 1 mg l⁻¹ 6-τ-τ-(dimethylallylamino)-purine (2iP), 3% sucrose and 0.7% agar. This is the first report on induction of somatic embryogenesis in Zantedeschia.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.     

The effect of parental genotype on initiation of embryogenic callus from elite maize (Zea mays L.) germplasm

Summary Embryogenic callus consisting of both Type 1, firm, compact, translucent, relatively slow growing callus and Type 2, highly friable, rapidly growing callus with well-formed somatic embryos, were observed in elite maize germplasm, notably B73 and hybrids with B73. Parental genotype is very important in the ability to identify and isolate embryogenic callus after 14 and 28 days in culture. A partial diallel analysis revealed that a large proportion of the genotypic variation was of the additive type although heterosis did positively increase culture response in most cases. A significant negative maternal effect for culture response was noted for inbred B73 from Reid-type germplasm while four lines sampled from Lancaster germplasm showed similar response whether used as male or female. Although significant media differences were observed in some genotypes, culture media did not preclude observation of Type 1 or Type 2 embryogenic cultures in this study after 14 and 28 days. Plants could be regenerated from all genotypes in this study after 14-days of culture, but not all genotypes were capable of sustained subculture and plant regeneration. Plant regeneration from Type 2 cultures of B73 and B73 hybrids has been obtained up to a year after initiation.     

Effect of benzylaminopurine on in vitro and in vivo root development in lentil, Lens culinaris Medik

The effect of benzylaminopurine (BAP) on the formation of roots from lentil shoots regenerated on media containing BAP was studied. Seedling shoot tips, first nodes and bractlets, and immature seeds cultured on the initiation media containing 2.25 or 0.225 mg/l of BAP regenerated multiple bud shoots. The regenerated shoots formed roots in percentages ranging from 4.6 to 39.9% on a rooting medium (R medium) containing 2 mg/l of indoleacetic acid. Rooting success on R medium depended upon the cytokinin used in the initiation media, its concentration, and the time elapsed during shoot formation on these media prior to transplanting regenerated shoots to R medium. In vivo study of root growth of lentil seedlings demonstrated the strong inhibitory effect of BAP on root growth reflected in a drastic reduction of the mitotic index of the root meristem. Received: 27 August 1996 / Revision received: 12 December 1996 / Accepted: 15 January 1997     

Effect of photoperiod and light intensity on in vitro propagation of Alocasia amazonica

Plantlets of Alocasia amazonica regenerated under a photon flux density (PFD) of 15 or 30 μmol m⁻² s⁻¹ showed better growth and development than those grown under higher PFDs. While chlorophyll a and chlorophyll b decreased, the number of stomata increased with increasing PFD. Photoperiods also affected plantlet growth and stomatal development. Highest growth was observed for the short photoperiod (8/16 h) and for equinoctial (12/12 h) light and dark periods. Very few stomata developed in the leaves of plantlets grown under a short photoperiod (8/16 h) and the number of stomata increased with increasing light period. In conclusion, both light intensity and photoperiod independently affect growth of A. amazonica and development of stomata, depending on the intensity and duration of light treatment.     

In vitro cultivation of an insect Microsporidian Tubulinosema ratisbonensis in mammalian cells

Tubulinosema ratisbonensis is a microsporidian pathogen of Drosophila melanogaster belonging to the family Tubulinosematidae. The microsporidia in this family mainly cause infections in invertebrate hosts, but two members of this family, Brachiola vesicularum and Brachiola algerae, have been found to cause infections in humans as well. Moreover, B. algerae can be transmitted to immunodeficient mice and grows in mammalian cell cultures. Thus, the examination of the opportunistic properties of other members of the family Tubulinosematidae is important. Spores of T. ratisbonensis, isolated from infected fruit flies, were used to inoculate mammalian and insect cell cultures. Parasite growth was only seen in human lung fibroblasts. No growth was seen in Vero cells or insect cell cultures. Comparison of growth kinetics at 31 degrees C and 37 degrees C showed that there were fewer and smaller parasitic foci in cultures incubated at 37 degrees C. Transmission electron microscopy revealed the typical ultrastructure of T. ratisbonensis, and scanning electron microscopy showed oval or slightly pyriform spores, with some spores having extruded their polar tubes. The PCR-amplified sequences of rDNA fragments from infected cell cultures were 100% identical to the original T. ratisbonensis rRNA sequence. As T. ratisbonensis is able to proliferate in mammalian cell cultures, it may have the opportunistic properties of other members of the family Tubulinosematidae.     

Endogenous hormone levels in habituated nucellar Citrus callus during the initial stages of regeneration

 Embryogenic nucellar callus cultures of different Citrus species and cultivars growing in hormone-free medium were transferred to medium containing either sucrose or glycerol as the only carbohydrate source. Glycerol has been reported to induce further development of Citrus somatic embryos, while in the presence of sucrose they continue to proliferate in an 'undifferentiated' manner. The endogenous hormone levels of the cultures were evaluated after 2 and 5  days to characterise the initial steps of embryo development. In most cases, differences among treatments were observed only after 5 days of culture. Higher cytokinin levels were found in most of the cultures transferred to the glycerol-containing medium. The effect of ageing sweet orange cultures on their endogenous hormone levels was determined by leaving them in the original culture medium without subculturing for 60 days. While no changes were observed in the free indoleacetic acid and gibberellin contents, lower levels of abscisic acid and cytokinins were found in the aged cultures than in those transferred at the normal interval, every 30 days. The endogenous hormone contents of Citrus callus of different genotypes were compared. Significant differences were observed in the levels of all hormones evaluated, even when the in vitro ontogeny of the different genotypes was very similar. Received: 10 February 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Ultrastructure of porcine embryos following development in vitro versus in vivo

The present study examined the ultrastructural appearance of porcine embryos from the four-cell stage to the blastocyst grown either in vivo or in vitro. Embryos were collected at slaughter from superovulated gilts and were fixed for transmission electron microscopy either immediately or after various periods of in vitro culture. In general, the morphology of in vivo and in vitro grown embryos was similar. In vivo grown four-cell stages contained dense fibrillar nucleoli. At the eight-cell stage the nucleoli possessed increasing amounts of chromatin and granules. In both stages the mitochondria were spherical or ovoid in shape and had only few cristae. In morulae and blastocysts the nucleoli were mainly of the fibrillogranular type, and the mitochondria were filamentous and possessed more cristae, of which many were tubular. Two major ultrastructural deviations were observed in about half of the in vitro cultured embryos. First, nucleolus-like structures were found outside the nuclei in the cytoplasm of blastomeres. These structures were spherical and composed of chromatin-like material containing characteristically a single large and several small vacuoles. The structures were frequently associated with profiles of smooth endoplasmic reticulum (SER). A second type of deviation was aggregates of SER appearing as spiral coils or multiangular complexes. Some embryos displayed both types of deviations. The physiological significance of these deviations remains speculative. They may be involved in the considerably reduced capability of porcine embryos to develop to piglets following in vitro culture.     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

联合应用大鼠血管内皮细胞和胰岛培养对胰岛功能的影响

目的 探讨通过体外共培养胰岛和血管内皮细胞能否改善胰岛的功能.方法 SD 大鼠分离纯化出胰岛细胞,分为两组：A 组胰岛单纯培养组,B 组胰岛和内皮细胞共培养组.从大鼠的胸主动脉分离纯化出血管内皮细胞,胰岛分离纯化后通过AO/PI 染色和胰岛素释放实验来判断两组胰岛的活性.结果 共培养组胰岛在7 d 内维持正常的形态,90﹪的胰岛通过AO/PI 染色显示良好的活性;胰岛素释放实验显示第7 天（2.21 ± 0.21）和第14 天（2.53 ± 0.21）共培养组和单纯培养组（1.94 ± 0.15,1.71 ± 0.19）刺激指数差异有统计学意义（P 〈 0.05）.结论 应用大鼠血管内皮细胞和胰岛共培养能够改善胰岛的存活及分泌功能.