Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Effects of carbon dioxide on the spatially separate electrogenic ion pumps and the growth rate in the hypocotyl ofVigna sesquipedalis

Carbon dioxide, introduced into the gas phase of the experimental chamber, has distinct effects on two spatially separate membrane potentials and the rate of elongation growth in hypocotyl segments ofVigna sesquipedalis Wight. Both membrane potentials (V _ps andV _px=the electric potential difference between the parenchyma symplast and the surface of the hypocotyl, and that between the parenchyma symplast and the xylem, respectively) hyperpolarized rapidly but transiently at the introduction of CO₂. Prolonged exposure of the hypocotyl to high concentrations of CO₂ (above 10%) caused depolarization of membrane potentials above the level before CO₂ introduction. When CO₂ was replaced with air, the membrane potentials exhibited a distinct depolarization response of transient nature. The growth rate of the hypocotyl segments exhibited similar responses to CO₂ as did the membrane potentials (the increase and the decrease of the growth rate were corresponded to the hyperpolarization and the depolarization, respectively), but these responses always followed the changes of the membrane potentials. The CO₂-induced maximum hyperpolarization ofV _ps and the maximum increase of the growth rate were closely correlated. All these responses were strictly dependent on aerobic metabolism. These results indicate that CO₂ may regulate elongation growth in two ways: by affecting the activity of the electrogenic ion pump via intracellular acidification, and also by acting via apoplastic acidification as a wall-loosening acid.Symbols and abbreviations V _sx electric potential difference between the surface (S) and the xylem (X) of the hypocotyl - V _px electric potential difference between the inside of a parenchyma cell (P) andX - V _ps electric potential difference betweenP andS - V _ps (CO₂, max) the maximum value of CO₂-induced hyperpolarization ofV _ps - GR(CO₂, max) the maximum value of CO₂-induced increase of the growth rate - IAA indole-3-acetic acid     

The effects of cadmium exposure on the cytology and function of primary cultures from rainbow trout

Cultured epidermal cells from explants of skin of rainbow trout were used to study the cytological and functional changes following sublethal exposure to cadmium stress. The aim was to develop diagnostic markers for ecotoxicology. Cultures were exposed to the pollutant for 48 h. Cell structural and cytological changes were established by light and electron microscopy. Metabolic alterations were detected by immunohistochemistry. The relation between the initiation of cellular alterations and cadmium concentrations was compared in cultures exposed in commercially-available serum-free and serum-containing medium. The expression of stress proteins (metallothionein and heat shock protein) was also studied. Rainbow trout epithelial cells exposed to cadmium showed typical morphological changes indicative of cell death by apoptosis. Sublethal exposure also resulted in cellular metabolic disturbances with increased deposits of glycogen. Increased melanization was also observed. These changes appeared at lower concentrations of cadmium when cells were exposed in serum-free media than in serum-containing media. Cadmium induced the expression of heat shock proteins but not of metallothioneins. The results broadly confirm in vivo findings for cadmium toxicity and suggest that this in vitro technique may have applications in aquatic toxicology. © 1998 John Wiley & Sons, Ltd.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary vasculogenesis is mediated, in part, by reduced responsiveness to endogenous angiogenic stimuli, including vascular endothelial growth factor A (VEGF-A)

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure prior to chick embryo incubation (GD 0) induces dilated cardiomyopathy, and reduces myocardial hypoxia, vascular endothelial growth factor A (VEGF-A) expression, and coronary vascularization. We investigated whether reduced coronary vascularization 1) occurs in the absence of changes in cardiac morphology and 2) is associated with altered secretion of VEGF-A and/or an antivasculogenic factor. METHODS: Chicken eggs were treated with control (corn oil) or TCDD (0.075-0.3 pmol of TCDD/gm) on GD 5. In vivo cardiac morphology and artery number were determined on GD 10, while in vitro vascular outgrowth and VEGF-A secretion were determined from cardiac explants on GD 6. Effects of recombinant VEGF-A (rcVEGF-A), soluble flt-1 (sFlt-1) receptor plus rcVEGF-A, and control conditioned media were assessed in TCDD explants, while effects of TCDD-conditioned media was assessed in control explants. RESULTS: TCDD reduced coronary artery number in vivo by 53 +/- 8% and induced a dose-related reduction in tube outgrowth in vitro, but had no effect on cardiac morphology. All TCDD doses reduced explant VEGF-A secretion equally (43 +/- 3%), compared to control. sFlt-1 blocked outgrowth in control cultures and blocked rcVEGF-A-mediated rescue of outgrowth in TCDD explants. Control conditioned media partially rescued outgrowth from TCDD explants, while conditioned media from TCDD explants had no effect on controls. CONCLUSIONS: TCDD inhibition of coronary vascularization can occur in the absence of changes in cardiac morphology and is associated with reduced VEGF-A secretion but not an antivasculogenic factor. Since control media only partly rescues TCDD's inhibitory effect, we suggest that TCDD-exposed endothelial cells are less responsive to vasculogenic stimuli.     

白桦下胚轴再生系统的建立

本文对白桦下胚轴的再生系统作了研究。讨论了不同的基本培养基、激素组成、培养条件及外植体的生理年龄在不同阶段产生的效应。通过诱导、分化和生根阶段, 得到了再生植株, 提出了最适的基本培养基、激素组成、培养条件及下胚轴外植体的生理年龄, 为白桦的遗传转化奠定了基础。     

Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh.

The aim of the study was to evaluate the influence of light conditions,physical state of the induction medium and the mutagenic treatment on theembryogenic ability of Arabidopsis thaliana (L.) immaturezygotic embryos differing in developmental stage. The efficiency of directsomatic embryogenesis (DSE) was analysed in a culture of immature zygoticembryos at an early (ES) and a late (LS) developmental stage. The efficiency ofDSE was scored as a percentage of the explants producing somatic embryos. Theexperiments indicated that the physical state of the induction medium (solid orliquid) did not influence the embryogenic ability of the cultured explants. Inthe cultures on both solid and liquid induction medium, the ES explantsproducedsomatic embryos with a frequency of 25.8–37.3% i.e. 2.5–3-timeslower than LS explants. However, an increase in the embryogenic ability of ESexplants (up to 69.8%) was observed when DSE was induced in darkness. Moreover,the stimulation of DSE efficiency in culture of ES explants was also observedafter mutagenic treatment. The chemical mutagens, MNH and EMS, applied forexplant treatment, both stimulated efficiency of somatic embryo formation inculture of ES explants. The most effective DSE induction was observed when MNHand EMS were applied in doses of 0.125–1.0 mM × 3h and0.05–0.2% × 18h, respectively. In these treatment combinations thefrequency of ES explants forming somatic embryos was found to be about 2 timeshigher than in the control culture.     

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane–derived vesicles in cell–cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 μm to 1 μm, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte–derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.     

Identification of emergent motion compartments in the amniote embryo

Abstract

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ～400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen's node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen's node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

Encapsulation of Immature Somatic Embryos of <i>Coffea arabica</i> L. for <i>in Vitro</i> Preservation

The present study aimed to develop a protocol for somatic embryogenesis and encapsulation of coffee embryos (Coffea arabica L.), for the conservation of genotypes with characteristics of commercial interest. Somatic embryos were induced from leaf explants in Murashige and Skoog medium (MS) supplemented with 1 mg · L⁻¹ of 2,4-dichlorophenoxiacetic acid (2,4-D) combined with 2 mg · L⁻¹ of benzyladenine (BA). Somatic embryos (SE) at the globular stage were encapsulated in a sodium alginate matrix; two treatments were tested: MS + 5 mg · L⁻¹ BA + 1 mg · L⁻¹ NAA + 3% (w/v) alginate, and MS + 7 mg · L⁻¹ BA + 5.7 mg · L⁻¹ indoleacetic acid (IAA) + 3% (w/v) alginate. Alginate was complexed with 100 mM calcium chloride (CaCl₂). Viability of the encapsulated SE was determined by staining with 0.01% fluorescein diacetate (FDA) after 0, 15, 30, and 45 days of storage at 4°C. Embryo viability was 100% in both treatments.