Effect of optimal stage of female gametophyte and heat treatment on in vitro gynogenesis induction in cucumber (Cucumis sativus L.)

Gynogenic plants of pickling cucumber were successfully produced by in vitro culture from unpollinated ovules. Haploids and spontaneous doubled haploids originated directly through embryogenesis. The cucumber ovaries were placed on induction media containing thidiazuron and cultured in the dark for 2-5 days, at 24°C, 28°C or 35°C. After induction, the material was transferred to regeneration media containing !-naphthaleneacetic acid and 6-benzylaminopurine. Results from the regeneration test indicated that the heat treatment increased the efficiency of gynogenesis in the optimal developmental stage of the female gametophyte. The highest number of embryos occurred following the35°C induction treatment. The maximum frequency of gynogenesis was 18.4%, while maximum plant regeneration was 7.1%. The flow cytometry analysis showed that 87.7% of the regenerants were haploid. On the basis of histological studies carried out on the female gametophyte, the best developmental stage for haploid induction seems to be the cellularization stage of embryo-sac formation, when the nuclei are already in position, the membranes have sometimes developed, and the cells are quite uniform in shape and structure.     

Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation—the Phase II conjugation reaction—of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3′-phosphoadenosine-5′-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.     

Structural change in the endoplasmic reticulum during the in situ development and in vitro fertilisation of wheat egg cells

The mechanism by which the sperm activates the egg cell of higher plants is largely unknown. Ca²⁺—implicated as a second messenger in the response of various plant cells to a wide range of stimuli—is a potential candidate for encoding the information brought by the sperm cell into the angiosperm egg. In higher plant cells the dominant calcium store appears to be the central vacuole; however, in the receptive wheat egg cell few vacuoles can be observed, thus it seems likely that the principal cell organelle performing a pivotal role in regulation of intracellular Ca²⁺ is the endoplasmic reticulum (ER). To examine this hypothesis, microinjection of the ER-specific fluorescent dicarbocyanine dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate [DiIC₁₆(3)], and low-light level CCD technology were used. Following the injection of an oil drop saturated with DiI, structural changes occurring in the ER during the in planta maturation of the female gamete were visualised. The ER was identified by chlorotetracycline labelling to be the main calcium store in the wheat egg cell. Structural changes occurring in the ER during the in planta maturation of the wheat female gamete led to a polarised ER network in the receptive egg cell. Within 3 min of in vitro sperm-egg fusion, a rapid, transient loss of continuity of the ER occurred, which underlines the significance in fertilisation of the ER structure in the female gamete of wheat.     

Periosteal microcirculatory action of chronic estrogen supplementation in osteoporotic rats challenged with tourniquet ischemia

AimsTransient ischemia of osteoporotic bones during elective orthopedic surgery or fracture repair carries risks for serious complications, and estrogen loss or replacement has a potential to influence ischemia–reperfusion-induced inflammatory activation. To clarify this, we investigated the periosteal inflammatory changes in a clinically relevant time frame in ovariectomized rats, an experimental model of postmenopausal bone loss. Furthermore, the effects of chronic estrogen supplementation on the postischemic local and systemic inflammatory reactions were assessed.Main methodsBilateral ovariectomy or sham operation was performed in 3-month-old female Sprague–Dawley rats. Five months later, estrogen replacement therapy with 17β-estradiol (20 μg^? 1 kg^? 1 day^? 1) or vehicle treatment was initiated. The microcirculatory inflammatory consequences of 60-min total hindlimb ischemia followed by 180-min reperfusion were examined 11 months after ovariectomy and were compared with those in 3-month-old animals.Key findingsThe osteoporosis that developed 5 months after ovariectomy was significantly ameliorated by estrogen replacement therapy. Both in ovariectomized and in non-ovariectomized animals, ischemia–reperfusion elevated the neutrophil adherence ~ 3-fold in the postcapillary venules of the periosteum (intravital microscopy), with an ~ 50–60% increase in intravascular neutrophil activation (CD11b; FACS analysis), an enhanced TNF-α release (ELISA) and periosteal expression of ICAM-1 (the endothelial ligand of CD11b; immunohistochemistry). Exogenous 17β-estradiol considerably reduced TNF-α release and the number of neutrophil–endothelial interactions in the periosteum, without affecting the CD11b and ICAM-1 expression changes.SignificanceOsteoporosis itself does not increase the magnitude of the limb ischemia–reperfusion-associated periosteal inflammatory reaction. Chronic estrogen supplementation, however, reverses osteoporosis and significantly ameliorates the microcirculatory consequences of transient ischemia.     

Soil microarthropods in non-intervention montane spruce forest regenerating after bark-beetle outbreak

We studied Oribatida and Collembola in an old-growth Norway spruce (Picea abies) forest that suffered a massive bark beetle (Ips typographus) outbreak in the 1990s and gradually decayed. It was left to regenerate naturally without human intervention. There was a high abundance of a few tolerant species and lower numbers of sensitive silvicolous ones. The most dominant species were Tectocepheus velatus, Platynothrus peltifer and Isotomiella minor. Although the details, which determine the identity of successful species, remain unknown, parthenogenesis, high reproduction rate and detrito- or detritofungivorous feeding were the common features of the most dominant species in our study. Trait assessment showed an overall predominance of parthenogenesis and high abundance of detritivorous oribatids. The soil functions connected with Oribatida and Collembola seem to be still affected by the bark-beetle outbreak and our results indicate that the disturbance caused important changes in the functioning of the whole soil ecosystem.     

Extraction of bioactive compounds from Alaria esculenta with pulsed electric field

Journal of Applied Phycology - There is a growing demand for bioactive compounds derived from green extraction methods from the food and cosmetic industries. Pulsed electric field (PEF)-assisted...     

Changes in sediment seed‐bank composition of invaded macrophyte communities in a thermal river

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Moderate heritability and low evolvability of sperm morphology in a species with high risk of sperm competition,the collared flycatcher Ficedula albicollis

Spermatozoa represent the morphologically most diverse type of animal cells and show remarkable variation in size across and also within species. To understand the evolution of this diversity, it is important to reveal to what degree this variation is genetic or environmental in origin and whether this depends on species’ life histories. Here we applied quantitative genetic methods to a pedigreed multigenerational data set of the collared flycatcher Ficedula albicollis, a passerine bird with high levels of extra‐pair paternity, to partition genetic and environmental sources of phenotypic variation in sperm dimensions for the first time in a natural population. Narrow‐sense heritability (h²) of total sperm length amounted to 0.44 ± 0.14 SE, whereas the corresponding figure for evolvability (estimated as coefficient of additive genetic variation, CV_a) was 0.02 ± 0.003 SE. We also found an increase in total sperm length within individual males between the arrival and nestling period. This seasonal variation may reflect constraints in the production of fully elongated spermatozoa shortly after arrival at the breeding grounds. There was no evidence of an effect of male age on sperm dimensions. In many previous studies on laboratory populations of several insect, mammal and avian species, heritabilities of sperm morphology were higher, whereas evolvabilities were similar. Explanations for the differences in heritability may include variation in the environment (laboratory vs. wild), intensity of sexual selection via sperm competition (high vs. low) and genetic architecture that involves unusual linkage disequilibrium coupled with overdominance in one of the studied species.     

Global ubiquity and local endemism of free‐living terrestrial protists: phylogeographic assessment of the streptophyte alga Klebsormidium

Despite considerable research attention during the last 10 years, the distribution and biogeography of protists remain as highly controversial issues. The presumably huge population sizes and unlimited dispersal capabilities should result in protist ubiquity. However, recent molecular investigations suggest that protist communities exhibit strong biogeographic patterns. Here, we examined the biogeographic pattern of a very common green algal genus Klebsormidium. We evaluated the geographic distribution of rbcL genotypes for 190 isolates sampled in six sampling regions located in Europe, North America and Asia. Measures of correlation between genetic and geographic distance matrices revealed a differential distribution pattern on two geographic levels. Globally, the populations were genetically homogeneous; locally, the genotypes were patchily distributed. We hypothesized that a local fine‐scale structuring of genotypes may be caused by various ecological factors, in particular, by the habitat differentiation of particular genotypes. Our investigations also identified a large number of new, previously unrecognized lineages. A total of 44 genotypes were identified and more than 66% of these were reported for the first time.