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Nowadays there are no well-established, standard methods in electron microscopy despite its 50-year history. An excessive variety of research objects prompt researchers to modify and improve methodological approaches to sample preparation. One of the difficult objects to study by electron microscopy is hydrophytic plants, for example, Phragmites au...
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... embedding medium (SPI Supplies 02660-AB) after dehydration [3]. Imbibition in the embedding medium is crucial, therefore, taking into account the peculiarities of the sample, it was used an inclined mixer. The vials with samples were positioned at an angle of 35 that ensures a layered displacement of the embedding medium relative to the sample (Fig. 2). Then the samples were placed into the flat-bottom capsules (Fig. ...
Citations
... It is highly resistant to biodegradation, due to which it biomagnifies quickly in the food chain by accumulating in the intercellular fluid of plants. It has been shown that BaP and 1,2-benzacenaphthene can be included in the metabolic chains of plants by binding to glucose and glutathione (GSH), penetrating into the space of the cell wall or cell organelles, causing ultrastructural damage and provoking an increase in the intensity of oxidative stress [6,7]. ...
... However, there are also similar changes in the structures of tissues of different species growing in PAH-contaminated soil. Thus, under the action of BaP in Hordeum sativum, degradation of the integumentary tissue of the roots and disturbance of the ordered arrangement of reduced cells of the primary cortex were observed [6]. Destruction of the epidermis and an increase in the size of the cortical cells with subsequent disruption of the originally concentric arrangement was found in Pisum sativum and Z. mays with the accumulation of polyarenes [30]. ...
... Histopathological evaluation was performed on tissue from the central part of the leaf (2 × 2 mm) and in the zone of root hairs (2 mm). The preparation of the histological specimens consisted of the following stages: plant tissue sampling, fixing, dehydration, embedding with polymerization of the prepared blocks, sectioning, and staining with toluidine blue [6]. Evaluation of the histological slides was performed using a Mikmed-6 microscope. ...
Benzo[a]pyrene (BaP) is noted as one of the main cancer-causing pollutants in human beings and may damage the development of crop plants. The present work was designed to explore more insights into the toxic effects of BaP on Solanum lycopersicum L. at various doses (20, 40, and 60 MPC) spiked in Haplic Chernozem. A dose-dependent response in phytotoxicity were noted, especially in the biomass of the roots and shoots, at doses of 40 and 60 MPC BaP and the accumulation of BaP in S. lycopersicum tissues. Physiological and biochemical response indices were severely damaged based on applied doses of BaP. During the histochemical analysis of the localization of superoxide in the leaves of S. lycopersicum, formazan spots were detected in the area near the leaf's veins. The results of a significant increase in malondialdehyde (MDA) from 2.7 to 5.1 times, proline 1.12-to 2.62-folds, however, a decrease in catalase (CAT) activity was recorded by 1.8 to 1.1 times. The activity of superoxide dismutase (SOD) increased from 1.4 to 2, peroxidase (PRX) from 2.3 to 5.25, ascorbate peroxidase (APOX) by 5.8 to 11.5, glutathione peroxidase (GP) from 3.8 to 7 times, respectively. The structure of the tissues of the roots and leaves of S. lycopersicum in the variants with BaP changed depending on the dose: it increased the intercellular space, cortical layer, and the epidermis, and the structure of the leaf tissues became looser.
... For microscopic examination of tissues and cells of H. vulgare, samples were taken from the middle part of the 2 nd or 3 rd leaf (2 × 2 mm) and the 2 mm of root (middle part) from the root hair zone. All stages of tissue preparation for anatomical and ultrastructural observations (contrasting, dehydration, fixing in epon -type polymerizing mixtures, preparation, and staining of semi-thin sections for light-optical study) were performed using the standard methodological techniques (Fedorenko et al., 2018). Semi-thin sections with a thickness of 0.5-1 μm for light-optical observation were stained with methylene blue, and examined with a light-optical microscope MikMed -6 (Russia) at a magnification of×100 and ×400 for anatomical understanding of plant tissues. ...
The constant use of zinc oxide nanoparticles (ZnO NPs) in agriculture could increase their concentration in soil, and cause a threat to sustainable crop production. The present study was designed to determine the role of spore-forming and metal-tolerant bacteria, and biochar in alleviating the toxic effects of a high dose of ZnO NPs (2000 mg kg −1) spiked to the soil (Haplic Chernozem) on barley (Hordeum sativum L). The mobile compounds of Zn in soil and their accumulation in H. sativum tissues were increased significantly. The addition of biochar (2.5% of total soil) and bacteria (10 10 CFU kg −1) separately and in combination showed a favorable impact on H. sativum growth in ZnO NPs polluted soil. The application of bacteria (separately) to the contaminated soil reduced the mobility of Zn compounds by 7%, due to loosely bound Zn compounds, whereas only biochar inputs lowered Zn mobile compounds mobility by 33%, even the combined application of biochar and bacteria also suppressed the soil Zn mobile compounds. Individual application of biochar and bacteria reduced the Zn plant uptake , i.e., underground parts (roots) by 44% and 20%, and in the above-ground parts of H. sativum plants by 39% and 13%, respectively, compared to ZnO NPs polluted soil treatments. Biochar, both separately and in combination with bacteria improved the root length by 48 and 85%, and plant height by 53 and 40%, respectively, compared to the polluted control. The root length and plant height decreased by 52 and 40% in ZnO NPs spiked soil compared clean soil treatments. Anatomical results showed an improvement in the structural organization of cellular -sub-cellular tissues of root and leaf. The changes in ultrastructural organization of assimilation tissue cells were noted all treatments due to the toxic effects of ZnO NPs compared with control treatment. The results indicate that metal-tolerant bacteria and biochar could be effective as a soil amendment to reduce metal toxicity, enhance crop growth, and improve soil health.
... To assess the impact of pollution on the ultrastructure in accordance with the accumulation of pollutants, background plants and samples of the site nos. 2 and 3 located in the prevailing wind direction were used. Sample preparation for light-optical and electron microscopy was carried out according to the method of plant tissue double fixation modified for the study of macrophyte plants (Fedorenko et al., 2018). Cross-sections at the roots were made at 3 and 6 cm from the growth apex. ...
The work is devoted to evaluation of the ability of Phragmites australis Сav. to indicate the soil pollution with heavy metals (HMs) and priority polycyclic aromatic hydrocarbons (PAHs) by studying changes in the plant’s ultrastructure. The concentration of Mn, Cu, Cr, Cd, Pb, Zn, Ni as well as 16 priority PAHs in hydromorphic soils and macrophyte plants (Phragmites australis Cav.) were increasing with distance decreasing to the power station and approaching to the direction of prevailing wind (northwest). The analyze of distribution of the studied pollutants in plants showed that the highest concentration have prevailed in the roots. A decrease in the diameter of the roots, and an increase in the thickness of the leaf blade was established. The transmission electron microscopy analysis showed that the ultrastructure of P. australis chloroplasts changed affected by accumulation of HMs and PAHs: a rise in the number of plastoglobules; a drop in the number of lamellae in granules, as well as changes in the shape, size, and electron density of mitochondria and peroxisomes. The most serious destructive violations of the main cellular organelles were noted for plants from the site within a 2.5 km from the emissions source and located on the predominant wind rose (north-west) direction. These macrophytes reflect spatial variations of pollutants metals in hydromorphic soils, therefore they are of potential use as bioindicators of environmental pollution.
... When the H. sativum reached the boot and head emergence phase, the samples were selected for microscopic analysis, and the root length and shoot height were measured. Light-optical microscopic examination of H. sativum roots: sampling, glutar-osmium fixation, contrast, dehydration, encapsulation in a polymerizing mixture, and staining of semi-thin sections; was performed using generally accepted methods [24]. Semi-thin sections with a thickness of 0.5-1 microns for light-optical observation were prepared on an EM UC6 ultramicrotome (Leica, Germany) and examined on a LOMO light-optical microscope (Russia) at magnification x100 and x400. ...
Cadmium is a phytotoxic element that has no known biological functions in plants. It can competitively replace such an important trace element as zinc. However, Zn is currently one of the most common anthropogenic pollutants. A model vegetation experiment was established to estimate the effects of soil pollution with Zn and Cd on the tissue-organ of the Hordeum sativum distichum. Adding 440 mg/kg of Zn (two approximately permissible concentrations) to the soil resulted in a 23% reduction in root length. Whereas the addition of 4 mg/kg of Cd (two approximately permissible concentrations) to the soil reduced the root length by 27% and shoot height by 15%. Microscopic examination showed anatomical changes in the structures of H. sativum roots grown on soil contaminated with heavy metals. A large degradation of the inner cell layers and a violation of the location of cells were found in H. sativum that grew with an excess of Cd. The revealed structural changes in the root tissues indicate a violation of the integrity and transport of nutrients by plants from the soil as a result of the toxic effect of Zn and Cd.
... The preparation of plant tissue samples for cellular structural and ultrastructural observation was performed according to the method developed by Fedorenko et al. [40] for light and TEM observations. A 1 mm leaf sample was obtained from the middle of a fresh leaf directly taken from the experimental plants. ...
The aim of the present work was to investigate the toxic effects of zinc oxide nanoparticles (ZnO NPs, particle size < 50 nm) on the physiological and anatomical indices of spring barley (Hordeum sativum L.). The results show that ZnO NPs inhibited H. sativum growth by affecting the chlorophyll fluorescence emissions and causing deformations of the stomatal and trichome morphology, alterations to the cellular organizations, including irregularities of the chloroplasts, and disruptions to the grana and thylakoid organizations. There was a lower number of chloroplasts per cell observed in the H. sativum leaf cells treated with ZnO NPs as compared to the non-treated plants. Cytomorphometric quantification revealed that ZnO NPs decreased the size of the chloroplast by 1.5 and 4 times in 300 and 2000 mg/L ZnO NP-treated plants, respectively. The elemental analysis showed higher Zn accumulation in the treated leaf tissues (3.8 and 10.18-fold with 300 and 2000 mg/L ZnO NPs, respectively) than the untreated. High contents of Zn were observed in several spots in ZnO NP-treated leaf tissues using X-ray fluorescence. Deviations in the anatomical indices were significantly correlated with physiological observations. The accumulation of Zn content in plant tissues that originated from ZnO NPs was shown to cause damage to the structural organization of the photosynthetic apparatus and reduced the photosynthetic activities.
... The preparations for analysis by microscope were done in accordance with the procedure suggested by Fedorenko et al. (2018). The preparations began with the cutting of the roots and above-ground parts of Phragmites australis Cav. in a size of about 1 mm. ...
The concentrations of ∑16 priority polycyclic aromatic hydrocarbons (PAHs) for soils, roots, and above-ground parts of reed (Phragmites australis Cav.) were determined on different monitoring plots located near the city of Kamensk-Shakhtinsky, southern Russia, where historically received industrial sewage and sludge. The total PAHs concentration in monitoring soil plots was significantly higher than those in the background site which situated at the distance of 2 km from the contamination source. Accordingly, the maximum accumulation was found for phenanthrene and chrysene among the 16 priority PAHs in most of the plant samples collected in the impact zone. The effects of PAHs’ pollution on changes of Phragmites australis Cav. cellular and subcellular organelles in the studied monitoring sites were also determined using optical and electron microscopy, respectively. The obtained data showed that increasing of PAHs contamination negatively affected the ultrastructural changes of the studied plants. Phragmites australis Cav. showed a high level of adaptation to the effect of stressors by using tissue and cell levels. In general, the detected alterations under the PAHs effect were possibly connected to changes in biochemical and histochemical parameters as a response for reactive oxygen species and as a protective response against oxidative stress. The obtained results introduce innovative findings of cellular and subcellular changes in plants exposed to ∑16 priority PAHs as very persistent and toxic contaminants.
... The preparation of plant tissue samples for anatomical observation was carried out according to the method described by Fedorenko et al. (2018). One mm-sized sample was collected and fixed using 2.5% glutaraldehyde/0.1 M phosphate buffer solution (PBS) at room temperature for 2 h. ...
The effects of bulk- and nano-CuO were monitored on barley (Hordeum sativum L.) in hydroponic conditions. The anatomical and cyto-/morphometric parameters of plants, exposed to both types of CuO in different doses (300 and 2000 mg/L) were recorded. The germination rate, root and shoot lengths decreased in a dose-dependent manner. Exposure to nano-CuO significantly increased Cu content in the H. sativum roots; however, the translocation rates of dissolved Cu were low and showed less accumulation in above-ground tissues. The differences between nano- and bulk-CuO treated plants were sufficiently evident, but at lower concentrations, these differences were non-significant. The relative seed germination inhibition was noted up to 11% and 22% under the high dose of bulk- and nano-CuO, respectively; however, at low dose, it was non-significant. The relative root length was reduced 3.6 fold by bulk- and 1.5 fold by nano-CuO, and shoot lengths decreased 1.6 fold by bulk- and 1.4 fold by nano-CuO under the high dose after growth of 30 days. It indicated more morphological effects on H. sativum caused by bulk- than the nano-CuO. The cytomorphometric analysis indicated the average cortex cell, total cortex, and total central cylinder areas of root cells and the average areas of chlorenchyma leaf cells were increased as compared to control in both bulk- and nano-CuO treated plants. It showed destructive effects of nano- and bulk-CuO on cellular organizations of H. sativum anatomy. Thus, at the low dose, the minimal effects of nano-CuO were observed than the bulk. Therefore, the finding could be interest for the safe application of nano-CuO.
... Preparation of plant tissue samples for transmission electron microscope (TEM) was carried out according to the method described by Fedorenko et al. (2018). Two millimeter-sized samples were collected from the middle part of the plant leaf and fixed using 2.5% glutaraldehyde/0.1 M phosphate buffer solution (PBS) at room temperature for 2 h. ...
The paper presents the results of the model experiment on spring barley (Hordeum vulgare L.) grown in polluted soil. The influence of separate and combined application of wood biochar and heavy metal-tolerant bacteria on morpho-physiological, anatomical and ultrastructural parameters of H. vulgare L. has been studied. The joint application of biochar and bacteria increased the shoot length by 2.1-fold, root length by 1.7-fold, leaf length by 2.3-fold and dry weight by threefold compared to polluted variant, bringing the plant parameters to the control level. The maximal quantum yield of photosystem II decreased by 8.3% in H. vulgare L. grown in contaminated soil, whereas this decrease was less in biochar (7%), bacteria (6%) and in combined application of bacteria and biochar (5%). As for the transpiration rate, the H. vulgare L. grown in polluted soil has shown a decrease in transpiration rate by 26%. At the same time, the simultaneous application of biochar and bacteria has led to a significant improvement in the transpiration rate (14%). The H. vulgare L. also showed anatomical (integrity of epidermal, vascular bundles, parenchymal and chlorenchymal cells) and ultrastructural (chloroplasts, thylakoid system, plastoglobules, starch grains, mitochondria, peroxisomes, ribosomes, endoplasmic reticulum, vacuoles) changes, revealed by light-optical and transmission electron microscopy of leaf sections. The effects were most prominent in H. vulgare L., grown in polluted soil but gradually improved with application of biochar, bacteria and their combination. The use of biochar in combination with metal-tolerant bacteria is an efficient tool for remediation of soils, contaminated with heavy metals. The positive changes caused by the treatment can be consistently traced at all levels of plant organization.
... The polymerization was carried out in a thermostat at a temperature of 37°C (1 day), 60°C (2 days). All stages of tissue preparation for morphological observation (contrasting, dehydration, encapsulation by polymerization, preparation and staining of semi-thin sections for light-optical study) were performed using standard methods (Usatov et al. 2004, Fedorenko et al. 2018. ...
Nowadays, nanotechnology is one of the most dynamically developing and most promising technologies. However, the safety issues of using metal nanoparticles, their environmental impact on soil and plants are poorly understood. These studies are especially important in terms of copper-based nanomaterials because they are widely used in agriculture. Concerning that, it is important to study the mechanism behind the mode of CuO nanoparticles action at the ultrastructural intracellular level. It is established that the contamination with CuO has had a negative influence on the development of spring barley. A greater toxic effect has been exerted by the introduction of CuO nanoparticles as compared to the macrodispersed form. A comparative analysis of the toxic effects of copper oxides and nano-oxides on plants has shown changes in the tissue and intracellular levels in the barley roots. However, qualitative changes in plant leaves have not practically been observed. In general, conclusions can be made that copper oxide in nano-dispersed form penetrates better from the soil into the plant and can accumulate in large quantities in it.
... The preparation of samples for microscopy was carried out according to an original technique developed for preparation and double fixation of plant tissue of the fresh samples of cattails [31,52]. Cross-sections at the roots of cattails were made at a distance of 3 and 6 cm from the growth apex, and the choice of distances from the growth apex of the adventitious roots was arbitrary. ...
... The plant samples of T. australis and T. laxmannii were characterized with an excess of the MPL [50]: Cr up to 19 times, Zn up to 4 times, Ni up to 4 times, and Cd up to 2 times. The excess of Cr in plant tissues, as noted above, is associated with the natural lithological specificity of this region [52]. The maximum concentration of Zn in cattails was observed in the roots and ranged from 174 mg/kg for T. australis to 195 mg/kg DW for T. laxmannii, exceeding its content in the vegetative and reproductive organs. ...
The aim of this study is to investigate the adaptation of two species of cattail Typha australis Schum. and Thonn. and Typha laxmannii Lepech. based on analysis of the morphological and anatomical features of their vegetative and generative organs to soil pollution with potentially toxic elements (PTE) in the riparian zones of the sea edge of the Don River delta (Southern Russia). Both species of the cattail are able to accumulate high concentrations of Ni, Zn, Cd, Pb and can be used for phytoremediation of polluted territories. The pattern of PTE accumulation in hydrophytes has changed on polluted soils of coastal areas from roots/rhizomes > inflorescences > stems to roots/rhizomes > stems ≥ inflorescences. The comparative morphological and anatomical analysis showed a statistically significant effect of the environmental stress factor by the type of proliferation in T. australis, and species T. laxmannii was visually in a depressed, deformed state with mass manifestations of hypogenesis. These deformations should be considered, on one hand, as adaptive, but on the other, as pathological changes in the structure of the spikes of the cattails. Light-optical and electron microscopic studies have shown that the degree and nature of ultrastructural changes in cattails at the same level of soil pollution are different and most expressed in the assimilation tissue of leaves. However, these changes were destructive for T. australis, but for T. laxmannii, these indicated a high level of adaptation to the prolonged technogenic impact of PTE.
