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Method for hydrophytic plant sample preparation for light and electron microscopy (studies on Phragmites australis Cav.).

ABSTRACT: 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 australis Cav. Traditional approaches to fixation and sample preparation do not give satisfactory results due to the peculiarities in structure and physiology of hydrophytic plants. The purpose of present research is modification description of the widespread method developed for double fixation of hydrophytic plant tissue for transmission electron microscopy. Suggested approach takes into account the features of hydrophyte plants. •The developed method allows improving the quality of plant samples by additional fixatives imbibition and removing of air bubbles from aerenchyma tissue using a vacuum.•The new step of sample preparation consisting in the layer-by-layer sample mixing in a special inclined mixer is applied for the embedding media penetrate sufficiently into the sample tissue.•The process of samples inclusion in polymeric resins is carried out in the flat-bottom capsules. Compare to standard conical capsules, flat-bottom capsules allow strictly defined orientation sample pieces, that is permit to produce a semi-thin and ultra-thin slices of perpendicular to the longitudinal structures of the plant. This is especially important to conduct an adequate comparative analysis of dimensions, shape, and electron density of fragments and parts of the studying samples.

SUBMITTER: Fedorenko GM

PROVIDER: S-EPMC6190527 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description:Interactions between introduced plants and soils they colonize are central to invasive species success in many systems. Belowground biotic and abiotic changes can influence the success of introduced species as well as their native competitors. All plants alter soil properties after colonization but, in the case of many invasive plant species, it is unclear whether the strength and direction of these soil conditioning effects are due to plant traits, plant origin, or local population characteristics and site conditions in the invaded range. Phragmites australis in North America exists as a mix of populations of different evolutionary origin. Populations of endemic native Phragmites australis americanus are declining, while introduced European populations are important wetland invaders. We assessed soil conditioning effects of native and non-native P. australis populations on early and late seedling survival of native and introduced wetland plants. We further used a soil biocide treatment to assess the role of soil fungi on seedling survival. Survival of seedlings in soils colonized by P. australis was either unaffected or negatively affected; no species showed improved survival in P. australis-conditioned soils. Population of P. australis was a significant factor explaining the response of seedlings, but origin (native or non-native) was not a significant factor. Synthesis: Our results highlight the importance of phylogenetic control when assessing impacts of invasive species to avoid conflating general plant traits with mechanisms of invasive success. Both native (noninvasive) and non-native (invasive) P. australis populations reduced seedling survival of competing plant species. Because soil legacy effects of native and non-native P. australis are similar, this study suggests that the close phylogenetic relationship between the two populations, and not the invasive status of introduced P. australis, is more relevant to their soil-mediated impact on other plant species.

Project description:Nutrient enrichment, particularly nitrogen, is an important determinant of plant community productivity, diversity and invasibility in a wetland ecosystem. It may contribute to increasing colonization and dominance of invasive species, such as Phragmites australis, especially during wetland restoration. Providing native species a competitive advantage over invasive species, manipulating soil nutrients (nitrogen) may be an effective strategy to control the invasive species and that management tool is essential to restore the degraded ecosystems. Therefore, we examined competition between Phragmites australis and Melaleuca ericifolia in a greenhouse setting with activated carbon (AC) treatments, followed by cutting of Phragmites shoots in nutrient-rich soils. Additionally, we evaluated the effect of AC on plant-free microcosms in the laboratory, to differentiate direct effects of AC on soil microbial functions from indirect effects. Overall, the objective was to test whether lowering nitrogen might be an effective approach for reducing Phragmites invasion in the wetland. The AC reduced Phragmites total biomass more significantly in repeated cut regime (57%) of Phragmites shoots compared to uncut regime (39%). Conversely, it increased Melaleuca total biomass by 41% and 68% in uncut and repeated cut regimes, respectively. Additionally, AC decreased more total nitrogen in above-ground biomass (41 to 55%) and non-structural carbohydrate in rhizome (21 to 65%) of Phragmites, and less total nitrogen reduction in above-ground biomass (25 to 24%) of Melaleuca in repeated cut compared to uncut regime. The significant negative correlation between Phragmites and Melaleuca total biomass was observed, and noticed that Phragmites acquired less biomass comparatively than Melaleuca in AC-untreated versus AC-treated pots across the cutting frequency. AC also caused significant changes to microbial community functions across Phragmites populations, namely nitrogen mineralization, nitrification, nitrogen microbial biomass and dehydrogenase activity (P???0.05) that may potentially explain changes in plant growth competition between Phragmites and Melaleuca. The overall effects on plant growth, however, may be partially microbially mediated, which was demonstrated through soil microbial functions. Results support the idea that reducing community vulnerability to invasion through nutrient (nitrogen) manipulations by AC with reducing biomass of invasive species may provide an effective strategy for invasive species management and ecosystem restoration.

Project description:The micro-morphology of leaf epidermises is valuable for the study of leaf development and function, as well as the classification of plant species. There have been few studies comparing different preparation and imaging methods for visualizing the leaf epidermis. Here, four specimen preparation methods were used to investigate the leaf epidermis morphology of Arabidopsis, radish, cucumber, wheat, rice, and maize, under an inverted basic light microscope (LM), a laser scanning confocal microscope (LSCM), or a scanning electron microscope (SEM). Optical microscope specimens were obtained using either the direct isolation method or the chloral hydrate-based clearing method. SEM images were obtained using a standard stage for conventional dehydrated samples or a Coolstage for fresh tissue. Different parts of epidermis peels were well focused under the LM. Investigation of samples cleared by chloral hydrate is convenient and autofluorescence of cell walls can be detected in rice. The resolution of images of conventional SEM leaf samples was generally higher than the Coolstage images at the same magnification, whereas local collapse and shrinkage were observed in leaves with high water content when using the conventional method. However, stomatal apparatuses of Arabidopsis, cucumber, radish, and maize deformed and showed poor appearance when using the Coolstage. Moreover, we usually used glutaraldehyde as an SEM fixative when using t-butanol for freeze-drying, though methanol is considered a better fixative in recent studies. In addition, fresh samples were not stable on the Coolstage. Thus, we compared four different t-butanol freeze-drying methods and two Coolstage methods. The dimension and morphology of tissues were compared using the six different methods. The results indicate that methanol fixative obviously reduced shrinkage of SEM samples compared with glutaraldehyde and formaldehyde alcohol acetic acid (FAA) fixatives. The use of methanol and a graded series of steps improved the preservation of samples. Preparing samples with optimal cutting temperature compound and observing at -30°C helped to increase the stability of Coolstage samples. In summary, our results provide an overview of the shortcomings and merits of four different methods, and might provide some information about choosing an optimal method for visualizing epidermal morphology.

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Method for hydrophytic plant sample preparation for light and electron microscopy (studies on Phragmites australis Cav.).

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