BACKGROUND: Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster's three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth. CONCLUSIONS AND SIGNIFICANCE: This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.

• MeSH
• jedle účinky léků   růst a vývoj   MeSH
• rostlinné proteiny metabolismus   MeSH
• smrk účinky léků   růst a vývoj   metabolismus   MeSH
• tetrazoliové soli farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Norsko MeSH
• Názvy látek
• rostlinné proteiny MeSH
• tetrazoliové soli MeSH
• triphenyltetrazolium MeSH Prohlížeč

We used carbon paste electrodes and a standard potentiostat to detect silver ions. The detection limit (3 Signal/Noise ratio) was estimated as 0.5 μM. A standard electrochemical instrument microanalysis of silver(I) ions was suggested. As a working electrode a carbon tip (1 mL) or carbon pencil was used. Limits of detection estimated by dilution of a standard were 1 (carbon tip) or 10 nM (carbon pencil). Further we employed flow injection analysis coupled with carbon tip to detect silver(I) ions released in various beverages and mineral waters. During first, second and third week the amount of silver(I) ions releasing into water samples was under the detection limit of the technique used for their quantification. At the end of a thirteen weeks long experiment the content of silver(I) ions was several times higher compared to the beginning of release detected in the third week and was on the order of tens of nanomoles. In subsequent experiments the influence of silver(I) ions (0, 5 and 10 μM) on a plant model system (tobacco BY-2 cells) during a four-day exposition was investigated. Silver(I) ions were highly toxic to the cells, which was revealed by a double staining viability assay. Moreover we investigated the effect of silver(I) ions (0, 0.3, 0.6, 1.2 and 2.5 μM) on guppies (Poecilia reticulata). Content of Ag(I) increased with increasing time of the treatment and applied concentrations in fish tissues. It can be concluded that a carbon tip or carbon pencil coupled with a miniaturized potentiostat can be used for detection of silver(I) ions in environmental samples and thus represents a small, portable, low cost and easy-to-use instrument for such purposes.

• Klíčová slova
• ecotoxicology, guppy (Poecilia reticulata), miniaturized carbon electrodes, silver, tobacco cells, voltammetry,
• Publikační typ
• časopisecké články MeSH

In this study, the influence of lead (II) ions on sunflower growth and biochemistry was investigated from various points of view. Sunflower plants were treated with 0, 10, 50, 100 and/or 500 μM Pb-EDTA for eight days. We observed alterations in growth in all experimental groups compared with non-treated control plants. Further we determined total content of proteins by a Bradford protein assay. By the eighth day of the experiment, total protein contents in all treated plants were much lower compared to control. Particularly noticeable was the loss of approx. 8 μg/mL or 15 μg/mL in shoots or roots of plants treated with 100 mM Pb-EDTA. We also focused our attention on the activity of alanine transaminase (ALT), aspartate transaminase (AST) and urease. Activity of the enzymes increased with increasing length of the treatment and applied concentration of lead (II) ions. This increase corresponds well with a higher metabolic activity of treated plants. Contents of cysteine, reduced glutathione (GSH), oxidized glutathione (GSSG) and phytochelatin 2 (PC2) were determined by high performance liquid chromatography with electrochemical detection. Cysteine content declined in roots of plants with the increasing time of treatment of plants with Pb-EDTA and the concentration of toxic substance. Moreover, we observed ten times higher content of cysteine in roots in comparison with shoots. The observed reduction of cysteine content probably relates with its utilization for biosynthesis of GSH and phytochelatins, because the content of GSH and PC2 was similar in roots and shoots and increased with increased treatment time and concentration of Pb-EDTA. Moreover, we observed oxidative stress caused by Pb-EDTA in roots where the GSSG/GSH ratio was about 0.66. In shoots, the oxidative stress was less distinctive, with a GSSG/GSH ratio 0.14. We also estimated the rate of phytochelatin biosynthesis from the slope of linear equations plotted with data measured in the particular experimental group. The highest rate was detected in roots treated with 100 μM of Pb-EDTA. To determine heavy metal ions many analytical instruments can be used, however, most of them are only able to quantify total content of the metals. This problem can be overcome using laser induced breakdown spectroscopy, because it is able to provide a high spatial-distribution of metal ions in different types of materials, including plant tissues. Data obtained were used to assemble 3D maps of Pb and Mg distribution. Distribution of these elements is concentrated around main vascular bundle of leaf, which means around midrib.

• Klíčová slova
• heavy metals, high performance liquid chromatography with electrochemical detection, laser induced breakdown spectroscopy, lead ions, phytoremediation, spectrometry, sunflower,
• Publikační typ
• časopisecké články MeSH

The aim of this work is to investigate sunflower plants response on stressinduced by silver(I) ions. The sunflower plants were exposed to silver(I) ions (0, 0.1, 0.5,and 1 mM) for 96 h. Primarily we aimed our attention to observation of basic physiologicalparameters. We found that the treated plants embodied growth depression, coloured changes and lack root hairs. Using of autofluorescence of anatomical structures, such aslignified cell walls, it was possible to determine the changes of important shoot and rootstructures, mainly vascular bungles and development of secondary thickening. Thedifferences in vascular bundles organisation, parenchymatic pith development in the rootcentre and the reduction of phloem part of vascular bundles were well observable.Moreover with increasing silver(I) ions concentration the vitality of rhizodermal cellsdeclined; rhizodermal cells early necrosed and were replaced by the cells of exodermis.Further we employed laser induced breakdown spectroscopy for determination of spatialdistribution of silver(I) ions in tissues of the treated plants. The Ag is accumulated mainlyin near-root part of the sample. Moreover basic biochemical indicators of environmentalstress were investigated. The total content of proteins expressively decreased withincreasing silver(I) ions dose and the time of the treatment. As we compare the resultsobtained by protein analysis - the total protein contents in shoot as well as root parts - wecan assume on the transport of the proteins from the roots to shoots. This phenomenon canbe related with the cascade of processes connecting with photosynthesis. The secondbiochemical parameter, which we investigated, was urease activity. If we compared theactivity in treated plants with control, we found out that presence of silver(I) ions markedlyenhanced the activity of urease at all applied doses of this toxic metal. Finally we studiedthe effect of silver(I) ions on activity of urease in in vitro conditions.

• Klíčová slova
• Biochemical marker, Heavy metals, Plant biosensor, Sensors, Silver,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH