Identification of Alleles of Puroindoline Genes and Their Effect on Wheat (Triticum aestivum L.) Grain Texture.

Grain hardness is one of the most important quality characteristics of wheat (Triticum aestivum L.). It is a significant property of wheat grains and relates to milling quality and end product quality. Grain hardness is caused by the presence of puroindoline genes (Pina and Pinb). A collection of 25 genotypes of wheat with unusual grain colour (blue aleurone, purple and white pericarp, yellow endosperm) was studied by polymerase chain reaction (PCR) for the diversity within Pina and Pinb (alleles: Pina-D1a, Pina-D1b, Pinb-D1a, Pinb- -D1b, Pinb-D1c and Pinb-D1d). The endosperm structure was determined by a non-destructive method using light transflectance meter and grain hardness by a texture analyser. Genotype Novosibirskaya 67 and isogenic ANK lines revealed hitherto unknown alleles at the locus for the annealing of primers of Pinb-D1. Allele Pinb-D1c was found to be absent from each genotype. The mealy endosperm ranged from 0 to 100% and grain hardness from 15.10 to 26.87 N per sample.

A long-term survey of heavy metals and specific organic compounds in biofilms, sediments, and surface water in a heavily affected river in the Czech Republic.

To assess the long-term anthropogenic load of the Bílina River (Czech Republic), the concentrations of heavy metals and specific organic compounds in different river ecosystem matrices (water, biofilms, and sediments) were determined. Although the current concentrations of pollutants in surface water are low, frequently below the limits of the quantitative analytical methods used, the river ecosystem is still heavily loaded by anthropogenic pollution, mainly from the chemical and mining industries. This was demonstrated by analyzing biofilms and sediments. These matrices are more accurate representatives of the actual situation in the river and do not depend on hydrological conditions or random variability in water quality. The results indicate that the middle and the lower parts of the river are heavily polluted by mercury, arsenic, vanadium, polychlorinated biphenyls, hexachlorobenzene, and dichloro-diphenyl-trichloroethane. As a tributary of the Elbe River, the Bílina River represents a significant risk for the development of quality in this major European river.

Validated method for bioactive lignans in Schisandra chinensis in vitro cultures using a solid phase extraction and a monolithic column application.

A simple and rapid method for determination of six lignans found in plant cell cultures of Schisandra chinensis was developed and validated. The lignans were extracted from plant samples with methanol and the extracts were effectively cleaned by solid-phase extraction using Strata C18-E (Phenomenex) cartridges. Chromatographic separation was carried out on a Chromolith Performance RP-18e monolithic column (100 x 4.6 mm, Merck) using an isocratic mobile phase of acetonitrile and water in a 50:50 (v/v) ratio. The eluent was monitored at 220 nm. The baseline separation of schizandrin, gomisin A, deoxyschizandrin, gamma-schizandrin, gomisin N and wuweizisu C was achieved in a relatively short time period (20 min), which was made possible by the relatively high flow rate of the mobile phase (2 mL/min). The lower limit of quantitation was 0.1 mg/L for schizandrin and gomisin A, 0.3 mg/L for deoxyschizandrin, gamma-schizandrin, and gomisin N and 1 mg/L for wuweizisu C. The analysis of spiked samples containing six lignans provided absolute recoveries between 93 and 101% in all cases. The validated method was successfully applied to the determination of lignans in embryogenic plant cell cultures of Schisandra chinensis.

Fully automated spectrometric protocols for determination of antioxidant activity: advantages and disadvantages.

The aim of this study was to describe behaviour, kinetics, time courses and limitations of the six different fully automated spectrometric methods--DPPH, TEAC, FRAP, DMPD, Free Radicals and Blue CrO5. Absorption curves were measured and absorbance maxima were found. All methods were calibrated using the standard compounds Trolox® and/or gallic acid. Calibration curves were determined (relative standard deviation was within the range from 1.5 to 2.5%). The obtained characteristics were compared and discussed. Moreover, the data obtained were applied to optimize and to automate all mentioned protocols. Automatic analyzer allowed us to analyse simultaneously larger set of samples, to decrease the measurement time, to eliminate the errors and to provide data of higher quality in comparison to manual analysis. The total time of analysis for one sample was decreased to 10 min for all six methods. In contrary, the total time of manual spectrometric determination was approximately 120 min. The obtained data provided good correlations between studied methods (R=0.97-0.99).

Silver(I) ions ultrasensitive detection at carbon electrodes-analysis of waters, tobacco cells and fish tissues.

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.

Phytohormones as important biologically active molecules--their simple simultaneous detection.

Phytohormones, their functions, synthesis and effects, are of great interest. To study them in plant tissues accurate and sensitive methods are required. In the present study we aimed at optimizing experimental conditions to separate and determine not only plant hormones but also their metabolites, by liquid chromatography coupled with a UV-VIS detector. The mixture we analyzed was composed of benzyladenine, kinetin, trans-zeatin, cis-zeatin, dihydrozeatin, meta-topolin, ortho-topolin, alpha-naphthalene acetic acid, indole-3-acetic acid, trans-zeatin-7-glucoside, trans-zeatin-O-glucoside, trans-zeatin-9-riboside, meta-topolin-9-riboside and ortho-topolin-9-riboside. We measured the calibration dependences and estimated limits of detection and quantification under the optimal chromatographic conditions (column: Polaris C(18); mobile phase: gradient starting at 2:98 (methanol:0.001% TFA) and was increasing to 55:45 during twenty minutes, and then decreasing for 10 min to 35:65, flow rate: 200 microL x min(-1), temperature: 50 degrees C, wavelength: 210 nm). The detection limits for the target molecules were estimated as tens of ng per mL. We also studied the effect of flax extracts on the phytohormones' signals. Recovery of aliphatic and aromatic cytokinins, metabolites of cytokinins and auxins were within the range from 87 to 105 %. The experimental conditions were tested on a mass selective detector. In addition we analysed a commercial product used for stimulation of roots formation in cuttings of poorly rooting plants. The determined content of alpha-naphthalene acetic acid was in good agreement with that declared by the manufacturer.

Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope.

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 µM and 10-50 µM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.

Affecting of aquatic vascular plant Lemna minor by cisplatin revealed by voltammetry.

Within the context of application of platinum derivates based effective cytostatics, we can suppose that these risk metals can get into aquatic ecosystems where they can show biologic availability for food chain. In the present work we report on investigation of affecting of duckweed (Lemna minor) by various doses of cisplatin (0, 5, 10, 20, 40, 80 and 160 microM) for 4 days. The toxic influence of cisplatin was evaluated on the basis of growth inhibition expressed as number of leaves, growth rate, and total amount of biomass. The result value of 96hEC50, calculated from growth inhibition with comparison of growth rates, was 6.93 microM. Moreover we aimed on determination of cisplatin content using differential pulse voltammetry. The highest content of cisplatin (320 ng g(-1) of fresh weight) was determined in plants treated by 80 microM at the second day of treatment. Plants protect themselves against heavy metals by means of synthesis of cysteine-rich peptides such as glutathione and phytochelatins. Thus thiol determination in the treated plants by means of Brdicka reaction followed. The marked increase in thiol concentration detected is associated with defence reaction of the plant against stress caused by cisplatin.

Electrochemical and spectrometric study of antioxidant activity of pomiferin, isopomiferin, osajin and catalposide.

The antioxidant properties of pomiferin, isopomiferin, osajin and catalposide are evaluated. The electrochemical behaviour of these compounds at a carbon paste electrode was studied using square wave voltammetry. Oxidative signals, optimized frequencies and appropriate pH acetate buffer conditions were determined. The detection limits (3S/N) for pomiferin, isopomiferin, osajin and catalposide were estimated to be 50 pg/ml, 800 pg/ml, 40 pg/ml and 10 ng/ml, respectively. Furthermore, spectrometric test was employed with 2,2-diphenyl-1-picrylhydrazyle (DPPH) to evaluate the antioxidant activities of these compounds. Based on the obtained results, the highest antioxidant activity measured by DPPH tests was found at pomiferin followed by isopomiferin. The activities of osajin and catalposide were undetectable. The protective effects of pomiferin, isopomiferin, osajin and catalposide on DNA exposed to oxygen radicals in vitro were also studied. Changes in height of oxidative signals for the four bases (guanine, thymine, adenine and cytosine) were measured for DNA exposed to oxygen radicals, generated by Fenton's reaction, non-oxidized ssDNA (50 microg/ml) displayed well developed signals; however, after oxidative damage the observed oxidative signals decreased. Significant protective effects were observed for pomiferin and osajin. Decreased effect was observed for isopomiferin while a further reduced protective effect was seen for DNA exposed to catalposide. Based on the obtained results, pomiferin had the highest antioxidant activity followed by isopomiferin, osajin and catalposide.

Influence of Cadmium(II) Ions and Brewery Sludge on Metallothionein Level in Earthworms (Eisenia fetida) - Bio- transforming of Toxic Wastes.

Metallothioneins belong to a group of intracellular, high molecular andcysteine-rich proteins whose content in an organism increase with increasing concentrationof a heavy metal. The aim of this work was to apply the electrochemical analysis for theanalysis of metallothioneins in earthworms exposed to cadmium ions and brewery sludge.Here we utilized adsorptive transfer technique coupled with differential pulse voltammetryBrdicka reaction to determine metallothionein in different biological samples. By meansthis very sensitive technique it was possible to analyze metallothionein in concentrationsbelow 1 µmol.l⁻1 with the standard deviation of 4-5%. We found out that the average MTlevel in the non-treated earthworms oscillated between 19 and 48 µmol.l-1. When weanalysed samples of earthworms treated by cadmium, we observed that the MT contentincreased with the exposition length and increase dose of cadmium ions. Finally, weattempted to study and compare the toxicity of the raw sludge and its leach by using ofearthworms. The raw brewery sludge caused the death of the earthworms quickly.Earthworms held in the presence of leach from brewery sludge increased their weight of147 % of their original weight because they ingested the nutrients from the sludge. Themetallothionein level changes markedly with increasing time of exposition and applieddose of toxic compound. It clearly follows from the obtained results that the MT synthesisis insufficient in the first hours of the exposition and increases after more than 24 h.

Multi-instrumental Analysis of Tissues of Sunflower Plants Treated with Silver(I) Ions - Plants as Bioindicators of Environmental Pollution.

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.

Electrochemical determination of Ag-ions in environment waters and their action on plant embryos.

We utilized liquid chromatography coupled with electrochemical detector (HPLC-ED) for analyzing of silver ions. The optimization of basic chromatographic parameters has been done. The detection limit (3 S/N) obtained were 20 nmol/dm(3). Influence of different interferences (anions and cations) on current response of silver ions has been described. Moreover, we used HPLC-ED to analyze waters of different purity including photographic emulsion, which naturally contained silver ions. We found out that content of silver ions in the emulsion was 1.57 x 0.03 mmol/dm(3). Moreover, we investigated influence of silver ions on early somatic embryos of Blue Spruce. We were interested in the issue how much silver ions can embryos uptake during four days long treatment. For this purpose, we used optimized HPLC-ED technique. The content increased with increasing treatment time and applied concentration. We also studied how silver ions can influence thiols content in the treated embryos. For these purposes we used adsorptive transfer stripping voltammetry in connection with differential pulse voltammetry--Brdicka reaction. It clearly follows from the obtained results that content of thiols increased with increasing treatment time and applied concentration.

Bottlenecks in bog pine multiplication by somatic embryogenesis and their visualization with the environmental scanning electron microscope.

Somatic embryogenesis (SE) is an important biotechnological technique used for the propagation of many pine species in vitro. However, in bog pine, one of the most endangered tree species in the Czech Republic, limitations were observed, which negatively influenced the development and further germination of somatic embryos. Although initiation frequency was very low-0.95 %, all obtained cell lines were subjected to maturation. The best responding cell line (BC1) was used and subjected to six different variants of the maturation media. The media on which the highest number of early-precotyledonary/cotyledonary somatic embryos was formed was supplemented with 121 µM abscisic acid (ABA) and with 6 % maltose. In the end of maturation experiments, different abnormalities in formation of somatic embryos were observed. For visualization and identification of abnormalities in meristem development during proliferation and maturation processes, the environmental scanning electron microscope was used. In comparison to the classical light microscope, the non-commercial environmental scanning electron microscope AQUASEM II has been found as a very useful tool for the quick recognition of apical meristem disruption and abnormal development. To our knowledge, this is the first report discussing somatic embryogenesis in bog pine. Based on this observation, the cultivation procedure could be enhanced and the method for SE of bog pine optimized.

Using of liquid chromatography coupled with diode array detector for determination of naphthoquinones in plants and for investigation of influence of pH of cultivation medium on content of plumbagin in Dionaea muscipula.

The interest of many investigators in naphthoquinones is due to their broad-range of biological actions from phytotoxic to fungicidal. The main aim of this work was to investigate the influence of different pH values of cultivation medium on naphthoquinone content in Dionaea muscipula. For this purpose, we optimized the simultaneous analysis of the most commonly occurring naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin) by high performance liquid chromatography coupled with diode array detector (HPLC-DAD). The most suitable chromatographic conditions were as follows: mobile phase: 0.1 mol l-1 acetic acid:methanol in ratio of 33:67 (%, v/v), flow rate: 0.75 ml min-1 and temperature: 42 degrees C. Moreover, we looked for the most suitable technique for preparation of plant samples (D. muscipula, Juglans regia, Paulownia tomentosa, Impatience glandulifera, Impatience parviflora, Drosera rotundifolia, Drosera spathulata and Drosera capensis) due to their consequent analysis by HPLC-DAD. It clearly follows from the results obtained that sonication were the most suitable technique for preparation of J. regia plants. We also checked the recoveries of the determined naphthoquinones, which were from 96 to 104%. Finally, we investigated the changes in content of plumbagin in D. muscipula plants according to different pH of cultivation medium. The content increased with increasing pH up to 5 and, then, changed gradually. The lower content of plumbagin at lower pH values was of interest to us. Therefore, we determined the content of this naphthoquinone in the cultivation medium, what has not been studied before. We discovered that the lower tissue content of plumbagin was due to secretion of this naphthoquinone into the cultivation medium.

Imaging of Norway spruce early somatic embryos with the ESEM, Cryo-SEM and laser scanning microscope.

This article describes the surface structure of Norway spruce early somatic embryos (ESEs) as a typical culture with asynchronous development. The microstructure of extracellular matrix covering ESEs were observed using the environmental scanning electron microscope as a primary tool and using the scanning electron microscope with cryo attachment and laser electron microscope as a complementary tool allowing our results to be proven independently. The fresh samples were observed in conditions of the air environment of the environmental scanning electron microscope (ESEM) with the pressure from 550Pa to 690Pa and the low temperature of the sample from -18°C to -22°C. The samples were studied using two different types of detector to allow studying either the thin surface structure or material composition. The scanning electron microscope with cryo attachment was used for imaging frozen extracellular matrix microstructure with higher resolution. The combination of both electron microscopy methods was suitable for observation of "native" plant samples, allowing correct evaluation of our results, free of error and artifacts.

Simultaneous femtomole determination of cysteine, reduced and oxidized glutathione, and phytochelatin in maize (Zea mays L.) kernels using high-performance liquid chromatography with electrochemical detection.

Thiol compounds such as cysteine (Cys), reduced (GSH) and oxidized (GSSG) gluathione, and phytochelatins (PCs) play an important role in heavy metal detoxification in plants. These thiols are biological active compounds whose function is elimination of oxidative stress in plant cells. The aim of our work was to optimise sensitive and rapid method of high-performance liquid chromatography coupled with electrochemical detector (HPLC-ED) for determination of the abovementioned thiol compounds in maize (Zea mays L.) kernels. New approach for evaluation of HPLC-ED parameters is described. The most suitable isocratic mobile phase for the separation and detection of Cys, GSH, GSSG and PC2 consisted of methanol (MeOH) and trifluoroacetic acid (TFA). In addition, the influence of concentrations of TFA and ratio of MeOH:TFA on chromatographic separation and detection of the thiol compounds were studied. The mobile phase consisting from methanol and 0.05% (v/v) TFA in ratio 97:3 (%; v/v) was found the most suitable for the thiol compounds determination. Optimal flow rate of the mobile phase was 0.18 ml min(-1) and the column and detector temperature 35 degrees C. Hydrodynamic voltammograms of all studied compounds was obtained due to the selection of the most effective working electrodes potentials. Two most effective detection potentials were selected: 780 mV for the GSSG and PC2 and 680 mV for determination of Cys and GSH. The optimised HPLC-ED method was capable to determine femtomole levels of studied compounds. The detection limits (3 S/N) of the studied thiol compounds were for cysteine 112.8 fmol, GSH 63.5 fmol, GSSG 112.2 fmol and PC2 2.53 pmol per injection (5 microl). The optimised HPLC-ED method was applied to study of the influence of different cadmium concentrations (0, 10 and 100 microM Cd) on content of Cys, GSH, GSSG and PC2 in maize kernels. According to the increasing time of Cd treatment, content of GSH, GSSG and PC2 in maize kernels increased but content of Cys decreased. Decreasing Cys concentration probably relates with the increasing GSH and phytochelatins synthesis.

Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

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.

Electrochemical determination of lead and glutathione in a plant cell culture.

In this work we established differential pulse anodic stripping voltammetry (DPASV) as the tool for analysis of lead in the plant cell culture. For the cultivation procedure, lead in Pb(II)-ethylenediaminetetraacetic acid (Pb-EDTA) chelate has been used. The detection limit of lead was found at 500 pM in phosphate buffer (pH 5.5), and 100 nM in prepared cells intracellular extract (20 pg Pb(II)/mg cells). For determination of cysteine-rich peptides, voltammetry in differential mode (DPV) in cobalt(III)-containing ammonia buffer (Brdicka reaction) was used. In this short communication, we present suitable voltammetric techniques for the physiological study of lead and thiols in plant cell culture.

Effects of various doses of selenite on stinging nettle (Urtica dioica L.).

The aim of this study was to investigate the effects of selenium (Se) on the growth, accumulation and possible mechanisms of Se transport in certain parts (roots, leaves, stamp and apex) of nettle (Urtica dioica L.) plants. Se was supplemented by one-shot and two repeated doses to the soil (2.0 and 4.0 mg Se per kg of substrate). Selenium content in roots increased linearly with dose and was significantly higher compared to other plant parts of interest. However, growth of the above-ground parts of plant as well as roots was slightly inhibited with increasing selenium concentration in comparison to the untreated plants. The content of phytochelatin2, a low molecular mass peptide containing a sulfhydryl group, correlated well with the Se content. This suggests a possible stimulation of synthesis of this plant peptide by Se.

Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection.

Easy, efficient and low demanding separation of mRNA from biological material is needed to study gene expression and to use in chip technologies. It is common knowledge that each mRNA molecule contains sequence of 25 adenines. This feature can be used for binding mRNA on the surface of the particles coated by thymine chains. The present work reports on suggesting and optimizing of mRNA separation and detection from biological material via paramagnetic microparticles coupled with electrochemical detection. Primarily we optimized cyclic and square wave voltammetric conditions to detect poly(A), which was used as standard to mimic behaviour of mRNA. Under the optimized square wave voltammetric conditions (frequency 280 Hz, accumulation time 200 s, supporting electrolyte and its temperature: acetate buffer 4.6 and 35 degrees C) we estimated detection limit down to 1 ng of poly(A) per ml. To enhance effectiveness and repeatability of isolation of nucleic acid automated approach for rinsing and hybridizing was proposed. We optimized the whole procedure and experimental conditions. Using automated way of isolation and under optimized conditions the yield of poly(A) (isolated concentration of poly(A)/given concentration of poly(A)*100) was approximately 75%. The suggested and optimized method for poly(A) isolation and detection was utilized for the analysis of brain tissues of patients with traumatic brain injury. The total amount of isolated mRNA varied from 40 to 760 g of mRNA per g of brain tissue. The isolation of mRNA from six samples per run was not longer than 2.5h. Moreover, we applied the optimized procedure on fully automated pipetting instrument to isolate mRNA. The instrument was successfully tested on the analysis of extracts from roots of maize plants treated with cadmium(II) ions.