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

Electroanalysis of Plant Thiols

Due to unique physico-chemical properties of –SH moiety thiols comprise widegroup of biologically important compounds. A review devoted to biological functions ofglutathione and phytochelatins with literature survey of methods used to analysis of thesecompounds and their interactions with cadmium(II) ions and Murashige-Skoog medium ispresented. For these purposes electrochemical techniques are used. Moreover, we revealedthe effect of three different cadmium concentrations (0, 10 and 100 μM) on cadmiumuptake and thiols content in maize plants during 192 hours long experiments usingdifferential pulse anodic stripping voltammetry to detect cadmium(II) ions and highperformance liquid chromatography with electrochemical detection to determineglutathione. Cadmium concentration determined in tissues of the plants cultivated innutrient solution containing 10 μM Cd was very low up to 96 hours long exposition andthen the concentration of Cd markedly increased. On the contrary, the addition of 100 μMCd caused an immediate sharp increase in all maize plant parts to 96 hours Cd expositionbut subsequently the Cd concentration increased more slowly. A high performance liquidchromatography with electrochemical detection was used for glutathione determination intreated maize plants after 96 and 192 hours of treatment. The highest total content of glutathione per one plant was 6 μg (96 h, 10 μM Cd) in comparison with non-treated plant (control) where glutathione content was 1.5 μg. It can be concluded that electrochemical techniques have proved to be useful to analyse plant thiols

Electrochemical Sensors for Detection of Acetylsalicylic Acid

Acetylsalicylic acid (AcSA), or aspirin, was introduced in the late 1890s and hasbeen used to treat a variety of inflammatory conditions. The aim of this work was to suggestelectrochemical sensor for acetylsalicylic detection. Primarily, we utilized square wavevoltammetry (SWV) using both carbon paste electrode (CPE) and of graphite pencilelectrode (GPE) as working ones to indirect determination of AcSA. The principle ofindirect determination of AcSA bases in its hydrolysis on salicylic acid (SA), which isconsequently detected. Thus, we optimized both determination of SA and conditions forAcSA hydrolysis and found out that the most suitable frequency, amplitude, step potentialand the composition and pH of the supporting electrolyte for the determination of SA was260 Hz, 50 mV, 10 mV and Britton-Robinson buffer (pH 1.81), respectively. The detectionlimit (S/N = 3) of the SA was 1.3 ng/ml. After that, we aimed on indirect determination ofAcSA by SWV CPE. We tested the influence of pH of Britton-Robinson buffer andtemperature on yield of hydrolysis, and found out that 100% hydrolysis of AcSA wasreached after 80 minutes at pH 1.81 and 90°C. The method for indirect determination ofAcSA has been utilized to analyse pharmaceutical drug. The determined amount of AcSA in the pharmaceutical drug was in good agreement with the declared amounts. Moreover, weused GPE for determination of AcSA in a pharmaceutical drug. Base of the results obtainedfrom stationary electrochemical instrument we used flow injection analysis withelectrochemical detection to determine of salicylates (SA, AcSA, thiosalicylic acid, 3,5-dinitrosalicylic acid and 5-sulfosalicylic acid – SuSA). We found out that we are able todetermine all of detected salicylates directly without any pre-treatment, hydrolysis and so onat units of femtomoles per injection (5 μl)

Determination of viability of regions of <i>Picea abies</i> embryos (vital staining).

<p>(<b>A</b>) A six week old cluster of ESEs was divided into six parts for subsequent cultivation. (<b>B</b>) Dependence of ESE viability on the cultivation time, which was detected by IA coupled with a fluorescence microscope. Embryos (~1 mg) used for the viability determination were prepared from six regions of the cluster. The cell material was mixed with fluorescein diacetate (FDA) and propidium iodide (PII), and the fluorescence was detected after 5 min by fluorescence microscopy. The percentage of green areas in the compact embryonal group of the embryos (marked by frames) was used to indicate live cells. Insets: upper inset–early somatic embryo (obtained from section 6b) stained with FDA/PI; bottom inset–early somatic embryo (obtained from section 1a) stained with FDA/PI. Average viability changes determined by (<b>C</b>) double FDA/PI staining and (<b>D</b>) esterase activity determination according to cultivation.</p

Determination of viability of early <i>Picea abies</i> somatic embryos in eight week old clusters (non-vital staining).

<p>(<b>A</b>) Schematic of the cluster structure of ESEs; (1) view from the top and (2) bottom, (a) inner part of cluster and (b) outer part of cluster. (<b>B</b>) Staining with 2,3,5-triphenyltetrazolium chloride (TTC). (<b>C</b>) Staining with acetocarmine; (a) embryos from the inner cluster, (b) embryos from the outer cluster.</p

The effect of spatial orientation of the clusters by transposition into new culture medium on the growth of the cluster area after 14 days of cultivation.

<p>(<b>A</b>) Six week old ESE clusters of clone 2/32 placed in the normal orientation or upside-down in the culture medium after 14 days of cultivation. The average area of a cluster was 190 mm² at the beginning of the experiment. The values represent the average of 5 clusters. (<b>B</b>) Two week old ESE clusters of clone 2/32 placed in the normal orientation or upside-down after 14 days of cultivation. The average area of a cluster was 22 mm² at the beginning of the experiment. The values represent the average of 5 clusters.</p