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.

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.

Application of computer imaging, stripping voltammetry and mass spectrometry to study the effect of lead (Pb-EDTA) on the growth and viability of early somatic embryos of Norway spruce (Picea abies /L./ Karst.).

Image analysis (IA) was used to determine the areas and circumferences of clusters of early somatic embryos (ESEs) of the Norway spruce (Picea abies /L./Karst.). Results obtained from IA were compared with the fresh weights of the ESE clusters and their esterase activities. The areas of the ESE clusters correlated well with both the increases in fresh weight (R2=0.99) of the ESEs and their esterase activities (R2=0.99). In addition, we studied the viability of the ESEs, which was determined by (a) double staining with fluorescein diacetate and propidium iodide (the resulting fluorescence was quantified by IA) and (b) determining esterase activity using a spectrofluorimetric detector. The results obtained with IA and esterase assay were comparable (the deviation between the tangents of the bisectors was 6.4%). IA was also used to study the effect of Pb-EDTA chelate (50, 250 and 500 microM) on the viability of the ESEs and on the growth of clusters. The presence of Pb-EDTA markedly slowed the growth of ESEs clusters (by more than 65% with 250 microM of Pb-EDTA after 288 h of cultivation) and decreased the viability of ESEs (by more than 30% with 500 microM of Pb-EDTA after 288 h of cultivation). The lead concentration in the ESEs was determined by differential pulse anodic stripping voltammetry and increased with the external lead concentration and the time of treatment from 100 to 600 pg Pb/100 mg of fresh weight of ESEs. Glutathione is a diagnostic marker of the influence of Pb-EDTA on ESEs and its content was determined by high-performance liquid chromatography coupled with mass spectrometry. The glutathione content changed linearly with treatment time and the applied external lead concentration. The highest glutathione content was obtained at 250 microM of Pb-EDTA after 192 h of cultivation.