Wpływ warunków świetlnych na stres oxydacyjny w kalusie kukurydzy

The aim of the first experiment was to study the effect of medium composition (MS and N6) on intracellular H₂O₂ content in maize genotype (two singlecross maize hybrids: K103 × K85 and KOC 9431). After 4 and 6 weeks of culture statistically significant genotypie differences in the increase of fresh mater were observed. For K103 × K85 genotype, the fresh matter was higher than KOC 9431. The MS medium was better for callus induction for both genotypes. The increase of K103 × K85 and fresh matter was accompanying the increase of H₂O₂ content. After 6 weeks of culture the K103 × K85 genotype accumulated the higher amount of H₂O₂ in 1 g fresh matter than KOC 9431. The higher inorganic nitrogen level (MS) stimulated hydrogen peroxide production in K103 × K85 callus and inhibited in KOC 9431 callus. In the second experiment we analysed the influence of light spectral on intracellular H₂O₂ level and the total content of phenols. K103 × K85 genotype was chosen as a more sensitive genotype to study light stress conditions. Blue- light-grown callus exhibited lower H₂O₂ level (86% control), than dark- (91%), red- (95%) and white-light-grown one (100%). The lower content of phenols was obtained in callus cultured under blue- (75% control) and red-light conditions (63% control).W pierwszym eksperymencie analizowano wpływ pożywki (MS i N6) na zawartość H₂O₂ w komórce dwóch genotypów kukurydzy: K103 × K85 i KOC 9431. Po 4 i 6 tygodniach zaobserwowano różnice w przyroście świeżej masy kalusów: dla genotypu K103 × K85 znacząco wyższą w porównaniu z genotypem KOC 9431. Skład pożywki MS był korzystniejszy dla obu genotypów niż skład pożywki N6. Wraz ze wzrostem masy akumulowany był nadtlenek wodoru. Oznaczany po 6 tygodniach 1g świeżej masy poziom H₂O₂ był znacznie wyższy dla K103 × K85. W drugim eksperymencie przeprowadzono badania wpływu rodzaju światła na poziom nadtlenku wodoru i fenoli. Do badań wybrano wrażliwszy genotyp K103 × K85. Rosnący w niebieskim świetle kalus gromadził niższy poziom H₂O₂ (86% kontroli) niż rosnący w ciemności (91%), w świetle czerwonym (95%) i białym (100%). Niższy poziom fenoli obserwowano podczas kultury w warunkach światła niebieskiego (75% kontroli) i czerwonego (63% kontroli)

The effect of endogenous hydrogen peroxide induced by cold treatment in the improvement of tissue regeneration efficiency

We propose that oxidative stress resulting from an imbalance between generation and scavenging hydrogen peroxide contributes to tissue regeneration efficiency during somatic embryogenesis of hexaploid winter wheat (Triticum aestivum cv. Kamila) and organogenesis of faba bean (Vicia faba ssp. minor cv. Nadwislanski). Endogenous hydrogen peroxide content and antioxidant capacity of cells were determined in initial explants and callus cultures derived from these explants. Regeneration-competent explants (immature embryos) contained more endogenous H2O2 than explants initiated from regeneration-recalcitrant tissue (mature wheat embryos and faba bean epicotyls). Higher H2O2 levels were observed despite the higher activity of antioxidative enzymes (superoxide dismutase and catalase) and the induction of their gene expression. Calli originating from immature embryos retained the capacity of the initial explants: high H2O2 production was observed during the whole culture period. Low temperature treatment (4°C) was found to be an effective factor, which improved both regeneration ability and H2O2 production. Exogenous application to the medium of H2O2 and catalase blocker (3-aminotriazole), but not FeEDTA and superoxide dismutase blocker (diethyldithiocarbamate), also resulted in the enhancement of regeneration efficiency. These results clearly indicate that plant regeneration is specifically regulated by endogenous H2O2 and by factors, which improve its accumulation. Moreover, a study of the activity of various SOD isoforms suggests that not only the absolute concentration of H2O2, but also its localisation might be responsible for controlling regeneration processe

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cell