Barbara Vilhar

Comet Assay measurements: a perspective

The Comet Assay or single cell gel electrophoresis assay is one of the very widely used assays to microscopically detect DNA damage at the level of a single cell. The determination of damage is carried out either through visual scoring of cells (after classification into different categories on the basis of tail length and shape) or by using different commercially available or public domain software (which automatically recognise the extent of damage). In this assay, the shape, size and amount of DNA within the ‘comet’ play important roles in the determination of the level of damage. The use of a software in particular also provides a range of different parameters, many of which might not be relevant in determining the extent of DNA damage. As a large number of factors could influence the shape, size, identification and determination of induced damage, which includes the scoring criteria, staining techniques, selection of parameters (whilst using the software packages) and appearance of ‘hedgehog’ or ‘clouds’, this article aims (a) to provide an overview of evolution of measurements of DNA damage using the Comet Assay and (b) to summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted whilst using this assay. It is suggested that judicious selection of different parameters, staining methods along with inter-laboratory validation and harmonisation of methodologies will further help in making this assay more robust and widely acceptable for scientific as well as regulatory studies.

Cytometrical Evidence That the Loss of Seed Weight in theminiature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm

“If you know a thing only qualitatively, you know it no more than vaguely. If you know it quantitatively—grasping some numerical measure that distinguishes it from an infinite number of other possibilities—you are beginning to know it deeply.” (C. Sagan, Billions and Billions, 1997). Using

Stimulatory effects of jasmonic acid on potato stem node and protoplast culture

The effect of jasmonic acid (JA), in a wide range of concentrations (0.01–100 μM), on the development of potato plantlets (Solanum tuberosum L.cv. Vesna) was investigated in order to discriminate between physiological and supraoptimal effects of this growth regulator in vitro. Concentrations from 0.1–1 μM significantly increased the length of developed plants. Application of JA in these concentrations resulted in a very well differentiated root system with many lateral branches. With increasing JA concentrations, the main roots shortened and thickened. Concentrations higher than 10 μM led to the compaction of the stem, roots, and root hairs, giving a stunted appearance to the plants. The effect of JA on cell wall regeneration and callus formation was observed in potato leaf protoplast culture. JA at concentrations from 0.01–1 μM stimulated cell division and microcalli development.

Selective significance of genome size in a plant community with heavy metal pollution

In eukaryotes, nuclear genome sizes vary by more than five orders of magnitude. This variation is not related to organismal complexity, and its origin and biological significance are still disputed. One of the open questions is whether genome size has an adaptive role. We tested the hypothesis that genome size has selective significance, using five grassland communities occurring on a gradient of metal pollution of the soil as a model. We detected a negative correlation between the concentration of contaminating metals in the soil and the number of vascular plant species. Analysis of genome sizes of 70 herbaceous dicot perennial species occurring on the investigated plots revealed a negative correlation between the concentration of contaminating metals in the soil and the proportion of species with large genomes in plant communities. Consistent with the hypothesis, these results show that species with large genomes are at selective disadvantage in extreme environmental conditions.

Genome size and the nucleolar number as estimators of ploidy level in Dactylis glomeratain the Slovenian Alps

Abstract. We studied five natural populations of Dactylis glomerata L. (Poaceae) growing at different altitudes in the south-eastern fringe of the Alps in northern Slovenia to determine the subspecies. The stomatal length, the pollen diameter and chromosome counts were consistent with the tetraploid taxon D. glomerata subsp. glomerata (2n=4x =28). Genome size was measured in 55 individuals. The mean 2C value was 8.6 pg DNA. The mean 2C values of populations growing at different altitudes showed only 2.1% variation, and no correlation was observed between altitude and genome size. In D. nk;glomerata subsp. glomerata eight nucleoli were observed in late telophase, indicating that the nucleolus-organising regions inherited from both diploid parent species are functional. We demonstrate that both genome size and the number of nucleoli may be used to determine the ploidy level as an alternative to chromosome counting.

Genome size of Adriatic seagrasses

Genome size (C-value) was measured in four species of Adriatic seagrasses with interphase-peak DNA image cytometry. The estimated 2C-value was 1.5 pg DNA for Zostera noltii (2n = 12), 1.2 pg for Zostera marina (2n = 12), 1.1 pg for Cymodocea nodosa(2n = 28) and 6.2 pg for Posidonia oceanica, using Pisum sativum(2C-value = 8.84 pg) as the calibration standard. Seagrass leaves were fixed in 4% buffered formaldehyde to mitigate stoichiometric error due to tannins and post-fixed in 3:1 methanol:acetic acid (MAA). DNA was stained with the Feulgen reaction after hydrolysis in 5 M HCl for 90 min at 20 ◦ C. Comparison of genome size of seagrasses with the data for other species of Alismatidae indicated that the ancestral genome of Alismatidae was relatively small.

The influence of jasmonic acid on biophysical properties of potato leaf protoplasts and roots.

SummaryJasmonic acid (JA) and its derivatives are a novel group of plant endogenous growth regulators. In our experiments some new data about the physiological effects of JA were obtained using electron paramagnetic resonance spectroscopy. Experiments were performed on potato plants (Solanum tuberosum L. cv. Vesna) grown in vitro. Different quantities of JA (0.1–10 μM) were added to the growth medium. Root samples of plants grown on media supplemented with JA showed a more rapid spin probe N-oxyl-2,2,6,6-tetramethyl piperidine reduction than the control plants, which is a possible indicator of altered root permeability. Samples of leaf protoplasts were probed with methyl ester of 5-doxyl-haxadecanoic acid. We observed a membrane fluidity decrease in protoplasts isolated from plants grown on higher concentrations of JA (1 and 10 μM).

Epithelial thickness and lipid droplets in the hepatopancreas of Porcellio scaber (Crustacea: Isopoda) in different physiological conditions.

We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.

Jasmonic acid stimulates development of rhizoids and shoots in fern leaf culture

Summary We studied the effects of jasmonic acid (JA) on rhizoid and adventitious shoot formation in leaf culture of the fern Platycerium bifurcatum (Cav.) C.Chr. Leaf explants were grown on modified MS medium without (control) or with 0.1, 1, 10, 100 μmol/L JA. JA promoted rhizoid development on leaves. Both the percentage of leaves with rhizoids and their number per explant increased in JA-treated leaves. Addition of JA to the medium enhanced shoot development on leaves. While on the control medium shoots arose mostly on the abaxial side of the leaf, JA induced shoot formation also on the adaxial side. Analysis of soluble proteins from 60-day-old JA-treated explants revealed several alterations in polypeptide patterns when compared with the control. These results strongly suggest that jasmonates may be involved in regulation of morphogenesis in the fern P. bifurcatum . Moreover, JA may interact with similar regulatory mechanisms in pteridophytes and angiosperms.

Active teaching of diffusion through history of science, computer animation and role playing

We developed and tested a lesson plan for active teaching of diffusion in secondary schools (grades 10–13), which stimulates understanding of the thermal (Brownian) motion of particles as the principle underlying diffusion. During the lesson, students actively explore the Brownian motion through microscope observations of irregularly moving small particles, a computer animation showing events at the molecular level and a role play simulating diffusion. The lesson plan follows the history of scientific discoveries, from Robert Browns description of particle motion to Albert Einsteins explanation of the phenomenon at the molecular level. Students are encouraged to learn towards conceptual change and to critically evaluate scientific models. Both secondary school teachers and students perceived the lesson as interesting and easy to understand. The lesson plan was developed through a universityschool partnership project.