Factors affecting protoplast isolation and cultivation of Cucumis spp.

Protoplasts of Cucumis anguria, Cucumis melo (3 accessions), Cucumis metuliferus and Cucumis sativus were isolated from leaves, growing apices, hypocotyls and calluses. Plants were cultured on 2 concentrations of sucrose. The effect of plant culture medium, explant age and explant type on protoplast viability were investigated. The protoplasts were cultured in 3 types of culture medium and at two temperatures. Optimal age range for protoplast isolation was 1-5 weeks depending on explant type and genotype. Viabilities ranging between 50 % and 80 % were obtained from all explants and genotypes. Increased concentration of sucrose had negative impact on viability of protoplasts. The highest level of regeneration achieved was callus, regenerated from leaf protoplasts of C. melo cv. ‘Charentais’ and C. melo ‘MR-1’. The lowest regeneration capability was observed in hypocotyls. Liquid LCM medium (B5 macro and microelements (1 g · l-1 CaCl2), B5 vitamins with 1g· l-1 myo-inositol, 2 mg· l-1 ascorbic acid, 0.8 mg· l-1 glycine, 20 mg· l-1 glutamine, 100 mg · l-1 casein hydrolysate, 70 g · l-1 mannitol, 10 g · l-1 sucrose, 5 g · l-1 glucose, 585 mg · l-1 MES, 5.37 μmol· l-1 NAA, 2.26 μmol· l-1 2,4-D, 2.22 μmol · l-1 BA) was optimal for protoplast regeneration. Agarose-solidified medium caused a decrease in the number of cell divisions (used in C. melo ‘PI 124112’). Cultivation at 25ºC resulted in a higher frequency of cell divisions (tested in C. metuliferus)

Haploid and mixoploid cucumber (Cucumis sativus L.) protoplasts – isolation and fusion

This paper reports on the isolation of haploid and mixoploid protoplasts in the genus Cucumis. The cucumber mixoploid plants (C. sativus L., 2x/4x; 2x = 14) were obtained after oryzalin treatments and the mesophyll protoplasts (2x/4x/8x) were isolated and cultivated by well known in vitro methods. The influence of oryzalin pretreatment on the average viability and density of  protoplasts was tested. The average viability as well as the density is signifi cantly influenced by the oryzalin concentration, whereas the time-span of the treatment doesn’t have significant impact on the density and the viability. Callus formation was the highest level of regeneration in the experiments described in our study. Furthermore the isolation and cultivation of the cucumber and muskmelon (C. melo L.; 2x = 24) haploid protoplasts from young-stage pollen grains were improved. Subsequently, somatic hybridization between mixoploid cucumber protoplasts and muskmelon mesophyll and callus protoplasts, and kiwano (C. metuliferus E. Meyer ex Naudin; 2x = 24) mesophyll protoplasts, by chemical fusion with polyethylene glycol (PEG) 6000 was performed for the first time. Heterofusants were observed and developed into micro colonies. Additionally, the gametosomatic hybridization between mixoploid cucumber protoplasts and pollen muskmelon protoplasts was performed for the first time. Heterofusants and the first cell division were observed, however, the regeneration stopped in this stage. In conclusion, the different ploidy, especially the mixoploid character of isolated protoplasts, has positive influence on protoplasts isolation and the following fusion as represented by a higher regeneration capacity. In addition, both types of protoplasts, haploid and mixoploid, represent a unique systems for biochemical, molecular and genetic experiments. Especially, the haploid protoplasts could be used during in vitro fertilization

Stretching the Rules: Monocentric Chromosomes with Multiple Centromere Domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel ``meta-polycentric'' functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes

Symposium Report The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

ABSTRACT This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b 5 . First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b 5 and 17a-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b 5 . The role of b 5 was also shown in vivo by selective hepatic knockout of b 5 from mice expressing CYP3A4 and CYP2D6; the lack of b 5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function

The peatland map of Europe

Based on the ‘European Mires Book’ of the International Mire Conservation Group (IMCG), this article provides a composite map of national datasets as the first comprehensive peatland map for the whole of Europe. We also present estimates of the extent of peatlands and mires in each European country individually and for the entire continent. A minimum peat thickness criterion has not been strictly applied, to allow for (often historically determined) country-specific definitions. Our ‘peatland’ concept includes all ‘mires’, which are peatlands where peat is being formed. The map was constructed by merging national datasets in GIS while maintaining the mapping scales of the original input data. This ‘bottom-up’ approach indicates that the overall area of peatland in Europe is 593,727 km². Mires were found to cover more than 320,000 km² (around 54 % of the total peatland area). If shallow-peat lands (< 30 cm peat) in European Russia are also taken into account, the total peatland area in Europe is more than 1,000,000 km2, which is almost 10 % of the total surface area. Composite inventories of national peatland information, as presented here for Europe, may serve to identify gaps and priority areas for field survey, and help to cross-check and calibrate remote sensing based mapping approaches

<scp>ReSurveyEurope</scp>: A database of resurveyed vegetation plots in Europe

AbstractAimsWe introduce ReSurveyEurope — a new data source of resurveyed vegetation plots in Europe, compiled by a collaborative network of vegetation scientists. We describe the scope of this initiative, provide an overview of currently available data, governance, data contribution rules, and accessibility. In addition, we outline further steps, including potential research questions.ResultsReSurveyEurope includes resurveyed vegetation plots from all habitats. Version 1.0 of ReSurveyEurope contains 283,135 observations (i.e., individual surveys of each plot) from 79,190 plots sampled in 449 independent resurvey projects. Of these, 62,139 (78%) are permanent plots, that is, marked in situ, or located with GPS, which allow for high spatial accuracy in resurvey. The remaining 17,051 (22%) plots are from studies in which plots from the initial survey could not be exactly relocated. Four data sets, which together account for 28,470 (36%) plots, provide only presence/absence information on plant species, while the remaining 50,720 (64%) plots contain abundance information (e.g., percentage cover or cover–abundance classes such as variants of the Braun‐Blanquet scale). The oldest plots were sampled in 1911 in the Swiss Alps, while most plots were sampled between 1950 and 2020.ConclusionsReSurveyEurope is a new resource to address a wide range of research questions on fine‐scale changes in European vegetation. The initiative is devoted to an inclusive and transparent governance and data usage approach, based on slightly adapted rules of the well‐established European Vegetation Archive (EVA). ReSurveyEurope data are ready for use, and proposals for analyses of the data set can be submitted at any time to the coordinators. Still, further data contributions are highly welcome.</jats:sec

IMAGE AS THE ELEMENTS OF ATTRACTIVENESS OF THE DESTINATIONS OF THE NATURE-ORIENTED TOURISM

Abstract NAVRÁTIL, J., PÍCHA, K., NAVRÁTILOVÁ, J., ŠVEC, R., DOLEŽALOVÁ, H.: Image as the elements of attractiveness of the destinations of the nature-oriented tourism. Acta univ. agric. et silvic. Mendel. Brun., 2012, LX, No. 4, pp. 281-288 The aim of this paper is to assess the relations between partial components of image of various touristically attractive locations. This research is focused on specifi c locations in vulnerable areas since the sustainable way of the tourism development concerns them in the highest manner and the touristic pressure on these locations permanently increases. The paper makes eff ort to extend the usual and nearly traditional understanding of the image in the tourism, which is usually related only to the problems of brand and/or the tourist destination. This is done through a survey realized at 26 selected locations with the aim to obtain 64 completely fi lled-in questionnaires in each location. Particular statements of respondents concerning the image of the visited location were summarized to the 20 categories. A multidimensional analysis was used to reveal the relations in partial answers. Authors have identifi ed an important number of elements of image of the tourist attractions. By means of the analysis authors have identifi ed diff erences between historic attractions and nature attractions. However, authors have identifi ed simultaneously both the natural type of the image of the destination and the culture-historical type of image of the destination in all studied locations. image, destination, nature-oriented tourism, PCA analysi

Impact of Water Level on Species Quantity and Composition Grown from the Soil Seed Bank of the Inland Salt Marsh: An Ex-Situ Experiment

The near elimination of inland salt marshes in Central Europe occurred throughout the 19th and 20th centuries, and the currently remaining marshes exist in a degraded condition. This work examines the impact of groundwater level on the growth of plants from a seed bank obtained from a degraded salt marsh in proximity to still existing one through an ex-situ experiment. An experimental tank was set up with the sample seed bank experiencing differing levels of water level. There were 1233 specimens of 44 taxa grown from the seed bank, of which 5 species were abundant, and 10 species are considered as halophytes. Only Lotus tenuis from halophytes was more abundant, and only five species of halophytes were represented by more than three individuals. The water level has a significant impact on the number of species (based on linear regression analysis) as well as species distribution among different water level treatments (a non-metric multidimensional analysis (nMDS) followed by linear regression). The results show a strong negative relationship between the average water level and the number of species. The water level did not affect the species composition of halophytes, but differences in individual species abundances were found among the halophytes. The species Bupleurum tenuissimum, Crypsis schoenoides, Melilotus dentatus, and Plantago maritima grew on the drier and non-inundated soils. Tripolium pannonicum, Spergularia maritima, and Lotus tenuis grew on both wet and dry soils. Trifolium fragiferum and Bolboschoenus maritimus were found in places with water stagnant at the soil level. Pulicaria dysenterica grew in inundated soil