Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba's ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

Combined QTL mapping and RNA-Seq profiling reveals candidate genes associated with cadmium tolerance in barley.

The high toxicity of cadmium (Cd) and its ready uptake by plants has become a major agricultural problem. To investigate the genetic architecture and genetic regulation of Cd tolerance in barley, we conducted quantitative trait loci (QTL) analysis in the phenotypically polymorphic Oregon Wolfe Barley (OWB) mapping population, derived from a cross between Rec and Dom parental genotypes. Through evaluating the Cd tolerance of 87 available doubled haploid lines of the OWB mapping population at the seedling stage, one minor and one major QTL were detected on chromosomes 2H and 6H, respectively. For chlorosis and necrosis traits, the major QTL explained 47.24% and 38.59% of the phenotypic variance, respectively. RNA-Seq analysis of the parental seedlings under Cd treatment revealed 542 differentially expressed genes between Cd-tolerant Rec and Cd-susceptible Dom genotypes. By analyzing sequence variations in transcribed sequences of the parental genotypes, 155,654 SNPs and 1,525 InDels were identified between the two contrasting genotypes and may contribute to Cd tolerance. Finally, by integrating the data from the identified QTLs and RNA-Seq analysis, 16 Cd tolerance-related candidate genes were detected, nine of which were metal ion transporters. These results provide promising candidate genes for further gene cloning and improving Cd tolerance in barley.

Phytotoxicity, cytotoxicity and genotoxicity evaluation of organic and inorganic pollutants rich tannery wastewater from a Common Effluent Treatment Plant (CETP) in Unnao district, India using Vigna radiata and Allium cepa.

The leather industry is a major source of environmental pollution in India. The wastewater generated by leather industries contains very high pollution parameters due to the presence of a complex mixture of organic and inorganic pollutants even after the treatment at a Common Effluent Treatment Plant (CETP) and disturbs the ecological flora and fauna. The nature, characteristics and toxicity of CETP treated wastewater is yet to be fully elucidated. Thus, this study aims to characterize and evaluate the toxicity of CETP treated tannery wastewater collected from the Unnao district of Uttar Pradesh, India. In addition to measuring the physico-chemical parameters, the residual organic pollutants was identified by GC-MS analysis and phytotoxicity, cytotoxicity and genotoxicity of the treated wastewater was evaluated using Vigna radiata L. and Allium cepa L. Results showed that the treated wastewater contained very high pollution parameters (TDS 3850 mg/L, BOD 680 mg/L, COD-1300 mg/L). GC-MS analysis revealed the presence of various types of residual organic pollutants including benzoic acid, 3-[4,-(T-butyl) Phenyl] furan-2-5-dione, benzeneacetamide, resorcinol, dibutyl phthalate, and benzene-1,2,4-triol. Further, toxicological studies showed the phytotoxic nature of the wastewater as it inhibited seed germination in V. radiata L. and root growth of A. cepa. Genotoxicity was evidenced in the root tip cell of A. cepa where chromosomal aberrations (stickiness, chromosome loss, C-mitosis, and vagrant chromosome) and nuclear abnormalities like micronucleated and binucleated cells were observed. Thus, results suggested that it is not safe to discharge these wastewater into the environment.

Dissecting the chromosomal composition of mutagen-induced micronuclei in Brachypodium distachyon using multicolour FISH.

Background and Aims: Brachypodium distachyon (Brachypodium) is a model species for temperate cereals and other economically important grasses. Its favourable cytogenetic features and advanced molecular infrastructure make it a good model for understanding the mechanisms of instability of plant genomes after mutagenic treatment. The aim of this study was to qualitatively and quantitatively assess the composition and origin of micronuclei arising from genomic fracture, and to detect possible 'hot spots' for mutagen-induced DNA breaks. Methods: Seeds of Brachypodium were treated with maleic hydrazide (MH) or X-rays. The structure of mutagen-induced micronuclei was analysed in root-tip meristematic cells using multicolour fluorescence in situ hybridization (mcFISH) with various repetitive (5S rDNA, 25S rDNA, telomeric, centromeric) and low-repeat [small and large pools of bacterial artificial chromosome (BAC) clones specific for chromosome Bd1] DNA sequences. Key Results: The majority of micronuclei derive from large, acentric fragments. X-rays caused more interstitial DNA breaks than MH. Double-strand breaks rarely occurred in distal chromosome regions. Bd1 contributed to the formation of more mutagen-induced micronuclei than expected from random chromosome involvement. Conclusions: mcFISH with chromosome-specific BAC clones offers insight into micronuclei composition, in so far as it allows their origin and formation to be determined more specifically. A reliable assay for micronuclei composition is crucial for the development of modern genotoxicity tests using plant cells. The combination of mutagenic treatments and well-developed cytomolecular resources in Brachypodium make this model species very promising for plant mutagenesis research.

Impact of Mutagens on DNA Replication in Barley Chromosomes.

Replication errors that are caused by mutagens are critical for living cells. The aim of the study was to analyze the distribution of a DNA replication pattern on chromosomes of the H. vulgare 'Start' variety using pulse 5-ethynyl-2'-deoxyuridine (EdU) labeling, as well as its relationship to the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and γ ray. To the best of our knowledge, this is the first example of a study of the effects of mutagens on the DNA replication pattern in chromosomes, as well as the first to use EdU labeling for these purposes. The duration of the cell cycle of the Hordeum vulgare 'Start' variety was estimated for the first time, as well as the influence of MH and γ ray on it. The distribution of the signals of DNA replication along the chromosomes revealed relationships between DNA replication, the chromatin structure, and DNA damage. MH has a stronger impact on replication than γ ray. Application of EdU seems to be promising for precise analyses of cell cycle disturbances in the future, especially in plant species with small genomes.

Genotoxicity effects of silver nanoparticles on wheat (Triticum aestivum L.) root tip cells.

The distribution and use of nanoparticles have rapidly increased over recent years, but the available knowledge regarding their mode of action, ecological tolerance and biodegradability remains insufficient. Wheat (Triticum aestivum L.) is the most important crop worldwide. In the current study, the effects of silver nanoparticles (AgNPs) obtained from two different sources, namely, green and chemical syntheses, on chromosomal aberrations and cell division were investigated. Wheat root tips were treated with four different AgNP concentrations (10, 20, 40 and 50 ppm) for three different exposure durations (8, 16 and 24 h), and the different concentrations of the nanoparticles were added to the tested grains until the root lengths reached 1.5-2 cm. For each concentration, the mitotic indexes (%) were obtained from an analysis of ~ 2000 cells. The treated root-tip cells exhibited various types of chromosomal aberrations, such as incorrect orientation at metaphase, chromosomal breakage, metaphasic plate distortion, spindle dysfunction, stickiness, aberrant movement at metaphase, fragmentation, scattering, unequal separation, scattering, chromosomal gaps, multipolar anaphase, erosion, and distributed and lagging chromosomes. These results demonstrate that the root tip cells of wheat can readily internalize the AgNPs and that the internalized AgNPs can interfere with the cells' normal function.

Biosynthesis of silver nanoparticles by leaf extract of Albizia saman (Jacq.) Merr. and their cytotoxic effect on mitotic chromosomes of Drimia indica (Roxb.) Jessop.

Silver nanoparticles synthesized using the leaf extract of Albizia saman (Jacq.) Merr. were tested for induction of cytogenetic abnormality in root tip cells of Drimia indica (Roxb.) Jessop (family Asperagaceae). The leaves are known to be rich in various phytochemicals like flavonoids, glycosides, saponins, steroids, tannins, and terpenoids, which may be responsible for bioreduction, biocapping, and stabilization of nanoparticles. The various instruments used for characterization include UV-VIS spectrophotometer, fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), X-Ray diffractometer (XRD), and high resolution transmission electron microscope (HR-TEM). The present study aims to evaluate the cytotoxic effect of biogenic silver nanoparticles on mitotic chromosomes by using root tip cells of D. indica. The root tips of D. indica was treated with suspensions of silver nanoparticles mixed in distilled water at different concentrations viz., 25, 50, 75, and 100% (w/v) for 6, 12, 18, and 24 h and then fixed in 1:3 ethanol: acetic acid following pre-treatment with 0.05% colchicine for cytological analysis. Silver nanoparticles induced a dose dependent decrease of mitotic index in root meristems. Furthermore, the treated meristem cells showed various types of chromosomal and mitotic aberrations such as anaphase bridge, sticky metaphase, lagging, or forward chromosome indicating genotoxic damage.

Association Analysis of Arsenic-Induced Straighthead in Rice (Oryza sativa L.) Based on the Selected Population with a Modified Model.

A rice physiological disorder makes mature panicle keep erect with empty grains termed as "straighthead." Straighthead causes yield losses and is a serious threat to rice production worldwide. Here, a new study of association mapping was conducted to identify QTL involved in straighthead. A subset of 380 accessions was selected from the USDA rice core collection and genotyped with 72 genome-wide SSR markers. An optimal model implemented with principle components (PCs) was used in this association mapping. As a result, five markers were identified to be significantly associated with straighthead. Three of them, RM263, RM169, and RM224, were consistent with a previous study. Three markers, RM475, RM263, and RM19, had a resistant allele associated with a decrease in straighthead rating (straighthead rating ≤ 4.8). In contrast, the two other marker loci RM169 and RM224 had a few susceptible alleles associated with an increase in straighthead rating (straighthead rating ≥ 8.7). Interestingly, RM475 is close to QTL "qSH-2" and "AsS" with straighthead resistance, which was reported in two studies on linkage mapping of straighthead. This finding adds to previous work and is useful for further genetic study of straighthead.

Brachypodium distachyon - A Useful Model in the Qualification of Mutagen-Induced Micronuclei Using Multicolor FISH.

Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the 'hot spots' and 'cold spots' of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.

Recreating Stable Brachypodium hybridum Allotetraploids by Uniting the Divergent Genomes of B. distachyon and B. stacei.

Brachypodium hybridum (2n = 30) is a natural allopolyploid with highly divergent sub-genomes derived from two extant diploid species, B. distachyon (2n = 10) and B. stacei (2n = 20) that differ in chromosome evolution and number. We created synthetic B. hybridum allotetraploids by hybridizing various lines of B. distachyon and B. stacei. The initial amphihaploid F1 interspecific hybrids were obtained at low frequencies when B. distachyon was used as the maternal parent (0.15% or 0.245% depending on the line used) and were sterile. No hybrids were obtained from reciprocal crosses or when autotetraploids of the parental species were crossed. Colchicine treatment was used to double the genome of the F1 amphihaploid lines leading to allotetraploids. The genome-doubled F1 plants produced a few S1 (first selfed generation) seeds after self-pollination. S1 plants from one parental combination (Bd3-1×Bsta5) were fertile and gave rise to further generations whereas those of another parental combination (Bd21×ABR114) were sterile, illustrating the importance of the parental lineages crossed. The synthetic allotetraploids were stable and resembled the natural B. hybridum at the phenotypic, cytogenetic and genomic levels. The successful creation of synthetic B. hybridum offers the possibility to study changes in genome structure and regulation at the earliest stages of allopolyploid formation in comparison with the parental species and natural B. hybridum.

Chromosome aberrations induced by zebularine in triticale.

Chromosome engineering is an important approach for generating wheat germplasm. Efficient development of chromosome aberrations will facilitate the introgression and application of alien genes in wheat. In this study, zebularine, a DNA methylation transferase inhibitor, was successfully used to induce chromosome aberrations in the octoploid triticale cultivar Jinghui#1. Dry seeds were soaked in zebularine solutions (250, 500, and 750 µmol/L) for 24 h, and the 500 µmol/L treatment was tested in three additional treatment times, i.e., 12, 36, and 48 h. All treatments induced aberrations involving wheat and rye chromosomes. Of the 920 cells observed in 67 M1 plants, 340 (37.0%) carried 817 aberrations with an average of 0.89 aberrations per cell (range: 0-12). The aberrations included probable deletions, telosomes and acentric fragments (49.0%), large segmental translocations (28.9%), small segmental translocations (17.1%), intercalary translocations (2.6%), long chromosomes that could carry more than one centromere (2.0%), and ring chromosomes (0.5%). Of 510 M2 plants analyzed, 110 (21.6%) were found to carry stable aberrations. Such aberrations included 79 with varied rye chromosome numbers, 7 with wheat and rye chromosome translocations, 15 with possible rye telosomes/deletions, and 9 with complex aberrations involving variation in rye chromosome number and wheat-rye translocations. These indicated that aberrations induced by zebularine can be steadily transmitted, suggesting that zebularine is a new efficient agent for chromosome manipulation.

Genome-Wide Analysis of the Expression of WRKY Family Genes in Different Developmental Stages of Wild Strawberry (Fragaria vesca) Fruit.

WRKY proteins play important regulatory roles in plant developmental processes such as senescence, trichome initiation and embryo morphogenesis. In strawberry, only FaWRKY1 (Fragaria × ananassa) has been characterized, leaving numerous WRKY genes to be identified and their function characterized. The publication of the draft genome sequence of the strawberry genome allowed us to conduct a genome-wide search for WRKY proteins in Fragaria vesca, and to compare the identified proteins with their homologs in model plants. Fifty-nine FvWRKY genes were identified and annotated from the F. vesca genome. Detailed analysis, including gene classification, annotation, phylogenetic evaluation, conserved motif determination and expression profiling, based on RNA-seq data, were performed on all members of the family. Additionally, the expression patterns of the WRKY genes in different fruit developmental stages were further investigated using qRT-PCR, to provide a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in strawberry.

Mutagenic potential of fine wastes from dimension stone industry.

The industrial treatment of dimension stones, such as marbles and granites, includes a stage of plate polishing, in which resins and abrasives are used, producing a fine grained waste with high moisture content. These wastes pass through decantation tanks in order to separate the solid and liquid phases. Until now, there is no knowledge about the mutagenic effects that this effluent can cause to organisms exposed to it. Thus, this study evaluated the mutagenic potential of dimension stone polishing wastes in onion root cells and fish erythrocytes. The onion seeds were germinated in Petri dishes with filter paper moistened in the liquid phase of the effluent. After germination, the onion roots were prepared for analysis of chromosomal aberrations in meristematic cells. The fishes were exposed during 72h to the solid phase of the effluent diluted in pure groundwater. Blood samples were used for counting of micronucleus and nuclear abnormalities. The onion seeds had similar germination and mitotic index in all treatments. However, it was observed in the seeds exposed to the polishing waste, numbers significantly higher of micronucleus, nuclear buds and other chromosomal aberrations when compared with the negative control. The fishes exposed to the waste showed numbers significantly higher of micronucleus when compared with the negative control. The fishes from all treatments showed significant increase in nuclear abnormalities when compared to the negative control. We concluded that the analysed wastes have mutagenic potential at the studied conditions; this effect can be related to the high content of phenolic compounds identified in the samples.

The phytoecdysteroid ß-ecdysone is genotoxic in Rodent Bone Marrow Micronuclei and Allium cepa L. Assays.

ETHNAOPHARMACOLOGIAL RELEVANCE: In South America, the ß-ecdysone ecdysteroid has been found in species of the genus Pfaffia Mart. Due to the similar morphology of its roots to the Panax ginseng C. A. Mey. (Korean ginseng), some species of this genus has been known as Brazilian ginseng and have been used as tonic and aphrodisiac, as well as for the treatment of diabetes and rheumatism. AIM OF THE STUDY: Here we report a cytogenotoxic evaluation of ß-ecdysone (a natural ecdysteroid found in plants) in Rodent Bone Marrow Micronuclei and Allium cepa Assays. MATERIALS AND METHODS: Three ß-ecdysone (pure) concentrations (based in human therapeutic dosage) were used in the Micronucleus Assay. The animals were treated during two consecutive days. Micronucleated cells were counted in 2000 polychromatic erythrocytes per animal. For A. cepa L. Assay, one ß-ecdysone concentration was analyzed. The onions bulbs were exposed for 24h. RESULTS: The Micronucleus Assay showed genotoxic effects for all treatments, expressed by an increase of micronucleated cells. In A. cepa L. Assay, cell abnormalities associated to the malfunction/non-formation of mitotic spindle (aneugenic effect) and chromosomal bridges (clastogenic effect) were observed. CONCLUSIONS: The results indicate a cytogenotoxic activity of ß-ecdysone. Therefore, the popular use of Pfaffia and others species containing ß-ecdysone should be considered with caution.

Genotoxic and mutagenic effects of sewage sludge on higher plants.

Sewage treatment yields sludge, which is often used as a soil amendment in agriculture and crop production. Although the sludge contains elevated concentrations of macro and micronutrients, high levels of inorganic and organic compounds with genotoxic and mutagenic properties are present in sludge. Application of sludge in agriculture is a pathway for direct contact of crops to toxic chemicals. The objective of this study was to compile information related to the genotoxic and mutagenic effects of sewage sludge in different plant species. In addition, data are presented on toxicological effects in animals fed with plants grown in soils supplemented with sewage sludge. Despite the benefits of using sewage sludge as organic fertilizer, the data showcased in this review suggest that this residue can induce genetic damage in plants. This review alerts potential risks to health outcomes after the intake of food cultivated in sewage sludge-amended soils.

Loss of heterozygosity of individual loci in Arabidopsis thaliana regenerants cultured with para-fluorophenylalanine.

Precise chromosome segregation is vital for speciation and hybrid formation. The aim of the work was to study the chromosomes behaviour and inheritance of maternal and paternal genomes in Arabidopsis regenerants de-rived from in vitro cultured cells on the medium with PFFA. The Arabidopsis thaliana model hybrid between Columbia and Landsberg erecta ecotypes was developed, which chromosomes were easy to distinguish using the 12 SSLP selected markers. Also, the influence of PFFA on the callus formation and regeneration of plants was analysed. 20 regenerated plants cultured with PFFA were derived, three of which were shown to loss the heterozygosity in six loci by DNA markers analysis. Different models are certainly required to understand how and when the mechanisms leading to proper chromosome segregation are established in species and hybrids.

Anther Culture in Eggplant (Solanum melongena L.).

The technique of in vitro anther culture is the most favorite to incite the production of plants from microspore through direct embryogenesis or regeneration from callus. Anther culture has been employed since 1980s in eggplant to obtain double-haploid plants from microspore derived embryos. From that time it has been refined and widely applied both at commercial level for a fast generation double-haploid parental lines of F1 hybrids, as well as for experimental studies as the complete homozygosis of the microspore-derived plants make more simply the genetic analysis. In this chapter, a step-by-step procedure is reported, taking into consideration all the aspects of the technique, including the growth condition of the anther donor plant, the in vitro regeneration of the androgenetic plantlets, their ploidy analysis, and the colchicine treatment to double the chromosome number of the haploids.

Cytogenetic studies of chromium (III) oxide nanoparticles on Allium cepa root tip cells.

The current study evaluates the cytogenetic effects of chromium (III) oxide nanoparticles on the root cells of Allium cepa. The root tip cells of A. cepa were treated with the aqueous dispersions of Cr2O3 nanoparticles (NPs) at five different concentrations (0.01, 0.1, 1, 10, and 100µg/mL) for 4hr. The colloidal stability of the nanoparticle suspensions during the exposure period were ascertained by particle size analyses. After 4hr exposure to Cr2O3 NPs, a significant decrease in mitotic index (MI) from 35.56% (Control) to 35.26% (0.01µg/mL), 34.64% (0.1µg/mL), 32.73% (1µg/mL), 29.6% (10µg/mL) and 20.92% (100µg/mL) was noted. The optical, fluorescence and confocal laser scanning microscopic analyses demonstrated specific chromosomal aberrations such as-chromosome stickiness, chromosome breaks, laggard chromosome, clumped chromosome, multipolar phases, nuclear notch, and nuclear bud at different exposure concentrations. The concentration-dependent internalization/bio-uptake of Cr2O3 NPs may have contributed to the enhanced production of anti oxidant enzyme, superoxide dismutase to counteract the oxidative stress, which in turn resulted in observed chromosomal aberrations and cytogenetic effects. These results suggest that A. cepa root tip assay can be successfully applied for evaluating environmental risk of Cr2O3 NPs over a wide range of concentrations.

Induction and identification of Stylosanthes guianensis tetraploids.

Stylosanthes guianensis is an elite and important forage legume species, which is extensively cultivated in tropical areas. Polyploid breeding via exposure to colchicine is a conventional and practical method to improve varieties of S. guianensis. Terminal buds of S. guianensis Reyan No.5 seedlings were treated with different concentrations of colchicine (0.00, 0.05, 0.10, 0.15, 0.20, and 0.25%) for 24, 48, and 72 h. Morphological and cytological variants were observed at a frequency of <96% among transplanted seedlings. The cytogenetic analysis of young leaf cells was conducted on all variants to identify their ploidy levels. The most efficient procedure for tetraploid production was the treatment of seedling apical buds with 20% colchicine for 48 h, with the tetraploid induction rate being 10%. This is a relatively simple and reliable method for the production of tetraploidy in S. guianensis.