Root Tip Meristem Cells Research Articles

Cytomolecular Analysis of Ribosomal DNA Evolution in a Natural Allotetraploid Brachypodium hybridum and Its Putative Ancestors-Dissecting Complex Repetitive Structure of Intergenic Spacers.

Nucleolar dominance is an epigenetic phenomenon associated with nuclear 35S rRNA genes and consists in selective suppression of gene loci inherited from one of the progenitors in the allopolyploid. Our understanding of the exact mechanisms that determine this process is still fragmentary, especially in case of the grass species. This study aimed to shed some light on the molecular basis of this genome-specific inactivation of 35S rDNA loci in an allotetraploid Brachypodium hybridum (2n = 30), which arose from the interspecific hybridization between two diploid ancestors that were very similar to modern B. distachyon (2n = 10) and B. stacei (2n = 20). Using fluorescence in situ hybridization with 25S rDNA and chromosome-specific BAC clones as probes we revealed that the nucleolar dominance is present not only in meristematic root-tip cells but also in differentiated cell fraction of B. hybridum. Additionally, the intergenic spacers (IGSs) from both of the putative ancestors and the allotetraploid were sequenced and analyzed. The presumptive transcription initiation sites, spacer promoters and repeated elements were identified within the IGSs. Two different length variants, 2.3 and 3.5 kb, of IGSs were identified in B. distachyon and B. stacei, respectively, however only the IGS that had originated from B. distachyon-like ancestor was present in the allotetraploid. The amplification pattern of B. hybridum IGSs suggests that some genetic changes occurred in inactive B. stacei-like rDNA loci during the evolution of the allotetraploid. We hypothesize that their preferential silencing is an effect of structural changes in the sequence rather than just the result of the sole inactivation at the epigenetic level.

Karyotype and genome size in Euterpe Mart. (Arecaceae) species.

Euterpe (Martius, 1823), a genus from Central and South America, has species with high economic importance in Brazil, because of their palm heart and fruits, known as açaí berries. Breeding programs have been conducted to increase yield and establish cultivation systems to replace the extraction of wild material. These programs need basic information about the genome of these species to better explore the available genetic variability. The aim of this study was to compare Euterpe edulis (Martius, 1824), Euterpe oleracea (Martius, 1824) and Euterpe precatoria (Martius, 1842), with regard to karyotype, type of interphase nucleus and nuclear DNA amount. Metaphase chromosomes and interphase nuclei from root tip meristematic cells were obtained by the squashing technique and solid stained for microscope analysis. The DNA amount was estimated by flow cytometry. There were previous reports on the chromosome number of Euterpe edulis and Euterpe oleracea, but chromosome morphology of these two species and the whole karyotype of Euterpe precatoria are reported for the first time. The species have 2n=36, a number considered as a pleisomorphic feature in Arecoideae since the modern species, according to floral morphology, have the lowest chromosome number (2n=28 and 2n=30). The three Euterpe species also have the same type of interphase nuclei, classified as semi-reticulate. The species differed on karyotypic formulas, on localization of secondary constriction and genome size. The data suggest that the main forces driving Euterpe karyotype evolution were structural rearrangements, such as inversions and translocations that alter chromosome morphology, and either deletion or amplification that led to changes in chromosome size.

Shoot Tip Meristem Cryopreservation of Hypericum Species.

Based on our long-standing experience with in vitro culture of Hypericum perforatum, a clonal multiplication system and vitrification-based cryopreservation protocols have been applied to several Hypericum species: H. humifusum L., H. annulatum Moris, H. tomentosum L., H. tetrapterum Fries, H. pulchrum L., and H. rumeliacum Boiss. The shoot tips were cryopreserved using a uniform procedure that includes pretreatment with abscisic acid (ABA), PVS3 cryoprotection, and direct immersion into the liquid nitrogen (LN). The freezing-tolerant Hypericum species were pre-exposed to the cold acclimation conditions performed by a 7-day exposure to 4 °C. The content of naphtodianthrones (hypericins) including hypericin, pseudohypericin, and their protoforms was quantified by HPLC. Ploidy of plants was determined by both flow cytometry of leaf tissue and chromosome counts of root tip meristematic cells. We have shown that the post-thaw recovery rate of the shoot tips, pretreated with 0.076 μM ABA for 7 days at room temperature, led to the post-cryogenic survival from 5 % in H. tomentosum to 21 % in H. annulatum. As compared to the untreated (control) plants, the content of hypericins in plants regenerated after cryopreservation remained unchanged or decreased in H. perforatum, H. humifusum, H. annulatum, H. tomentosum, H. tetrapterum, and H. rumeliacum. However, the pre-exposition of the freezing-tolerant H. perforatum to cold acclimation prior to excision of the shoot tips has improved the post-thaw recovery to 45 % and resulted in threefold increase of the total hypericin content.

Ultrastructural changes in onion (Allium cepa L.) root tip meristem cells treated with Selol and sodium selenate (IV)

Investigations on the antimitotic and anticancer effect of Selol have been carried out for more than a decade. In a previous work we analysed ultrastructural changes in onion root tip meristem cells (the Allium test), their division activity (the mitotic index), and proportion of division phases (the phase index). Our study revealed inhibition of cell divisions in response to both compounds; however, the effect of sodium selenate (IV) on cells was more toxic, involving frequent clastogenic aberrations. The aim of the present work was to compare the effect of both compounds (organic and inorganic), containing selenium with oxidation number at +4, i.e. Selol and sodium selenate (IV), on ultrastructure of plant cells of the Allium test, and their concentration in plant cells. Analysis of ultrastructural changes showed high toxicity of sodium selenate (IV), with degradation of cytoplasm, nucleus and cell organelles. Treatment with Selol did not cause such dramatic changes in ultrastructure. It resulted in gra...

Toxicity assessment of industrial wastewaters reaching Dandugan Oya, Sri Lanka using a plant based bioassay

Industrial waste may contain complex chemical mixtures with potential cytotoxic and genotoxic effects. The Dandugan Oya, a water canal located in the Western Province of Sri Lanka receives industrial waste from multiple sources. In the present study potential toxicity of selected industrial wastewaters reaching the Dandugan Oya, and the downstream water was assessed using a plant based bioassay with onion ( Allium cepa L. var. ascalonicum ) as the test organism. Of the physico-chemical characteristics tested, temperature, pH, biochemical oxygen demand, chemical oxygen demand, cadmium and chromium levels of the wastewaters collected during three sampling occasions in the year 2012 were within the national tolerance limits specified for the discharge of industrial effluents into inland surface waters. The exposure of A. cepa bulbs to wastewater and downstream water from the Dandugan Oya resulted in the reduction of root growth (24 – 62 %) and mitosis (31 – 55 %), induction of micronuclei (up to 0.6 %), nuclear abnormalities (3 - 14 folds) and chromosomal aberrations (3 - 21 folds) in the root tip meristematic cells compared to those exposed to the control and the upstream water, indicating cytotoxic and genotoxic effects. No significant difference between the control and the upstream water was found in relation to the measured biological effects (p > 0.05). The present study revealed that the tested wastewaters contained cyto-genotoxic contaminants and, the inherent dilution/detoxification capacity of Dandugan Oya during the study period was not adequate to eliminate the toxic effects in the downstream water. In addition to the conventional physico-chemical analyses, inclusion of suitable bioassays as additional assessments in water quality monitoring programmes could alert cyto-genotoxic impacts in wastewater receiving inland surface waters. J.Natn.Sci.Foundation Sri Lanka 2015 43 (2): 153-163

Cytogenetic effects of99technetium on meristematic cells of root tips ofVicia fabaL. andstatistical comparison

In this study, cytogenetic effects of 99technetium (99Tc) on meristematic cells of root tips belonging to Vicia faba L. have been investigated. Seeds of the plant were prepared and kept in 99Tc standard for different time periods: 1/12, 1/4, 1/2, 1, 2, 3, 4, 5, 6 and 12 h. Seeds treated with 99Tc were sprouted and the root tips obtained were prepared for microscopic examination. Some abnormalities e.g. chromosome breaking, chromosome dispersion, bridge chromosomes, chromosome adherence and ring chromosomes were observed. Abnormalities seen for each treatment depended on the time period. The variety and number of abnormalities usually increased with increased treatment time. The results obtained were evaluated statistically.

Hypoxia-Induced Programmed Cell Death in Root-Tip Meristematic Cells of Triticum aestivum L.

Abstract In this study, wheat (Triticum aestivum L.) roots were treated with hypoxic water. The staining of cell preparations with DAPI revealed morphological changes of the cells such as nuclear condensation, deformation and fragmentation. Under TEM, cellular membrane shrinkage and breakage, chromatin condensation and apoptotic-like bodies were displayed. The number of mitochondria increased dramatically; their cristae were damaged; the interior became a cavitation and only some flocculent materials were distributed. Indirect immunofluorescence staining indicated that cytochrome C diffused from mitochondria to nucleoplasm and cytoplasm. TUNEL positive nuclei indicated double strand breaks of DNA. DAB staining was used for the identification of hydrogen peroxide and examination showed that the longer the treating time, the darker the staining of the meristematic zones of the roots which suggested the increased accumulation of these Reactive Oxygen Species (ROS). The elevation of hydrogen peroxide production was paralleled with the increase of SOD and POD activities. A negative correlation between the exposure time under hypoxia and the contents of soluble proteins was found. No obvious effect of hypoxia on MDA was established. The obtained results demonstrate that hypoxia causes programmed cell death in the root-tip meristematic cells of Triticum aestivum L. which is most probably attributed to the accumulation of large amounts of ROS.

Cell synchronization and isolation of chromosomes from Chinese fir root tips for flow cytometric analysis.

To optimize treatment parameters to synchronize the cell cycle of Chinese fir root tip meristem cells. Optimal mitotic indices were achieved by treating seedlings with 1.25 mM hydroxyurea for 10 h, incubating for 7 h without chemical treatment to release the hydroxyurea block, and then treating emerging roots with 0.05 % colchicine for 16 h. The mitotic index of synchronized Chinese fir root tips was over 30 %. Suspensions of intact chromosomes were prepared using a simple slicing procedure. The chromosome preparations were suitable for flow cytometric characterization and sorting. The chromosome peaks of the observed flow karyotype resembled the predicted flow karyotype calculated from the Chinese fir chromosome size.

Differential sensitivity of barley (Hordeum vulgare L.) to chlorpyrifos and propiconazole: Morphology, cytogenetic assay and photosynthetic pigments

The present investigation was performed to evaluate the effects of an insecticide and fungicide, namely, chlorpyrifos (CP) and propiconazole (PZ) on barley (Hordeum vulgare L. variety Karan-16). The seeds were treated with three concentrations of CP and PZ, i.e., 0.05%, 0.1% and 0.5% for 6 hours after different pre-soaking durations of 7, 17 and 27 hours. Different pre-soaking durations (7, 17 and 27 h) represent three phases of the cell cycle i.e., G1, S and G2, respectively. Double distilled water and ethyl methane sulfonate were used as negative and positive controls, respectively. As compared to their respective controls, treated root tip meristematic cells of barley showed significant reductions in the germination percentage, seedling height, mitotic index and comparative increase in chromosomal aberrations against both the pesticides, and the magnitude was higher in CP. After treatment with the pesticides, chlorophyll and carotenoid contents increased up to 0.1% but reduced at 0.5% and the decrease was more prominent in CP as compared to PZ. In treated cells, fragmentation, stickiness, bridges, multipolar anaphase and diagonal anaphase were observed as aberrations. As compared to control, chromosomal aberrations were higher in CP as compared to PZ. The results of the present study concluded that CP induced chromosomal aberrations were more frequent than PZ; hence it has higher probability to cause genotoxicity in barley.

Copper-induced root growth inhibition of Allium cepa var. agrogarum L. involves disturbances in cell division and DNA damage.

Copper (Cu) is considered to be an indispensable microelement for plants. Excessive Cu, however, is toxic and disturbs several processes in the plant. The present study addressed the effects of ionic Cu (2.0 µM and 8.0 µM) on mitosis, the microtubule cytoskeleton, and DNA in root tip cells of Allium cepa var. agrogarum L. to better understand Cu toxicity on plant root systems. The results indicated that Cu accumulated in roots and that root growth was inhibited dramatically in Cu treatment groups. Chromosomal aberrations (for example, C-mitosis, chromosome bridges, chromosome stickiness, and micronucleus) were observed, and the mitotic index decreased during Cu treatments at different concentrations. Microtubules were one of the target sites of Cu toxicity in root tip meristematic cells, and Cu exposure substantially impaired microtubule arrangements. The content of α-tubulin decreased following 36 h of exposure to 2.0 µM or 8.0 µM of Cu in comparison with the control group. Copper increased DNA damage and suppressed cell cycle progression. The above toxic effects became more serious with increasing Cu concentration and prolonged exposure time.

In situobservation of multiple levels of intermediate chromatin fibers on metaphase chromosomes ofTriticum aestivumL.

Metaphase chromosomes of root-tip meristematic cells of wheat (Triticum aestivum L.) were carefully examined under the transmission electron microscope. It was obvious that only in one section of a metaphase cell, besides 10 and 2530 nm fibers, a serial of intermediate chromatin fibers between 30 nm and metaphase chromosome such as 5060, 100–120 and 200–300 nm, could be identified on three metaphase chromosomes in the cell. The higher degrees of chromatin fibers were helically coiled from the lower levels of chromatin fibers and at last, 750~800 nm metaphase chromosomes were formed from 200–300 nm fibers. It was concluded that the 30 nm chromatin fibers exist truly in the metaphase chromosomes which are hierarchically organized from the 10 nm ones and there are at least 4 levels of helical coils from 30 nm to metaphase chromosomes.

Nucleolus disassembly and distribution of segregated nucleolar material in prophase of root-tip meristematic cells in Triticum aestivum L.

This paper presents details of the process of nucleolar disassembly, studied by conventional transmission electron microscopy (TEM) in wheat root cells. In early prophase, chromatin condensation and irregular nucleolar morphology are observed, with many small particles appearing around the nucleolus. In middle prophase, the nucleolus radiates outwards; in late prophase, the fine structure of the nucleolus disappears and nucleolar material diffuses away. Using ?en bloc? silver-staining to distinguish between nucleoli and chromatin, we observed that the dispersed nucleolar material aggregates around the chromatin, forming a sheath-like perichromosomal structure that coats the chromosomes in late prophase.

Effects of lead on the morphology and structure of the nucleolus in the root tip meristematic cells of Allium cepa L.

To study the toxic mechanisms of lead (Pb) in plants, the effects of Pb on the morphology and structure of the nucleolus in root tip meristematic cells of Allium cepa var. agrogarum L. were investigated. Fluorescence labeling, silver-stained indirect immunofluorescent microscopy and western blotting were used. Fluorescence labeling showed that Pb ions were localized in the meristematic cells and the uptake and accumulation of Pb increased with treatment time. At low concentrations of Pb (1–10 μM) there were persistent nucleoli in some cells during mitosis, and at high concentration (100 μM) many of the nucleolar organizing regions were localized on sticky chromosomes in metaphase and anaphase cells. Pb induced the release of particles containing argyrophilic proteins to be released from the nucleus into the cytoplasm. These proteins contained nucleophosmin and nucleolin. Pb also caused the extrusion of fibrillarin from the nucleus into the cytoplasm. Western blotting demonstrated the increased expression of these three major nucleolar proteins under Pb stress.

PI3K is involved in nucleolar structure and function on root-tip meristematic cells of Triticum aestivum L.

In this study, wheat (Triticum aestivum L.) seeds were used to detect the effect of wortmannin, a specific inhibitor of PI3K, on the nucleolar structure and function. When the germinated seeds were treated with wortmannin, it was shown that the root growth was suppressed and the mitotic index was decreased. The inhibition effects were positively correlated with the concentrations of the drug. The observations of light and transmission electron microscopy revealed that the nucleolar morphology became irregular and their fine structure disappeared. Some granules with a size range of 0.05–0.30μm diffused from the nucleoli and gradually moved to the nucleoplasm between or around the chromatin. Indirect immunofluorescence staining indicated that B23 shuttled from the nucleoli to the nucleoplasm, or even, to the cytoplasm. RT-PCR technique demonstrated that the expression of C23 was severely down-regulated. Our results suggest, for the first time, that wortmannin treatment can not only damage nucleolar structure, but also inhibit its function, implying that PI3K is involved in nucleolar structure and function.

Cytotoxic and genotoxic effects of two medicinal species of Verbenaceae

Lantana camara L. and Lippia alba (Mill.) N. E. Br. are two important species of Verbenaceae family and are commonly used in folk medicine in many countries of Central and Southern America. The aim of this study was to investigate, for the first time, the cytotoxic and genotoxic effects of aqueous extracts from leaves of both species on Lactuca sativa (lettuce) root tip meristem cells using a cytogenetic approach. Seeds of lettuce were separately treated during 72h with different concentrations of L. camara and L. alba aqueous extracts (5, 10, 20 and 30g/L). The percentage of germination, root development and cellular behavior were analyzed and the results showed that the highest concentration of aqueous extracts reduced the mitotic index, the seeds germination and the root development of lettuce. The extracts also induced chromosome aberrations and cellular death in roots cells of L. sativa. The cytogenotoxicity of L. camara and L. alba extracts was comparatively described.

Mitotic activity, submicroscopic organization and respiration of meristematic cells of root tips of Allium cepa L. treated with dacarbazine (DTIC)

Studies were carried out on the effect of dacarbazine (DTIC) on meristematic cells of root tips of <em>Allium</em> cepa L. Noticeable mitodepression was observed. It depended upon time of exposure and DTIC concentration. Mitodepression was caused by an inhibition of DNA synthesis and blocking of the G<sub>2</sub> phase. DTIC caused also disturbances of the karyokinesis and cytokinesis, similar as in case of colchicine effect. In the experimental conditions inhibition of cell respiration was noted, as also changes in the tissue ultrastructure. These changes were typical of the effect of respiratory inhibitors.

Highly active lanthanum doped ZnO nanorods for photodegradation of metasystox

La-doped ZnO nanorods with different La contents were synthesized by microwave assisted method and characterized by various sophisticated techniques such as XRD, UV–Vis., EDS, XPS, SEM and TEM. The XRD patterns of the La-doped ZnO indicate hexagonal crystal structure with an average crystallite size of 30nm. It was found that the crystallite size of La-doped ZnO is much smaller as compared to pure ZnO and decreases with increasing La content. The photocatalytic activity of 0.5mol% La-doped ZnO in the degradation of metasystox was studied. It was observed that degradation efficiency of metasystox over La-doped ZnO increases up to 0.5mol% doping then decreases for higher doping levels. Among the catalyst studied, the 0.5mol% La-doped ZnO was the most active, showing high photocatalytic activity for the degradation of metasystox. The maximum reduction of concentration of metasystox was observed under static condition at pH 8. Reduction in the Chemical Oxygen Demand (COD) of metasystox was observed after 150min. The cytotoxicological studies of meristematic root tip cells of Allium cepa were studied. The results obtained indicate that photocatalytically degraded products of metasystox were less toxic as compared to metasystox.

Cariotipo de Espeletiopsis muiska

RESUMENObjetivo. Estudiar el ciclo celular y realizar la descripción cromosómica de Espeletiopsis muiska, utilizando células meristemáticas de ápices radicales obtenidos a través de cultivo in vitro de embriones en medio MS suplementado con AIB. Materiales y métodos. Se colectaron ápices radicales a diferentes horas del día y se contaron en ellos las células presentes en cada fase mitótica, se determinó la duración del ciclo celular y la hora de mayor actividad mitótica. La descripción de los cromosomas y la elaboración del cariotipo de la especie, se realizó utilizando un protocolo que permitió la obtención de cromosomas metafásicos. Resultados. Respecto a la duración del ciclo celular, se encontró que la interfase comprende el 94.65% y la mitosis el 5.35% del total del ciclo celular. Entre las fases mitóticas la profase tiene mayor duración, 1.92%, mientras que la anafase es la más corta, 0.96%. En cuanto a la hora mitótica, las células meristemáticas de los ápices radicales presentaron mayor actividad entre las 10:30 y las 11:15. Con el estudio citogenético se encontraron 19 pares de cromosomas con longitudes e índices centroméricos similares y cariotipo asimétrico. Conclusiones. El estudio detallado de los cromosomas, permitió determinar un complemento cromosómico 2n=38+2B, dos cromosomas con microsatélites terminales y uno con constricción secundaria.

Variability of stomata and 45S and 5S rDNAs loci characteristics in two species ofAnthoxanthumgenus:A. aristatumandA. odoratum(Poaceae)

Diploid Anthoxanthum odoratum and tetraploid A. aristatum were compared with respect to stomatal guard cell lengths, and stomatal density at adaxial and abaxial surfaces of the lamina. Further, the genome size of both species was determined by flow cytometry, and the number as well as the chromosomal distribution of 5S and 45S rDNAs were examined using FISH with ribosomal DNA (rDNA) probes. The average length of stomatal guard cells in A. odoratum was shown to be greater than that for A. aristatum, but the ranges overlapped. Moreover, reduction in stomatal frequency was found at higher ploidy levels.The genome size was 6.863 pg/2C DNA for A. aristatum and 13.252 pg/2C DNA for A. odoratum. A. aristatum has four sites of 5S rDNA in its root-tip meristematic cells, whereas A. odoratum has six. Both species have six sites of 45S rDNA. Chromosomal localization of the rDNA varied, which suggests that chromosome rearrangements took place during Anthoxanthum genome evolution.

Mutagenicity and genotoxicity assessment of industrial wastewaters

The genotoxicity of industrial wastewaters from Jajmau (Kanpur), was carried out by Ames Salmonella/microsome test, DNA repair-defective mutants, and Allium cepa anaphase-telophase test. Test samples showed maximum response with TA98 strain with and without metabolic activation. Amberlite resins concentrated wastewater samples were found to be more mutagenic as compared to those of liquid-liquid extracts (hexane and dichloromethane extracts). The damage in the DNA repair defective mutants in the presence of Amberlite resins concentrated water samples were found to be higher to that of liquid-liquid-extracted water samples at the dose level of 20 μl/ml culture. Among all the mutants, polA exhibited maximum decline with test samples. Mitotic index (MI) of root tip meristematic cells of A. cepa treated with 5, 10, 25, 50, and 100 % (v/v) wastewaters were significantly lower than the control. Complementary to the lower levels of MI, the wastewaters showed higher chromosomal aberration levels in all cases investigated.