Root Tips Of Vicia Faba Research Articles

Mutagenic effects of chromium trioxide on root tip cells of Vicia faba

In this study on the mutagenic effects of different concentrations of chromium trioxide (CrO(3)) on Vicia faba root tip, micronucleus assay and chromosome aberration assay were used to determine the mitotic indexes, micronucleus aberration rate and chromosome aberration rate of Vicia faba root tip cells. The results showed that the effects of CrO(3) concentration on the mitotic indexes were complicated. CrO(3) increases the micronucleus aberration rate of Vicia faba root tip cells. It was found that within certain range of CrO(3) concentration the micronucleus aberration rate increased systematically with increased concentration of CrO(3), but that the micronucleus aberration rate decreased at higher level of CrO(3) and that CrO(3) also caused various types of chromosome aberration at a rate which increased systematically with increased concentration of CrO(3). We concluded that CrO(3) has significant mutagenic effect on Vicia faba root tip cells.

Genotoxicity of hydrated sulfur dioxide on root tips of Allium sativum and Vicia faba

Genotoxicity of sulfur dioxide (SO 2) and its hydrates (bisulfite and sulfite) in human lymphocytes and other mammalian cells have been found earlier in our laboratory. In the present studies, we used Allium stavium and Vicia faba cytogenetic tests, which are the highly sensitive and simple plant bioassays. A mixture of sodium bisulfite and sodium sulfite (1:3), at various concentrations from 1×10 −4 to 2×10 −3 M was used for the treatment. Genotoxicity was expressed in terms of anaphase aberration (AA) frequencies in the Vicia-AA test and in terms of micronuclei (MCN) frequencies in both Vicia-MCN test and Alllium-MCN test. On average, the results showed a 1.7–3.9-fold increase of AA frequencies and a 3.5–4.5-fold increase of MCN frequencies in Vicia root tips as compared with the negative control. Similarly, results of Allium-MCN test also showed a significant increase in MCN frequencies in the treated samples. In addition, pycnotic cells (PNC) appeared in Allium root tips of treated groups. The frequencies of MCN, AA and PNC increased dose-dependently and the cell cycle delayed at the same time in bisulfite treated samples. Results of the present study suggest that the Vicia and Allium cytogenetic bioassays are efficient, simple and reproducible in genotoxicity studies of bisulfite.

Effect of the Two Biofertilizers Rhizobacterien and Phosphoren on Cell Cycle and Mitotic Division in Vicia faba.

The present investigation has been conducted to study the mutagenic effect of the 2 biofertilizers Rhizobacterien and Phosphoren on mitotic division in root tips of Vicia faba, addition to the cell image was used to estimate and analyze the gray values of these cells. The 2 applied biofertilizers induced a significant and/or highly significant increase in mitotic activity. Proliferation activity was activated with the increase of the biofertilizers treatments. Moreover, the percentage of cells having 2C-DNA content (G 0 /G 1 ) decreased than those of control, while the percentage of the cells 3C (S phase) and 4C (G 2 , M) increased than control at cell image analysis. On the other hand, the biofertilizers induced several chromosomal aberrations in mitotic cells.

Evaluation of Genotoxic and Cytotoxic Properties of Pesticides Employed in Italian Agricultural Practices

In a program coordinated by the Italian Ministry of Works, we tested in vitro four pesticides widely employed in a developed agricultural region of central Italy. The four commercial agents were chosen on the basis of their diffusion in agricultural practice, knowledge of their active principle(s), and scant availability of data concerning their toxic and genotoxic activity. The agents were Cirtoxin, Decis, Tramat Combi (TC), and Lasso Micromix (LM). All substances were tested in three in vitro systems: Chinese hamster ovary (CHO) cells, a metabolically competent hamster cell line (Chinese hamster epithelial liver; CHEL), and root tips of Vicia faba (VF). The cytotoxic and genotoxic end points challenged were micronuclei and root tip length (RTL) in VF and mitotic index (MI), proliferation index (PI), cell survival (CS), cell growth (CG), cell cycle length (CCL), sister chromatid exchanges, chromosomal aberrations, and single-cell gel electrophoresis, or comet assay, in CHEL and CHO cells. Tested doses ranged from the field dose up to 200× the field dose to take into account accumulation effects. On the whole, tested agents appear to induce genotoxic damage only at subtoxic or toxic doses, indicating a low clastogenic risk. MI, PI, CS, CG, RTL, and CCL appear to be the less sensitive end points, showing no effects in the presence of a clear positive response in some or all of the other tests. Using cytogenetic tests, we obtained positive results for TC and LM treatments in CHO but not in CHEL cells. These data could be accounted for by postulating a detoxifying activity exerted by this cell line. However, cytogenetic end points appear to be more sensitive than those referring to cytotoxicity.

Localisation of Aluminium in Root Tips of Zea mays and Vicia faba

Monocot species and dicot species differ widely in the composition of their cell walls, namely in the pectin content, and thus in potential Al-binding sites. The effect of these differences on cellular localisation as well as on tissue distribution of Al was compared in root tips of Zea mays and Vicia faba . The localisation of Al was assayed by Laser Microprobe Mass Analysis (LAMMA) after freeze-substitution. The radial mobility of Al was much lower in the roots of Vicia faba . In root tips of Zea mays , Al had reached the stele already after 60 min while it was confined to the rhizodermis and outer cortex cells in Vicia faba , indicating a stronger binding of Al in the cell walls of the dicoe. This binding, however, had no influence on intracellular distribution. Within 60 min intracellular Al was detectable in both species. Nevertheless, by far the highest Al concentrations were always measured in the cell wall.

Evaluation of antimutagenic and desmutagenic effects of humic and fulvic acids on root tips ofVicia faba

Humic acids (HA) and fulvic acids (FA) of different origin were evaluated for their antimutagenic and/or desmutagenic activity on Vicia faba germinating seeds treated with the mutagen compound maleic hydrazide (MH). Both HA and FA were tested at two different concentrations, 20 and 200 mg L -1 , either alone or together with 10 mg L -1 of MH. Two experimental procedures were used to investigate the occurrence and nature of the antimutagenic or desmutagenic activity. In procedure 1, HA or FA and MH were interacted for 24 h and then added to the seeds. In procedure 2, the seeds were first treated with HA or FA for 6 days and then transferred to plates containing the MH solution, so that no interaction occurred between HA or FA and MH outside the seeds. The evaluation of the genotoxic activity was done by counting both micronuclei and aberrant anatelophases in root tip cells. Results obtained by using procedure 1 showed a clear desmutagenic action of HA and FA used at both concentrations, whereas results of procedure 2 indicated no antimutagenic effect of HA or FA.

Adaptation to alkylation damage in DNA measured by the comet assay.

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.

Cell cycle synchronization in plant root meristems.

The analysis of structure and metabolism of a cell at a defined phase of cell cycle is often difficult because cell cycle progression in somatic tissues is asynchronous and only a fraction of cells are cycling. An elegant solution to obtain populations of cells enriched for single stage of the cell cycle is to impose the synchrony artificially. Different systems have been used to obtain synchronized populations of plant cells, including suspension-cultured cells, leaf mesophyll protoplasts and root tip meristems. Root tips have been frequently used in a variety of studies ranging from chromosome analysis to cell cycle and its regulation. Seedlings with actively growing roots may be obtained in most plant species, they are easy to handle, the experimental system is well defined, reproducible and can be easily modified for different species. This paper describes a protocol for cell cycle synchronization in root tips of Vicia faba, which is based on the use of DNA synthesis inhibitor hydroxyurea [18]. Modifications of the protocol for Pisum sativum, Medicago sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, and Zea mays are also given. Flow cytometric data indicate that about 90% of root tip cells are synchronized. On average, mitotic indices exceeding 50% are obtained with the method. Synchronized cells may be accumulated at metaphase using a mitotic spindle inhibitor to achieve metaphase indices exceeding 50%.

Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle.

We describe a protocol for flow cytometry analysis of cell cycle in plants using indirect immunolabelling staining and Vicia faba, Pisum sativum and Zea mays root tip cells as model systems. The protocol is based on simultaneous analysis of two fluorescent signals. The first, obtained after staining with propidium iodide, is used to quantify nuclear DNA content. The second, obtained after indirect immunofluorescent staining of bromodeoxyuridine (BrdUrd), is used to quantify the amount of BrdUrd incorporated into nuclear DNA. In an attempt to standardize the procedure, the effects of various conditions for partial DNA denaturation using HCl, as well as of BrdUrd concentration and incorporation time on flow cytometry DNA/BrdUrd content analysis have been studied. Maximum BrdUrd-linked fluorescence was observed after a 30 min pulse with 10 microM BrdUrd and after DNA denaturation with 1.5 N HCl (final concentration) for 30 min at 25 degrees C. Under these conditions, DNA content histograms with relatively small coefficient of variation (< 4%, full peak) could be obtained. To avoid non-specific staining of cytoplasm and cell walls, the protocol involves the use of nuclei isolated from formaldehyde-fixed tissues. Fixed isolated nuclei are stable and may be stored in hexylene glycol 0.75 M at 4 degrees C for prolonged periods prior to actual staining and analysis.

In vivo and in vitro promutagen activation by Vicia faba of thiocarbamate herbicides molinate and butylate to products inducing sister chromatid exchanges in human lymphocyte cultures

Molinate and butylate treatments for 4 h of Vicia faba root tip meristems, showed that both thiocarbamate herbicides increased significantly SCE frequency. Direct treatments of molinate and butylate on human lymphocytes applied 24 h after the beginning of culture did not induce SCE. When S10 extracts of the Vicia roots, treated for 4 h with molinate and butylate (in vivo activation) were added to lymphocytes (24 h after of the beginning of culture), SCE were induced in a concentration–response manner. The in vitro assays, in which molinate and butylate was added at 48 h lymphocyte cultures for 4 h, showed a negative response, however, in the treatment where the S10 metabolic mix was added the SCE frequencies were significantly different to the control, and the concentration–response relationship was not observed with molinate, but it was obtained with butylate. The results showed that both herbicides needed the V. faba metabolism to produce SCE in human lymphocyte culture.

Mutagenicity (micronucleus test in Vicia faba root tips), polycyclic aromatic hydrocarbons and heavy metal content of sediments collected in Tiber river and its tributaries within the urban area of Rome

Sediments collected in Tiber river and in its main tributary water courses within the urban area of Rome were tested for mutagenicity by means of Vicia faba root tips micronucleus (MN) test. Representative samples were scored for micronucleus generating events (chromosome/chromatid loss and fragments) too. Sediments were assayed for content of the thirteen most important chemicals of polycyclic aromatic hydrocarbon (PAH) group and for some heavy metal ions (Cd, Cr, Cu, Ni, Pb, Zn). Samples were collected in four tributary rivers (Prima Porta, Acqua Traversa, Aniene and Magliana) just before their confluence with Tiber river and at different stations along the Tiber river itself upstream and downstream the sites of confluence of the sampled tributaries. All samples were collected in July 1992. An alarming level of mutagenicity was reached in most of the tested stations, with an effect comparable to an X-rays exposure up to 0.4 Gy. Chemical analysis showed that the total amount of identified PAHs ranged from 4.5 to 625.2 ng/g of dry matrix in the different stations and the total amount of heavy metals ranged from 130 to 570 ppm. Tiber mutagenicity is likely to be mainly due to local factors such as the confluence of a small polluted tributary rather than to large scale effect due to an upstream–downstream relationship.

Treatment ofVicia fabaroot tip cells with specific inhibitors to cyclin‐dependent kinases leads to abnormal spindle formation

Many events during cell division are triggered by an evolutionary conserved regulator, the cyclin-dependent kinase (Cdk). Here we used two novel drugs, the purine analogues bohemine and roscovitine, to study the role of Cdks in cell cycle progression and microtubule organisation in Vicia faba root tip cells. Both drugs inhibited the activity of immunopurified Vicia faba and alfalfa Cdc2-kinase. The transcript levels of an A- and B-type cyclin, as well as of the cdc2 genes, declined in treated root tips, while the mRNA level of a D-type cyclin gene was not affected. An observed transient arrest at the G1/S and G2/M regulatory points indicated that inhibition of the Cdc2-kinase had an effect on both transitions. In contrast to the regular bipolar spindle in untreated cell, in drug-treated metaphase cells abnormally short and dense kinetochore microtubule fibres were observed. These microtubules were randomly arranged in the vicinity of the kinetochores and connected the chromosomes. Thus, the chromosomes were not aligned on the metaphase plate but were arranged in a circle, with kinetochores pointing inwards and chromosome arms pointing outwards. gamma-Tubulin, which plays a role in microtubule nucleation, also localised to the centre of the monopolar spindle. The observed abnormalities in mitosis, after inhibition of Cdc2-kinase by specific drugs, suggest a role for this enzyme in regulating some of the steps leading to a bipolar spindle structure.

The genotoxicity of selected heterocyclic nitrogen compounds: A quantitative structure‐activity relationship study

The genotoxicity of 13 heterocyclic nitrogen compounds was evaluated by micronuclei test of Vicia faba root tip cell. All these chemicals are genotoxic. A meaningful Quantitative Structure‐Activity Relationship (QSAR) model was proposed by using the quantum chemistry descriptors: The above equation is significant because of its great R2 and F values. It may reveal the mutagenic mechanism to some extent and can be used to describe the genotoxicity of 13 tested chemicals. The main key steps are metabolic course and mutagenic reaction. The chemical with higher q+ and lower Elumo has greater genotoxicity.

Monitoring the genotoxicity of Lake Taihu, using two kinds of micronucleus tests

Mutagenicity of surface water in Lake Taihu was monitored by genotoxicological methods. The goal is to determine the situation concerning the contamination by mutagens, their distribution in the lake, and the potential effect on public health. In this study, the micronucleus test of Vicia faba root tip, a standard monitoring technique now in China, was carried out to make a preliminary screening of environmental mutagens in 39 representative sites of the lake. Highly significant differences in MCN permillage (average number of micronuclei per 1000 cells) and PI (pollution index) values were found among these sites. From the results, a rough distribution of the contamination could be obtained: mainly concentrated in the north-west area of the lake from several tributaries flowing into the lake. In order to provide further evidence, organic extracts from four entrances and one outlet of the five major tributaries were examined by the micronucleus test of human peripheral lymphocytes. The results were in accord with those of the micronucleus tests using Vicia faba. The tests also revealed that the intensity and mechanism of the pollutants were diverse in the different sources.

Sister chromatid exchanges in Vicia faba induced by arsenic-contaminated drinking water from Zimapan, Hidalgo, Mexico

Sister chromatid exchanges (SCE) in Vicia faba root tips were used to examine well water containing high levels of arsenic. The increased amount of arsenic was contained in well water from different towns of Zimapan, Hidalgo, Mexico. Treatments of 3 h were applied followed by the differential staining technique of Tempelaar et al. (Mutation Res. 103 (1982) 321–326). Concentrations of arsenic from 0.267 up to 1.070 mg/l were determined by colorimetry in the polluted samples used for this study. These values were above the permissible limit of 0.05 mg/l in drinking water. In all cases, except one in which the As concentration was 0.021, the arsenic-contaminated water produced significant increases of SCE compared with the control ( p<0.001) and a concentration–response relationship was observed. The SCE potency factor of 33 per mg/l of arsenic was calculated as the slope of a common regression line, pooling data previously obtained in the Comarca Lagunera and the results observed in Zimapan.

Effects of dNTPs on the SCE frequency in plant root tip cells

The effects of deoxynucleoside triphosphates (dNTPs) on the frequency of sister-chromatid exchange (SCE) have been studied in plant root tip cells. Treatment with dATP caused a dose-dependent decrease in SCE frequency, while dGTP, caused a dose-dependent increase in SCE frequency both in Hordeum vulgare and in Vicia faba root tip cells. Treatment with dCTP and dTTP significantly increased SCE frequency in H. vulgare root tip cells, but significantly decreased SCE frequency in V. faba root tip cells. The SCE induced by treatment with dGTP could be totally reversed by treatment with an equal concentration of any of the other three dNTPs (dATP, dCTP and dTTP) both in H. vulgare and in V. faba. Treatment with a mixed equal concentration of dATP, dCTP, dGTP and dTTP did not alter the SCE frequency. These results suggest that dNTP pool imbalance is an SCE-affecting factor in plant cells.

Heavy metal content and mutagenic activity, evaluated by Vicia faba micronucleus test, of Tiber river sediments

Tiber river sediment samples, collected in October 1995, were tested for mutagenicity by the micronucleus test in Vicia faba root tips. Four stations were studied within the urban area of Rome: (1) Castel Giubileo, at the entry of the urban area; (2) Ponte Tor di Quinto, immediately after the confluence of the tributary river Aniene; (3) Ponte Sublicio, in the middle of the city; and (4) Ponte della Magliana, immediately outside Rome, 20 km from the sea. Since no significant increase in micronucleus frequency was observed in any of the tested stations compared to control (while in previous campaigns mutagenic activity was observed in some of the same stations), it can be assumed an interesting recovery from mutagenic pollution in the 2 last years. The samples were analysed for pH value, nitrogen, organic matter and carbonate content, and the concentration of some potentially mutagenic heavy metal ions (Zn, Cd, Ni, V, Cu) was assessed. In all samples, a concentration of heavy metals higher than unpolluted areas was observed. However, the alkaline pH measured should keep them as non-bioavailable elements.

Cytotoxicity of Insecticide Temik 15G (Decarb) in Mitotic and Meiotic Cells of Vicia faba Plant.

The effect of Temik on both mitosis and meiosis was studied. The insecticide decreased the mitotic activity of Vicia faba root tip cells and produced different kinds of abnormalities. These kinds of abnormalities were nearly identical in both mitosis and meiosis. Stickiness was the most common type of these abnormalities resulted after all treatments.Disturbance mitotic phases which is a mitotic poisons effect were also present with considerable percentage. Such phenomena indicate the toxicity of Temik and its low mutagenic potential.

Effect of Hypoxia on Nucleoli in Excised Root Tips of Vicia faba Immunoelectron microscopy using anti-DNA antibodies.

Effect of hypoxia on the localization of intranucleolar DNA was examined in excised root tips of Vicia faba with immunoelectron microscopy using anti-DNA antibodies. In actively growing roots, the fibrillar centers (FCs) appeared as low electron-opaque patches. The immunoelectron microscopic study indicated that the FCs were heavily labeled and significantly strong label was also appreciated in the region around FCs. When excised root tips were subjected to a hypoxic condition which was attained by boiling distilled water for 10 min and sealed with “Parafilm” after cooling, area of the FCs relative to the whole area of nucleolus gradually increased with exposure time. At the 4th hr under hypoxia, chromatin-like compact fragments were sometimes seen in the developed FCs. These fragments were heavily labeled with anti-DNA antibodies as condensed chromatin. The label, on the other hand, extremely decreased in the region around FCs. Thus, evidence is presented suggesting that rDNA engaged in transcription deep in the DFC temporarily form condensed chromatin fragments in the FCs when the cells are exposed to hypoxia.

Effect of Hypoxia on Nucleoli in Eucised Root Tips of Vicia faba: An Electron Microscopic Study.

Actively growing root tips of Vicia faba excised to about 5 mm length were exposed to hypoxia and the structure of nucleoli were examined under electron microscopy. The nucleolar segregation was first discerned under hypoxia. Then, the fibrillar centers (FCs) enlarged at the 4th hr, accompanying many unusual granules about 35 nm in diameter (35 nm granules). Most FCs appeared as a channel-like configuration at the 4th and 8th hr. The 35 nm granules became ambiguous toward the 8th hr. In parallel with development of the FCs, the GC dispersed into the space between dense chromatin masses. Then, collapse of the DFC followed and eventually, the FCs coated with thin layer of the DFC were exposed. In the control specimens, the FCs also developed a little. However, no 35 nm granule arose in the FCs and no conspicuous degradation of the nucleoli occurred. These results strongly suggest that the hypoxic shock lowers the rate of rRNA synthesis to retract active rRNA genes from the DFC, leaving some nucleolar proteins in the FCs as the 35 nm granules.