Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cent. Eur. J. Biol.• 5(5) • 2010 • 633-640 DOI: 10.2478/s11535-010-0048-7 Central European Journal of Biology Genotoxicity of barley stripe mosaic virus in infected host plants Research Article Larisa I. Andronic*, Anatol G. Jacota, Valeriu V. Bujoreanu, Tatyana B. Grigorov Institute of Genetics and Plant Physiology of the Academy of Sciences of Moldova, MD 2002 Chisinau, Republic of Moldova Received 26 January 2010; Accepted 09 April 2010 Abstract: A comparative study of the effect of barley stripe mosaic virus (BSMV) and gamma irradiation on mitotic divisions in barley (Hordeum vulgare L.) roots was performed by evaluating the mitotic index (MI), micronucleus (MN) frequency and sister chromatid exchanges (SCE). Results indicate that, similarly to gamma irradiation at doses of 100, 150 and 250 Gy, BSMV reduces the mitotic activity, increases the micronucleus frequency and the rate of SCE and promotes the formation of C-metaphases. In root meristematic cells of the three barley cultivars studied (Galactic, Sonor and Unirea), the mitotic index of infected plants was found to be 52.5, 54.48 and 64.17%, respectively, lower than the uninfected control. An increase in frequency of sister chromatid exchanges was observed in all the experimental variants. In treatments involving viral infection alone or in combination with gamma irradiation chromosomes with three and more chromatid exchanges were observed, while their percentage in the control or in treatments with gamma irradiation alone was reduced. The results of the study indicate that in plants derived from irradiated seeds, BSMV produces an effect that is correlated nonlinearly with the radiation dose applied. Cytological analysis of mitotic divisions in barley roots revealed the genotoxicity of BSMV infection. Keywords: B arley stripe mosaic virus • Gamma radiation • Genotoxicity • Hordeum vulgare L. • Micronuclei • Mitotic index • Mitotic division • Sister chromatid exchange © Versita Sp. z o.o. 1. Introduction In studying genotoxicity of various factors, a number of parameters characterizing mitotic divisions are used, such as mitotic index, micronucleus frequency, and sister chromatid exchange (SCE) rate [1]. Mitotic index may be used as a direct indicator of the genotype response to various factors representing the organism’s mechanism of adaptation at the cellular level ensuring maintenance of homeostasis in cellular systems. Mitotic index is an important criterion used in evaluating proliferation processes [2]. Sister chromatid exchange assay is considered to be one of the most sensitive tests in assessing genotoxicity [3]. Evaluating features of the proliferation of vegetative cells, including meristematic cells, involves analyzing various plant responses to treatments [4]. Meristematic cells respond rapidly and actively to endogenous and exogenous factors [5]. The conservative nature of proliferation processes allows various mitotic 633 division parameters to be used in monitoring studies. Cytogenetic studies carried out by a number of authors suggest that barley, a genetically well studied crop plant, has successfully been used as a convenient and sensitive test system [6]. Barley carries seven pairs of chromosomes on which a large number of genes and gene mutations have been localized and described. Barley has been successfully used in genotoxicity studies [1]. Barley is a host plant for barley stripe mosaic virus (BSMV), a pathogen which has been shown to produce genetic effects on the host plant, such as induction of triploids and aneuploids in barley [7] and wheat [8]. A number of studies have recognized the contribution of viral infection to somatic and meiotic recombination enhancement, resulting in rearrangements which could be transmitted to the next generation [9,10]. An increase in somatic recombination is considered a general response of plants to various factors, including pathogens [11]. * E-mail: [email protected] Unauthenticated Download Date | 8/12/17 1:03 PM L.I. Andronic et al. The increased genetic flexibility resulting from recombination might facilitate adaptation of plant populations to stressful environments. Assessing proliferative processes of cells during viral infection can contribute to the understanding of the complex interaction between viruses and host plants. The objective of the present study is the cytogenetical evaluation of mitotic divisions in barley cells infected with BSMV alone or in association with gamma irradiation. 2. Experimental Procedures 2.1 Biological material and exposure schedule Three cultivars of spring barley (Hordeum vulgare L., 2n=14), Galactic, Sonor and Unirea, were used in the study. All seeds were initially tested for the presence of virus infection by immunosorbent electron microscopy (ISEM) [12]. Seeds confirmed to be non-infected were used in further experiments. Barley seeds were exposed to the following doses of gamma radiation: 100 Gy, 150 Gy and 250 Gy. RXM-γ-20 installation with Co60 as source of radiation was used to produce gamma rays. Barley plants from both irradiated seeds and untreated seeds were mechanically inoculated with a barley stripe mosaic virus (BSMV) extract. Plants derived from nonirradiated, healthy seeds that had been inoculated with distilled water and presented negative results upon virological examination served as the control. All three investigated cultivars Galactic, Sonor and Unirea are susceptible to BSMV and displayed external symptoms at 10-14 days after mechanical inoculation. 2.2 Virological analysis Control plants at the two- to three-leaf stage were mock inoculated with distilled water. Leaves of the plants from experimental lots, at the same stage, were infected two times at two-day intervals with BSMV inoculum using carborundum powder. Inoculum was prepared by grinding leaf tissue in distilled water (1:2). Initially, the extract was prepared from field-grown barley plants that expressed symptoms specifically to those induced by BSMV and presented positive response to ISEM test using polyclonal anti-BSMV serum diluted 1:50 [12]. Further work involved material collected from plants showing external disease symptoms and yielded positive results through ISEM. 2.3 Cytological analysis To study the induction of sister chromatid exchanges (SCE) upon treatment with gamma irradiation alone or in combination with BSMV infection, the frequency of SCEs in mitotic chromosomes from root meristems was analyzed using differential incorporation of 5-Brom-2’-deoxyuridin (BrdU). Seeds were germinated in the dark until rootlets reached 2–3 cm in length. The BrdU incorporation was performed during two cycles of cell division by exposure to a mixture comprised of 100 µM BrdU, 0.1 µM 5-fluorodeoxyuridine (FdUrd) and 5 µM uridine (Urd). The procedure of material fixation, maceration and staining was followed as previously described [13]. For cytological analysis of mitotic index (MI) and micronucleus (MN) test, rootlets that had reached 2–3 cm in length were subjected to direct fixation in an acetic acid: alcohol (3:1) solution. The samples were then rinsed in distilled water, mounted onto a slide, macerated and stained with acetocarmine or 5% Giemsa (for micronucleus evaluation). MI was expressed as the number of dividing cells per 100 scored cells and MN frequency was expressed as the number of cells with MN per 1000 scored cells, resulting from 10 separate seedlings for each group. The criteria for the identification micronucleus were as follow: (i) the MN had to be smaller than one-third of the main nuclei; (ii) the MN could not touch the main nuclei; (iii) the MN should have the same color and staining intensity as the main nuclei. 2.4 Statistical analysis The statistical processing of data was carried out using the software package Statgraphics Plus for Windows (version 2.1; Microsoft Corp., Redmond, WA, USA) and Microsoft Excel. The contribution of variation sources was computed following the ANOVA test results [14]. 3. Results 3.1 Mitotic index and micronucleus frequency in barley root meristems under viral infection and gamma radiation According to the results of the cytological study, the mitotic index (MI) in the genotypes under study was 10.44 in the cv. Galactic, 12.39 in cv. Sonor and 12.7 in cv. Unirea. The mitotic index of seed material collected from plants infected with BSMV and/or exposed to gamma radiation was lower than the mean value of MI in the control (Table 1). The largest decrease in the mitotic index of cv.Galactic (69% lower than in the control) occurred in the treatment where BSMV was used in combination with gamma a radiation dose of 250 Gy. A similar decrease in the MI was observed in treatments of gamma radiation alone at a dose of 250 Gy. Exposure to gamma radiation produced a dose-dependent effect. The impact of gamma radiation at doses of 100 Gy and 634 Unauthenticated Download Date | 8/12/17 1:03 PM Genotoxicity of barley stripe mosaic virus in infected host plants a) b) c) Figure 1. Interphase Interphases analyzed Mitotic Micronucleus (number) index value frequency Galactic Control Virus 100 Gy 100 Gy + virus 150 Gy 150 Gy + virus 250 Gy 250 Gy + virus 1683 1405 1800 1830 1890 1795 2405 2069 10.44±1.34 4.96±0.60*** 5.65±0.58** 6.82±1.25* 5.60±0.89** 5.88±1.69* 4.27±0.71*** 3.23±0.94*** 0.233±0.037 0.633±0.059*** 0.390±0.010*** 0.363±0.025** 0.863±0.163*** 0.983±0.194*** 1.167±0.050*** 1.223±0.133*** 1.42 1.11 0.42 1.93 Sonor meristematic cells in barley (Hordeum vulgare L.) rootlets: (a) normal interphases in rootlets of the control plants (x 850); (b) an interphase with a micronucleus (indicated by an arrow) in rootlets of barley plants, cv. Galactic, infected with barley stripe mosaic virus (x 700); (c) a micronucleus (indicated by an arrow) in meristematic cells of rootlets of barley cv. Galactic irradiated with gamma rays at a dose of 150 Gy and infected with barley stripe mosaic virus (x 1000). Control Virus 100 Gy 100 Gy + virus 150 Gy 150 Gy + virus 250 Gy 250 Gy + virus 2039 2737 2345 2263 2657 2340 2683 2014 12.39±0.93 5.64±1.04*** 5.93±0.37*** 6.33±0.43*** 5.66±0.79*** 5.90±1.55*** 4.63±0.49*** 4.59±0.66*** 0.140±0.131 0.620±0.115** 0.477±0.124* 0.510±0.098** 0.687±0.154** 0.793±0.191** 0.933±0.146*** 1.136±0.125*** 0.37 0.43 0.99 Unirea 150 Gy was milder in the case of virus-infected plants (the difference between treatments was statistically significant at P≤0.05). The same pattern in mitotic index modification was observed in the other two cultivars, Sonor and Unirea. In the cv. Sonor, virus infection caused a reduction in the frequency of mitotic cells similar to that produced by gamma radiation at doses of 100 Gy and 150 Gy. Upon irradiation of barley seeds with gamma rays at a dose of 250 Gy, the mitotic index value decreased 62%, practically to the same extent as in the treatment using gamma irradiation at 250 Gy in combination with virus infection (63%). In the case of cv. Unirea, BSMV tended to enhance the mitodepressive effect of gamma radiation at a dose of 150 Gy and to reduce the effect of gamma rays at a dose of 250 Gy. Reductions in the mitotic index in various treatments (experimental variants) were accompanied by an increase in the micronucleus frequency (Table 1, Figure 1). In all three barley varieties studied, viral infection alone induced a higher number of micronuclei than viral infection in combination with gamma radiation at a dose of 100 Gy. Treatment with gamma irradiation at a dose of 150 Gy resulted in approximately the same frequency of micronuclei as exposure to gamma radiation in combination with viral infection. Gamma irradiation at a dose of 250 Gy caused the frequency of micronuclei to increase 5-fold over the control in Galactic, 6.7-fold in Sonor and 4.3-fold in Unirea. The frequency of Control Virus 100 Gy 100 Gy + virus 150 Gy 150 Gy + virus 250 Gy 250 Gy + virus 1482 1661 1972 1895 1724 1966 2383 1980 12.70±0.79 4.55±0.51*** 5.16±0.11*** 5.51±0.76*** 6.09±0.85*** 4.91±0.25*** 3.47±0.66*** 5.06±0.53*** 0.267±0.083 0.727±0.032*** 0.356±0.006* 0.573±0.210* 0.700±0.069*** 0.853±0.071*** 1.143±0.222*** 1.433±0.132*** 0.60 0.42 1.52 Treatment Table 1. C-metaphase (‰) Frequency of micronuclei in roots of barley plants derived from gamma-irradiated seeds and infected with barley stripe mosaic virus. *; **; *** - significant difference from the control at P≤0.05; 0.01; 0.001 635 Unauthenticated Download Date | 8/12/17 1:03 PM L.I. Andronic et al. micronuclei in virus-infected plants derived from seeds irradiated with gamma rays at a dose of 250 Gy was similar to that induced by gamma radiation alone. Micronuclei are extranuclear chromatinic bodies resulting from chromosome breaks or aneuploidy [1]. Examination of cytological preparations obtained using the acetocarmine staining technique showed micronuclei to be well-defined chromatinic structures located in close proximity to nuclei and characterized by wide variation in size. The presence of micronuclei is considered to be an indicator of genome instability [15]. The occurrence of C-metaphases was observed in barley plants infected with BSMV (Table 1, Figure 2). In Galactic and Sonor, C-metaphases were visualized in plants subjected to a combination of viral infection and gamma radiation at doses of 150 and 250 Gy, whereas in Unirea they only occurred in treatments with simultaneous application of viral infection and gamma radiation at a dose of 250 Gy. Abnormalities of the C-mitosis type are believed to be due to the division spindle restructuring [16]. C-mitoses may result in chromosome number doubling. In the absence of cytokinesis, polyploid or bi- and polynucleate cells may form. In the case of phragmoplast and cell wall formation, cells with different chromosome numbers may arise [17]. The possibility of aneuploids being induced by BSMV in wheat and barley has a) Figure 2. C-metaphase b) in rootlets of the barley cv. Unirea: (a) infected with barley stripe mosaic virus; (b) irradiated with gamma rays (150 Gy) and infected with barley stripe mosaic virus (x 1200). previously been described [7,8]. Cytological studies of mitotic and meiotic divisions have shown BSMV particles in direct association with microtubules [18], which may explain the direct effect of the virus on components of the spindle apparatus. 3.2 A study of sister chromatid exchanges in barley (Hordeum vulgare L.) under viral infection and gamma radiation Analysis of sister chromatid exchanges (SCE) in three spring barley cultivars (Sonor, Unirea and Galactic) made it possible to determine the number of SCEs per cell or chromosome in the case of infection with BSMV, irradiation with gamma rays (at doses of 100, 150 and 250 Gy), as well as in the case of viral infection of plants derived from gamma-irradiated seeds. The results of the SCE test indicated that under normal conditions an average of 5.6–6.1 SCEs per cell occur. Both gamma radiation and viral infection increased the frequency of SCEs (Table 2). In the case of viral infection, the highest percentage of SCEs induced by BSMV was observed in the cv. Unirea (a nearly 55.4% increase over the control, at P≤0.001). The effect produced by gamma radiation varied according to the dose applied to the seeds as well as according to the genotype analyzed. The frequency of SCEs in the case of exposure to gamma radiation at a dose of 100 Gy increased by 40.1% and 33.2% over the control in barley Sonor and Unirea, respectively, whereas the frequency of SCEs in cv. Galactic only increased by 8.23% (P≤0.05). It is only in the case of cv. Galactic that gamma radiation at a dose of 150 Gy produced a more mitodepressive effect (statistically confirmed) than gamma radiation at a dose of 100 Gy. In the cv. Unirea, the rate of sister chromatid exchanges per cell varied around 7.2 and tended to decrease. When viral infection was combined with gamma radiation treatment, the effect on the rate of SCEs varied according to the genotype and the gamma radiation dose applied. Genotype analyzed Treatment Sonor Unirea Galactic Control 6.128±0.337 5.561±0.259 6.108±0.265 Virus 8.359±0.299*** 8.641±0.286*** 8.703±0.312*** 100 Gy 8.585±0.273*** 7.410±0.284*** 6.611±0.240* 100 Gy + virus 8.946±0.287*** 7.973±0.314*** 8.028±0.244*** 150 Gy 8.846±0.295*** 7.270±0.339*** 7.973±0.269*** 150 Gy + virus 8.385±0.281*** 8.081±0.246*** 9.054±0.274*** 250 Gy 10.081±0.318*** 9.540±0.358*** 8.081±0.291*** 250 Gy + virus 10.971±0.305*** 8.811±0.321*** 8.838±0.306*** Table 2. The rate of sister chromatid exchanges (per cell) induced by BSMV in meristematic cells of roots of healthy and irradiated barley plants. *; *** - the differences from the control are statistically significant at P ≤ 0.05; 0.001 636 Unauthenticated Download Date | 8/12/17 1:03 PM Genotoxicity of barley stripe mosaic virus in infected host plants In plants of the cv. Sonor derived from seeds irradiated with gamma rays at a dose of 100 Gy, BSMV infection caused a larger increase in the SCE frequency. The rate of SCEs induced by viral infection in Sonor irradiated with gamma rays at a dose of 150 Gy was close to that in the treatment where infection alone was applied. The same tendency of reduction in SCE frequency was also observed for the cv. Unirea with infection combined with 250 Gy treatment. In Galactic, BSMV infection caused an increase in SCE rate compared to gamma irradiation alone. Analysis of SCEs in terms of distribution of exchanges per chromosome revealed a tendency for dispersion to increase in the case of experimental variants (P≤0.5) (Figure 3). Examination of the spectrum of sister chromatid exchanges indicated that the number and type of SCEs are affected by both factors analyzed. While in the control plants the rate of SCEs per chromosome averaged 0.40–0.46, in some experimental variants (treatments) it reached 0.69–0.73 (in Galactic and Sonor irradiated with gamma rays at a dose of 250 Gy). A study of SCE distribution has, in general, revealed a tendency for the frequency of chromosomes with three and more exchanges to increase, especially in treatments using viral infection alone or in combination with gamma radiation (Figures 3 and 4). An increase in mean frequency of SCEs in the case of viral infection, as well as in the case of gamma irradiation, occurs predominantly due to multiple chromatid exchanges whose percentage under normal conditions is reduced (0.28–0.48 per cell). In experimental variants, the percentage of multiple exchanges increased substantially, reaching its maximum in the cv. Sonor upon infection of BSMV in combination with irradiation by gamma rays at a dose of 250 Gy. For this treatment, the proportion of chromosomes with multiple exchanges was found to increase 12.7-fold relative to that in the control. Application of ANOVA revealed that barley stripe mosaic virus, like gamma radiation, causes the frequency of sister chromatid exchanges to increase. Analyses Source of variance indicate that effect on SCEs is dependent on the plant genotype (3.6%), virus (6.1%) and radiation dose (13.4%) (Table 3). In the aggregate, the degree of influence of radiation against the background of viral infection may increase or decrease, depending on the genotype and the dose applied. The most powerful influence was found to be exerted by gamma radiation as dependent on the dose applied (13.4%). a) b) c) d) Figure 3. M etaphase plates in meristematic cells of roots of H. vulgare, cv. Unirea: (a) control, (b) barley stripe mosaic virus, (c) gamma irradiation 150 Gy, (d) gamma irradiation 150 Gy + virus (x 1200). a) b) c) d) e) f) g) Figure 4. M etaphase chromosomes in Hordeum vulgare: (a,b) chromosomes without chromatid exchanges, (c) schematic representation of a metaphase chromosome without chromatid exchanges, (d) a chromosome with a chromatid exchange in the long arm, (e) a chromosome with a chromatid exchange in each arm, (f) chromosomes with multiple chromatid exchanges, (g) schematic representation of a metaphase chromosome with a chromatid exchange. Contribution of the source of Degree of Sum of variance, % freedom squares S2 F test P A: Virus 6.06 1 1.37672 1.37672 71.241 0.0000 B: Genotype 3.65 2 0.829628 0.4148129 21.47 0.0000 C: Radiation dose 13.42 3 3.04913 1.01638 52.50 0.0000 Interaction AB 0.3 2 0.0832031 0.0416016 2.38 0.0930 Interaction AC 3.95 3 0.898543 0.299514 17.15 0.0000 Interaction BC 3.01 6 0.684898 0.11415 6.53 0.0000 Interaction ABC 1.35 6 0.3059 0.0509834 2.92 0.0080 913 22.7279 Total Table 3. Analysis of variance of the SCE frequency in barley plants, healthy and infected with BSMV. 637 Unauthenticated Download Date | 8/12/17 1:03 PM L.I. Andronic et al. Cytogenetic studies suggest that BSMV infection, like gamma radiation, causes the number of SCEs to change significantly. The effect of gamma rays on DNA is well understood, and their ability to induce sister chromatid exchanges in a dose-effect manner has been described previously [19]. These findings have been confirmed in our previous studies. The ability to increase the frequency of SCEs in conditions of viral pathogenesis is indicative of the genotoxic effect of the pathogen involved. Genotoxic effects have also been observed in infection by some animal viruses, such as hepatitis B virus or hepatitis C virus [20]. BSMV appears to have genotoxic effects, as evidenced by an increase in sister chromatid exchanges, the rate of which, in most cases, is dependent on multiple exchanges. 4. Discussion Various factors, predominantly chemical agents, have been shown to inhibit mitosis in crop plants, including barley. A decrease in mitotic index has been reported in barley plants exposed to sulfur dioxide [21], oxygenated compounds [22], and some insecticides and fungicides [17]. It is believed that variation in the mitotic index values directly reflects the intensity of physiological and biochemical reactions whereas the rhythm of variation is dependent on the rate of enzymatic reactions [23]. A decrease in value of mitotic index with exposure to various factors attests to the mitodepressive nature of these factors, which inhibit cell divisions [24]. Hidalgo and coworkers [25] explain the antimitotic effect of herbicides (propham and chlorpropham) in Allium cepa as inhibiting the activity of enzymes, including DNA polymerase. A reduction in mitotic index may result from inhibition of DNA synthesis in S phase of the cell cycle [26] or cell blocking in the G1 period [27]. Inhibition of mitosis may also result from retention of cells in G2 [16]. According to Yi et al. [21], a decline in proliferation processes occurring in barley roots exposed to sulfur dioxide may also be due to DNA degradation. The effect of mitodepressive factors on mitoses is believed to be stronger during S phase of the cell cycle [6]. Sister chromatid exchanges (SCEs) were first described in plants by Kihlman and Kronborg [28] by employing the Giemsa staining technique [29]. According to many reports, SCEs are brought about by irreparable double-strand breaks occurring during DNA replication, as has already been suggested by Wolff and coworkers [30]. SCEs can also arise from disturbances in the double-strand break repair process [31]. SCE genesis is considered to be conditioned by DNA replication events, in particular, those of replicative fork formation during which sister chromatid recombination may occur [32]. According to the replication model of sister chromatid exchange proposed by Painter [33], replication and repair of DNA double-strand breaks occurring at the boundary of two replicon clusters proceeding asynchronously. A similar situation may result from changes in the rate of replication process as well as from modifications in the structure of the DNA molecule. A study of SCEs in Saccharomyces cerevisiae revealed three mechanisms involved in the generation of chromatid exchanges: induction of DNA double-strand breaks, decline in the replication process, and reduction of DNA-polymerase activation processes [34]. In cells exposed to UV rays, X-rays, 4-nitroquinoline 1-oxide or methyl methanesulfonate, activation of genes RAD55, RAD57 and RAD54 involved in repair of DNA breaks has been observed. This phenomenon does not occur spontaneously, contrary to the observation that under normal conditions some chromatid exchanges do occur. Results indicate that different mechanisms are responsible for spontaneous and induced SCE generation. Although the mechanism of SCE generation is still unclear [35], it is well established that SCE genesis is associated with DNA lesions. According to many reports, plant viruses are capable of inducing various genetic effects [36-40], including modification of the sister chromatid exchange rate [36,41]. SCE analyses have demonstrated the mutagenic effect of the bean common mosaic virus in bean plants that varies according to the stage of viral pathogenesis. In the case of natural virosis occurring in later stages of ontogenesis, this effect has not been statistically confirmed [36]. The SCE test has also been used to analyze the mutagenic activity of the maize rough dwarf virus in some maize lines sensitive to this pathogen [41]. Results indicate that BSMV infection separately or in combination with gamma radiation causes a decline in the mitotic process (produces a mitodepressive effect), thus inhibiting the cell proliferation process.Cell division blocking is accompanied by mitotic abnormalities, such as micronuclei and C-metaphases. The effects of gamma irradiation are represented by a sinusoidal curve where complexity is specific to the genotype analyzed. A similar nonlinear effect was observed by Geraskin and coworkers [42] upon irradiation of barley seeds with low doses of gamma rays (10–1,000 mGy). The impact of radiation on genetic material is well understood. Rogakou and coworkers [43] explain the genesis of double-strand breaks under the influence of radiation as being the direct result of histone H2A phosphorylation at the level of serine, while suggesting the existence of other centers sensitive to this factor. Gamma irradiation 638 Unauthenticated Download Date | 8/12/17 1:03 PM Genotoxicity of barley stripe mosaic virus in infected host plants of stem cells deficient in the protein kinase Chk1 gene disrupts the G2/M transition checkpoints [44]. Data from cytogenetic analysis of mitotic divisions regarding the induction of DNA damage have been confirmed through assessment of chlorophyll mutations. It is worth noting that albino plantlets were not observed in any of the treatments. Chlorophyll mutations, manifesting themselves as lack of chlorophyll pigments, were predominantly observable in barley plants of cv. Unirea in the plot involving viral infection alone or in combination with gamma irradiation at a dose of 150 Gy. The possibility of inducing chlorophyll mutations in barley plants using various treatments have been described previously [42]. BSMV infection produces effects similar to those induced by gamma rays. It appears the effect of viral infection is comparable to irradiation doses of 150 or 250 Gy, depending on the genotype analyzed. Combining viral infection with radiation treatment has demonstrated nonlinear effects, determined by the interaction of the three factors of genotype, infection, and radiation dose. Viral infection has also been found to be involved in the induction of abnormalities associated with division spindle aberrations, such as C-mitoses, which reflects the aneugen nature of the pathogenic agent under study. A cytological study of mitotic divisions in infected barley roots indicates a mitodepressive effect of viral infection and genotoxicity of BSMV. Acknowledgements The authors extend their sincere thanks to Mr. G.K. Lakhman for translating the manuscript into English. References [1] Maluszynska J., Juchimiuk J., Plant genotoxicity: a molecular cytogenetic approach in plant bioassays, Arh. Hig. Rada Toksikol., 2005, 56, 177-184 [2] Kozak M.F., The mitotic rhythms in representatives of soybean Glycine L., Cytologiâ i Genetika, 2004, 38, 7-12, (in Russian) [3] Tucker J.D., Auletta A., Cimido M.C., Dearfield K.L., Jacobson-Kram D., Tice R.R., et al., Sister chromatid exchange: second report of the GeneTox Program, Mutat. Res., 1993, 297, 101-180 [4] Ivanov V.B., Cell proliferation in plants, Itogy nauki i tekhniki, Ser. Tsitologia, VINITI, Moscow, 1987, (in Russian) [5] Dmitrieva S.A., Minnibaeva F.V., Gordon L.Kh., Mitotic index of meristematic cells and growth of Pisum sativum roots exposed to modulators of the inositol series, Cytologiâ, 2006, 48, 475-479, (in Russian) [6] Singh P., Srivastava A.K., Singh A., Cell Cycle Stage Specific Application of Cypermethrin and Carbendazim to Assess the Genotoxicity in Somatic Cells of Hordeum vulgare L., Bull. Environ. Contam. Toxicol., 2008, 81, 258-261 [7] Sandfaer J., Barley stripe mosaic virus and the frequency of triploids and aneuploids in barley, Genetics, 1973, 73, 597-603 [8] Linde-Laursen I., Siddiqui K.A., Triploidy and aneuploidy in virus infected wheat, Triticum aestivum, Hereditas, 1974, 76, 152-154 [9] Kovalchuk I., Kovalciuc O., Kalck V., Boyko V., Filkowski J., Heinlein M., et al., Pathogen-induced systemic plant signal triggers DNA rearrangements, Nature, 2003, 423, 760-762 [10] Boyko A., Kathiria P., Zemp F.J., Yao Y., Pogribny I., Kovalchuk I., Transgenerational changes in the genome stability and methylation in pathogeninfected plants (Virus-induced plant genome instability), Nucl. Acids Res., 2007, 35, 1714-1725 [11] Lucht J.M., Mauch-Mani B., Steiner H.Y., Metraux J.P., Ryals J., Hohn B., Pathogen stress increases somatic recombination frequency in Arabidopsis, Nat. Genet., 2002, 30, 311-314 [12] Milne R.G., Lesemann D.-E., Immunosorbent electron microscopy in plant virus studies, Meth. Virol., 1984, 8, 85-101 [13] Panda K.K., Patra J., Panda B.B., Induction of sister chromatid exchanges by heavy metal salts in root meristem cells of Allium cepa, Biol. Plantarum, 1996, 38, 555-561 [14] Clewer A.G., Scarisbrick D.H., Practical statistics and experimental design for plant and crop science, John Wiley & Sons Ltd., Chichester, 2001 [15] Il’inskikh N.N., Micronucleus analysis and cytogenetic instability, Tomsk University Press, 1992, (in Russian) [16] El-Ghamery A.A., El-Nahas A.I., Mansour M.M., The action of atrazine herbicide as an indicator of cell division on chromosomes and nucleic acids content in root meristems of A. cepa and V. faba, Cytologia, 2000, 65, 277-287 [17] Landis W.G., Gorsuch J.W., Hughes J.S., Anthony M.L., (Eds.), Environmental toxicology and risk assessment, vol. 2, Gorsuch, Dwyer, Ingersoll, La Point, 1993 [18] Mayhew D.E., Carroll T.W., Barley Stripe Mosaic Virions Associated with Spindle Microtubules, Science, 1974, 185, 957-958 639 Unauthenticated Download Date | 8/12/17 1:03 PM L.I. Andronic et al. [19] Bozsakyová E., Wsólová L., Chalupa I., Spontaneous and gamma-ray-induced sister chromatid exchanges in patients with carcinoma of cervix uteri, Int. J. Radiat. Biol., 2005, 81, 177-185 [20] Ucur A., Palanduz S., Cefle K., Ozturk S., Tutkan G., Vatansever S., et al., Sister chromatid exchange and mitotic index in patients with cirrhosis related to hepatitis B and C viruses and in chronic carriers, Hepato-Gastroenterology, 2003, 50, 2137-2140 [21] Yi H., Lui J., Zheng K., Effect of sulfur dioxide hydrates on cell cycle, sister chromatid exchange, and micronuclei in barley, Ecotox. Envir. Safe., 2005, 62, 421-426 [22] Sang N., Xin X., Municipal landfill leachate induces cytogenetic damage in root tips of Hordeum vulgare, Ecotox. Envir. Safe., 2006, 63, 469-473 [23] Butorina A.K., Do Niû Tien, The circadian mitotic rhythms in mung bean Vigna radiata (L.) R.Wilczek, Cytologiâ, 2008, 50, 729-733, (in Russian) [24] Pandey R.M., Cytotoxic effects of pesticides in somatic cells of Vicia faba L., Tsitologia i Genetika, 2008, 42,13-18 [25] Hidalgo A., Gonzalez-Reyes J.A., Navas P., Garcia-Herdugo G., Abnormal mitosis and growth inhibition in Allium cepa root induced by propham and chloropropham, Cytobios, 1989, 57, 7-14 [26] Sudhakar R., Ninge Gowda K.H., Venu G., Mitotic abnormalities induced by silk dyeing industry effluents in the cells of Allium cepa, Cytologia, 2001, 66, 235-239 [27] Schneidermann M.H., Dewey W.C., Highfield D.P., Inhibition of DNA synthesis in synchronized Chinese hamster cell treated in G1 with cycloheximide, Exp. Cell Res., 1971, 67, 147-155 [28] Kihlman B.A., Kronborg D., Sister chromatid exchange in Vicia faba. I. Demonstration by a modified fluorescent plus Giemsa (FPG) technique, Chromosoma, 1975, 51, 1-10 [29] Perry P., Wolff S., New Giemsa method for the differential staining of sister chromatids, Nature, 1974, 251, 156-158 [30] Wolff S., Bodycote J., Painter R.B., Sister chromatid exchanges induced in Chinese hamster cells by U.V. irradiation at different stages of cell cycle: the necessity for cell to pass through S, Mutat. Res., 1974, 25, 73-81 [31] Ishii Y., Bender M., Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet lightinduced sister-chromatid exchanges in Chinese hamster cells, Mutat. Res., 1980, 79, 19-32 [32] Kato H., Mechanisms of sister chromatid exchanges and the relation to production of chromosomal aberrations, Chromosoma, 1977, 59, 179-191 [33] Painter R.B., A replication model for sister-chromatid exchange, Mutat. Res., 1980, 70, 337-341 [34] Dong Z., Fasullo M., Multiple recombination pathways for sister chromatid exchange in Saccharomyces cerevisiae: role of RAD1 and the RAD52 epistasis group genes, Nucl. Acids Res., 2003, 31, 2576-2585 [35] Wilson D.M., Thompson L., Molecular mechanisms of sister-chromatid exchange, Mutat. Res., 2007, 616, 11-23 [36] Andronic L., Study of the mutagenic effect of viral infection based on the analysis of sister chromatid exchanges, Proceedings of National Conference on genetics, biotechnology and crop improvement, (17-18 February, 2005, Chisinau), 2005, 17-20, (in Romanian) [37] Bujoreanu V., Chiriac Gh., Plant viruses as possible inducers of genotypic variability in plants, Proceedings of National Conference on genetics, biotechnology and crop improvement, (9-10 November, 1994, Chisinau), 1994, 8-10, (in Romanian) [38] Chiriac Gh.I., Andronic L., Bujoreanu V.V., Marii L., Features of crossing-over in virus-infected tomato, Cent. Eur. J. Biol., 2006, 1, 386-398 [39] Mock R.J., Stokes I.E., Jullesfie A.J., Effect of sugarcane mosaic virus infection in parental stock on panicle and seed production of virus-free F2 progeny in Sorghum (Sorghum bicolor), Plant Dis., 1985, 69, 310-312 [40] Yukhimenco A.I., Voloshchuk S.I., Girco V.S., Winter wheat viruses as biological stress factors inducing genetic variation, Proceedings of 11th congress of the federation of European societies of plant physiology, (7–11 September 1998, Varna, Bulgaria), Bulgarian J. Plant. Physiol., 1988, 222 [41] Nemčinov L.G., Genetic variability in maize under conditions of viral pathogenesis, PhD thesis, Institute of Genetics and Cytology of the National Academy of Sciences of Belarus, Republic of Belarus, 1990, (in Russian) [42] Geraskin S., Oudalova A., Kim J., Dikarev V., Dikareva N., Cytogenetic effect of low dose gammaradiation in Hordeum vulgare seedlings: non-linear dose-effect relationship, Radiat. Environ. Bioph., 2007, 46, 31-41 [43] Rogakou E.P., Boon C., Redon C., Bonner W.M., Megabase chromatin domains involved in DNA doublestrand breaks in vivo, J. Cell. Biol., 1999, 146, 905-916 [44] Liu Q., Guntuku S., Cui X.S., Matsuoka S., Cortez D.,Tamai K., et al., Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint, Gene Dev., 2000, 14, 14481459 640 Unauthenticated Download Date | 8/12/17 1:03 PM