Coupled Restriction Enzyme Digestion-random Amplification Research Articles

Cumin Seed Oil Induces Oxidative Stress-Based Antifungal Activities on Fusarium graminearum.

In this study, the antifungal activity of cumin seed oil (CSO) was tested on Fusarium graminearum. (i) Minimum inhibitory concentrations (MICs) and related concentrations (IC75, IC50, and IC25) were detected; (ii) toxicity was evaluated by a water-soluble tetrazolium salt-1 (WST-1) assay; (iii) genomic/epigenomic alterations were evaluated by the coupled restriction enzyme digestion-random amplification (CRED-RA) method; (iv) oxidative stress was investigated by CAT expression, catalase activity, and DCF-DA staining; (v) deoxynivalenol biosynthesis was evaluated by tri6 expression; (vi) and potential effects of CSO on wheat were tested by a water loss rate (WLR) assay. MIC, IC75, IC50 and IC25 values were detected at 0.5, 0.375, 0.25, and 0.125 mg mL-1. In WST-1 assays, significant decreases (p < 0.001) were detected. Genomic template stability (GTS) related to methylation differences ranged from 94.60% to 96.30%. Percentage polymorphism for HapII/MspI values were as 9.1%/15.8%. CAT (oxidative stress-related catalase) and tri6 (zinc finger motif transcription factor) gene expressions were recorded between 5.29 ± 0.74 and 0.46 ± 0.10 (p < 0.05). Increased catalase activity was detected (p < 0.05) by spectrophotometric assays. DCF-DA-stained (oxidative stressed) cells were increased in response to increased concentrations, and there were no significant changes in WLR values. It was concluded that CSO showed strong antifungal activity on F. graminearum via different physiological levels.

Effects of Selenium on DNA Methylation and Genomic Instability Induced by Drought Stress in Wheat (Triticum aestivum L.)

The main purpose of the study was to clarify the effect of selenium (Se) on DNA damage and DNA methylation in wheat (Triticum aestivum L.) plants exposed to polyethylene glycol (PEG)-induced drought stress under in vitro tissue culture. Random amplified polymorphic DNA (RAPD) and coupled restriction enzyme digestion-random amplification (CRED-RA) were utilized to explain the DNA damage grade and variations in DNA methylation patterns, respectively. The outcomes indicate that drought stress gives rise to a rise in RAPD profile variations (as DNA damage) and a decrease in genomic template stability (GTS) rate and DNA methylation changes. According to the RAPD data, the greatest GTS value was computed at 56.9% (5% PEG 6000), and the lowest GTS value was 41.2% (15% PEG 6000), demonstrating the adverse effects of PEG 6000. However, DNA damage can be reduced by treatment with sodium selenate (2, 4, and 6 µM of Na2SeO4) together with PEG (5%, 10%, and 15% PEG 6000)-induced water deficits. Moreover, according to CRED-RA analysis, PEG-induced DNA methylation rates were changed after treating different doses of Se. These data demonstrate that Se dose-dependently modulates both DNA damage and methylation alterations induced by drought in wheat.

Modeling DNA Methylation Profiles and Epigenetic Analysis of Safflower (Carthamus tinctorius L.) Seedlings Exposed to Copper Heavy Metal.

Heavy metals are chemical elements with high density that can be toxic or poisonous even at low concentrations. They are widely distributed in the environment due to industrial activities, mining, pesticide use, automotive emissions and domestic wastes. This study aimed to investigate the toxic effects of copper (Cu) heavy metal on safflower plants in terms of genetic and epigenetic parameters. Safflower seeds were exposed to different concentrations of Cu heavy metal solution (20, 40, 80, 160, 320, 640, 1280 mg L-1) for three weeks, and changes in the genomic template stability (GTS) and methylation pattern in the root tissues were analyzed using PCR and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques. The results indicated that high doses of Cu have genotoxic effects on the genome of safflower plants. Epigenetic analysis revealed four different methylation patterns, with the highest total methylation rate of 95.40% observed at a 20 mg L-1 concentration, and the lowest rate of 92.30% observed at 160 mg L-1. Additionally, the maximum percentage of non-methylation was detected at 80 mg L-1. These results suggest that changes in the methylation patterns can serve as an important mechanism of protection against Cu toxicity. Furthermore, safflower can be used as a biomarker to determine the pollution in soils contaminated with Cu heavy metal.

Investigation of antifungal activities of myrcene on Fusarium reference strains.

Antifungal effects of myrcene, the plant-based naturel compound, were investigated on Fusarium graminearum PH-1 and Fusarium culmorum FcUK99 references, for the first time. Minimum inhibitory concentration (MIC) and half of MIC (MIC50) of both Fusarium strains against myrcene were found as 25µg/µl and 12.5µg/µl, respectively. MIC50 application decreased the cell viabilities in the ratios of 34.90% and 33.91% in PH-1 and FcUK99, respectively (p < 0.01). The significantly increased catalase (CAT) activity was recorded in MIC50 treated strains (p < 0.01). Apoptosis-like process and cellular oxidative stress were also monitored with acridine orange/ethidium bromide (Ao/Eb) dual staining and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) staining. The genomic template stability (GTS) percentages were calculated as 79% for PH-1 and 71% for FcUK99 via random amplified polymorphic DNA (RAPD). Methylation polymorphism values were calculated as 53.8% and 50.6% in PH-1 and 40.4% and 39% in FcUK99 for HapII and MspI, respectively by coupled restriction enzyme digestion-random amplification (CRED-RA). Methylation-sensitive amplified polymorphism (MSAP) revealed that myrcene caused both type I and type III epigenetic modifications in both genomes. MIC50 dose caused up to 13.86 ± 0.42-fold changes in the expressions of cat, mst20, and stuA, whereas downregulation in tri5 was recorded. Myrcene application did not change the retrotransposon movement in both species by the amplifying of idiomorphic retrotransposon patterns through inter-retrotransposon polymorphism-polymerase chain reaction (IRAP-PCR). This study demonstrated that myrcene is an effective compound in the management of phytopathogenic Fusarium species by causing morphological, genetic, epigenetic, and cellular alterations, and has a potential to utilize as an antifungal agent.

TERPINOLENE IS AN EFFECTIVE ESSENTIAL OIL COMPOUND TO PROTECT Hordeum vulgare L. FROM Fusarium avenaceum (Fr.) Sacc.

Fusarium avenaceum (Fr.) Sacc. is an important phytopathogen. Fight against F. avenaceum includes primarily fungicide usage. However, novel strategies are needed in a struggle with F. avenaceum. In this study, terpinolene was used against F. avenaceum as an antimicrobial agent, and the harmlessness of terpinolene was tested on two contrast barley genotypes, Hordeum vulgare L. cv. Cervoise and H. vulgare L. cv. Premium. Firstly, minimum inhibitory concentration (MIC) and half inhibitory concentration (IC50) were detected as 6 and 3 µg µL-1. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was informative about the effects of terpinolene on enniatin and zearalenone biosynthesis in F. avenaceum. Terpinolene leads to the potential decreased enniatin and zearalenone biosynthesis in F. avenaceum. However, no significant differences were recorded for gene expression of aurofusariun biosynthesis. In barley, electroconductivity (EC), catalase (CAT) activity, coupled restriction enzyme digestion-random amplification (CRED-RA), and qRT-PCR assays were tested. No significant ion leakage differences (p&amp;gt;0.05) were detected. Similarly, CAT activity and water loss rate (WLR) values in barley sets were not affected (p&amp;gt;0.05) by terpinolene treatment in majority of experiment sets. Relatively low levels of genomic template instability (75-85%) and epigenetics changes (11-20.6%) were recorded in barley due to terpinolene treatment. WRKY6, WRKY24, and WRKY41 expressions were not significantly changed. The findings showed that terpinolene could be accepted as a potential plant protective agent against phytopathogenic fungi.

Iron toxicity-induced DNA damage, DNA methylation changes, and LTR retrotransposon polymorphisms in Zea mays

The impact of Fe2+ (iron)toxicity on genomic instability, DNA methylation status, and Long Terminal Repeat Retrotransposons (LTR RTs) polymorphisms on Zea mays is unknown. We investigated the toxicity of Fe2+ using Random Amplified Polymorphic DNA (RAPD), Coupled Restriction Enzyme Digestion-Random Amplification (CRED-RA) and Inter Retrotransposon Amplified Polymorphism (IRAP) assays in Zea mays seedlings, respectively. The results indicated that each dose of FeSO4 (50, 100, 200, and 300 mg/L) had a reducing effect on Genomic Template Stability (GTS) and increasing in RAPD pattern changes (DNA damage). The value of DNA methylation rised gradually depending on FeSO4 doses. Moreover, five LTR RTs (Wltr2105, Nikita-N57, Sukkula, Nikita-E2647, and Stowaway) of the maize genome revealed polymorphism in all FeSO4 doses. Furthermore, the present study indicated that there is a relationship between DNA methylation alterations and LTR RTs mobilization. It was concluded that iron caused DNA methylation changes as well as genotoxic damage in the maize genome. Also, considering the increase in some LTR RTs polymorphism we can say that it may be a part of the defense mechanism of the plant during stress

Investigation of DNA Methylation Level in Wheat Genome Exposed to Vanadium by Using CRED-RA Technique

Vanadium (V) has become a serious pollutant due to its widespread use in industry. In this study, DNA methylation changes in Triticum aestivum L. 'Çetinel 2000' seeds were determined by CRED-RA (Coupled Restriction Enzyme Digestion-Random Amplification) analysis. DNA damage and genomic instability were determined by Simple Repeat Inter-Sequence Polymorphism (ISSR) analysis. Vanadium solutions (4.4 mM, 6.6 mM, 8.8 mM) and boric acid (BA) solutions (4 mM and 8 mM) were used. According to CRED-RA analysis, it was observed that V caused more changes in the DNA methylation level in wheat genome compared to BA application. All doses of V caused DNA damage and decreased genomic template stability (GTS). It was determined that the genomic stability was maintained in BA doses applied with V. Genomic stability was maintained at BA doses administered with V. As a result, BA may be an alternative to reduce genotoxic damage to V stress in plants.

Application of Zinc Oxide Nanoparticles and Plant Growth Promoting Bacteria Reduces Genetic Impairment under Salt Stress in Tomato (Solanum lycopersicum L. ‘Linda’)

Salinity is an edaphic stress that dramatically restricts worldwide crop production. Nanomaterials and plant growth-promoting bacteria (PGPB) are currently used to alleviate the negative effects of various stresses on plant growth and development. This study investigates the protective effects of different levels of zinc oxide nanoparticles (ZnO-NPs) (0, 20, and 40 mg L−1) and PGPBs (no bacteria, Bacillus subtilis, Lactobacillus casei, Bacillus pumilus) on DNA damage and cytosine methylation changes in the tomato (Solanum lycopersicum L. ‘Linda’) seedlings under salinity stress (250 mM NaCl). Coupled Restriction Enzyme Digestion-Random Amplification (CRED-RA) and Randomly Amplified Polymorphic DNA (RAPD) approaches were used to analyze changes in cytosine methylation and to determine how genotoxic effects influence genomic stability. Salinity stress increased the polymorphism rate assessed by RAPD, while PGPB and ZnO-NPs reduced the adverse effects of salinity stress. Genomic template stability was increased by the PGPBs and ZnO-NPs application; this increase was significant when Lactobacillus casei and 40 mg L−1 of ZnO-NPs were used.A decreased level of DNA methylation was observed in all treatments. Taken together, the use of PGPB and ZnO-NPs had a general positive effect under salinity stress reducing genetic impairment in tomato seedlings.

Protection of Phaseolus vulgaris L. from Herbicide 2,4-D Results from Exposing Seeds to Humic Acid

Bean (Phaseolus vulgaris L.) is the world’s most significant and basic legume crop for people diet. The intense use of the herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) in bean planting areas can lead to a number of toxicological issues. To prevent such damages, humic acid (HA) may be used to increase plant development by improving nutrient uptake and to play a protecting role against stresses by regulating the antioxidative system of plants. Hence, the aim of the current study was to investigate the impacts of HA on DNA damage levels and DNA methylation changes against 2,4-D stress in the bean. HA treatments were applied to bean seedlings, and the 2,4-D was sprayed on the three-to-four-leaf stages at 2,4-D dose. We used random amplified polymorphic DNA (RAPD) for determining the changes in DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) for DNA methylation changes. According to results, while the genomic template stability (GTS) decreased in the 2,4-D (5, 10, 20 and 40 mg/L) treatments, this value increased comparatively in the 2,4-D applied together with HA (0, 2, 4, 6, 8 and 10 mg/L) treatments. In CRED-RA patterns, as the HA doses were increased, there was generally a decrease in polymorphism rates (DNA methylation changes) caused by 2,4-D applications. Our results have clearly demonstrated that HA has a curative effect up to a level against genotoxic and DNA methylation changes caused by 2,4-D.

Herbisit Stres Korumasında Putresin: Buğdayda Genomik Kararsızlığı Azaltma ve DNA Metilasyon Değişiklikleri

Wheat is one of the most consumed and important food in worldwide. During its growing season, weeds around the cultivated areas grow rapidly and inhibit the normal growth and development, stable yield and quality of wheat seriously. The prevention and removal of weeds are achieved by herbicide treatments. Dicamba is one of the herbicides that is used in agricultural areas which may represent a potential genotoxic risk to off-target crops. The present study was aimed to evaluate the effect of dicamba (0.2, 0.4 and 0.6 ppm) which caused to destabilize of genomic template stability (GTS) and DNA methylation changes in Triticum aestivum L. seedlings by RAPD (Randomly Amplified Polymorphic DNA) and CRED-RA (Coupled Restriction Enzyme Digestion-Random Amplification) techniques, respectively. Also, Full Methylation Ratio and Methylation Ratio were computed according to data of CRED-RA patterns. It was determined that the damage raised with an increasing dose of dicamba. To minimalize the genotoxic effects of dicamba, putrescine (0.01, 0.1 and 1 ppm), a kind of polyamine, were used. Especially, 1 ppm of putrescine was the best concentration to revert the stress-exposed wheat seedlings. Polyamines are positively charged organic cations and hence they interact with negatively charged macromolecules such as DNA and RNA and stabilize them. The results of this experiment have clearly shown that putrescine could be used effectively to protect wheat seedlings from the effects of dicamba on DNA damage and DNA methylation changes, also RAPD and CRED-RA could be used as ideal techniques to get reliable and accurate results.

Investigation of Camphor Effects on Fusarium graminearum and F. culmorum at Different Molecular Levels.

Fusarium graminearum and F. culmorum are phytopathogens, which cause destructive diseases in cereals. Epidemics of these phytopathogens are caused by mycotoxin contamination and the reduction of crop quality. In this study, the alteration due to in vitro camphor treatment on F. culmorum 9F and F. graminearum H11 isolates was investigated in terms of epigenetic, cellular, and transcription levels. Camphor with different concentrations (0.2, 0.4, 0.8, 1, 2, and 4 µg/µL) was applied to potato dextrose agar (PDA) growth media. The minimum inhibitory concentration (MIC) and the half maximal inhibitory concentration (IC50) were calculated as 2 and 1 µg/µL, respectively. hog1, mst20, CAT, POD, mgv1, stuA, and tri5 genes, which are related to various cellular processes and pathogenesis, were examined by qPCR assay. qPCR analysis showed that camphor treatment leads to the downregulation of tri5 expression but the upregulation of the remaining genes. Apoptosis and oxidative stress were confirmed via acridine orange/ethidium bromide (AO/EB) and dichlorofluorescin diacetate (DCF-DA) staining, respectively. Moreover, coupled restriction enzyme digestion-random amplification (CRED-RA) assay, used for DNA methylation analysis, was carried out to evaluate epigenetic alterations. The decrease in genomic template stability (GTS) values, which resulted due to the alterations in random amplified polymorphic DNA (RAPD) profiles caused by camphor treatment, were detected as 97.60% in F. culmorum 9F and 66.27% in F. graminearum H-11. The outer and inner methylated cytosine profiles are determined by CRED-RA assay as type I–IV epigenetic alterations. The outcomes indicated that camphor could lead to alterations at several molecular levels of F. graminearum and F. culmorum.

Salicylic acid and nitric oxide alleviate high temperature induced oxidative damage in Lablab purpureus L plants by regulating bio-physical processes and DNA methylation

Salicylic acid (SA) and sodium nitroprusside (SNP, NO donor) modulates plant growth and development processes and recent findings have also revealed their involvement in the regulation of epigenetic factors under stress condition. In the present study, some of these factors were comparatively studied in hyacinth bean plants subjected to high temperature (HT) environment (40–42 °C) with and without exogenous application of SA and SNP under field condition. Exogenous application of SA and SNP substantially modulated the growth and biophysical process of hyacinth bean plants under HT environment. Exogenous application of SA and SNP also remarkably regulated the activities of antioxidant enzymes, modulated mRNA level of certain enzymes, improves plant water relation, enhance photosynthesis and thereby increasing plant defence under HT. Coupled restriction enzyme digestion-random amplification (CRED-RA) technique revealed that many methylation changes were “dose dependent” and HT significantly increased DNA damages as evidenced by both increase and decrease in bands profiles, methylation and de-methylation pattern. Thus, the result of the present study clearly shows that exogenous SA and SNP regulates DNA methylation pattern, modulates stress-responsive genes and can impart transient HT tolerance by synchronizing growth and physiological acclimatization of plants, thus narrowing the gaps between physio-biochemical and molecular events in addressing HT tolerance.

Alleviative Role of Β-Estradiol Against 2,4-Dichlorophenoxyacetic Acid Genotoxicity on Common Bean Genome

2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the several herbicides that widely used to prevent development of Taraxacum officinale and broadleaf weeds on cereal crops culture. However, it is known that 2,4-D can cause genetic damage to plants at low concentrations as well as produce observable physiological effects. There is no report investigating the effect of mammalian hormones in crops against the applied 2,4-D. Therefore, the present study was aimed at investigating levels of DNA damage, changes in DNA methylation and DNA stability in common bean (Phaseolus vulgaris) exposed to 2,4-D and determine whether β-estradiol has any effect. RAPDs (Randomly Amplified Polymorphic DNA) and CRED-RAs (Coupled Restriction Enzyme Digestion-Random Amplification) techniques were used to define the DNA damage levels and changes in the pattern of DNA methylation. The obtained data demonstrated that 2,4-D led to an increase in RAPDs profile changes (DNA damage), and a reduction in genomic template stability (GTS). The effects caused by 2,4-D were decreased after application with different concentrations of β-estradiol. The results of this study clearly show that β-estradiol could be used function effectively to prevented from the genetic and epigenetic changes caused by 2,4-D herbicide in common bean.

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in Phaseolus vulgaris.

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.

Aluminum-Induced Changes on DNA Damage, DNA Methylation and LTR Retrotransposon Polymorphism in Maize

Aluminum (Al) toxicity is a kind of abiotic stresses that causes several damages on plants. In this study, Al-induced DNA damages, DNA methylation alterations and long terminal repeat retrotransposons (LTR RTs) polymorphisms were evaluated in Zea mays seedlings, using random amplified polymorphic DNA (RAPD), coupled restriction enzyme digestion–random amplification (CRED–RA) and inter-retrotransposon amplified polymorphisms assays, respectively. Our results fairly proved that all doses of \(\hbox {AlCl}_{3}\) (200, 400, 600 and 800 \(\upmu \)M) caused a reduction in genomic template stability and an increase in RAPD band frequencies (DNA damage). Furthermore, there was an increase in DNA methylation ratio based on CRED–RA results. Five LTR RTs (Nikita-E2647, N57 (Nikita), Sukkula, WLTR 2105 and Stowaway) that were determined in barley genome were investigated in maize genome. Polymorphism was determined under Al stress based on the used LTR primers. It was suggested that this mobilization and methylation status under Al stress might be a part of the defense mechanism in stress.

5-Aminolevulinic acid improves DNA damage and DNA Methylation changes in deltamethrin-exposed Phaseolus vulgaris seedlings

Deltamethrin, synthetic type II pyrethroid, is one of the most widely used pesticide in agriculture. Intense use of deltamethrin can cause permanant or temporary damages in nontarget plant species. In this study, we aimed to determine DNA methylation change and DNA damage level in Phaseolus vulgaris seedlings subjected to different concentrations of deltamethrin (0.02, 0.1 and 0.5 ppm). Coupled Restriction Enzyme Digestion-Random Amplification (CRED-RA) was performed to analyze the changes of DNA methylation as well as Randomly Amplified Polymorphic DNA (RAPD) was used for genotoxic influences estimation and genomic stability. The results showed that deltamethrin caused to increase in RAPD profile changes (DNA damage) and reduce in Genomic Template Stability (GTS). GTS declined markedly in relation to increasing concentration of deltamethrin applied. The lowest GTS value (71.4%) observed in 0.5 ppm deltamethrin treatment. Also, DNA hypermethylation was occurred in all treatments. Moreover, alleviative effect of 5-aminolevulinic acid (ALA) (20, 40 and 80 mg/l), one of the plant growth regulators, was tested against the 0.5 ppm deltamethrin. Adverse effects of deltamethrin on GTS decreased after ALA treatments, especially 20 mg/l concentration. As a result, we concluded that ALA has a strong anti-genotoxic agent against deltamethrin and it could be an alternative chemical to reduce genetic damage in plants under deltamethrin stress conditions.

Analyses of somaclonal variation in endosperm-supported mature embryo culture of rye (Secale cereale L.)

ABSTRACTGenetic variations, and especially somaclonal variations, are undesirable in genetic transformation. In this study, random amplified polymorphic DNA (RAPD) and coupled restriction enzyme digestion-random amplification (CRED-RA) markers were used for detection of the variation in calli that were obtained from endosperm-supported mature embryo of rye on Murashige and Skoog (MS) medium containing different auxins (2,4-D, dicamba and picloram) at a range of different concentrations (2, 4, 6, 8, 10 and 12 mg/L). High level of auxins caused a reduction in the genomic template stability (GTS) value. While the highest GTS was observed in the calli maintained on MS medium with 2 mg/L dicamba (98.4%), the calli maintained on MS medium with 12 mg/L picloram were found to show the least GTS (81.7%) when RAPD patterns were analysed. Epigenetic changes were more frequent and variable than genetic changes when compared to RAPD and CRED-RA results. DNA hypermethylation was observed at higher concentrations of 2,4-D and picloram, whereas DNA hypomethylation was observed in dicamba. These results indicate that RAPD and CRED-RA techniques can be used for detection of somaclonal variation in in vitro cultures, which is a fundamental step in plant genetic transformation.

Static magnetic field induced epigenetic changes in wheat callus.

Deoxyribonucleic acid (DNA) is always damaged by endogenous and exogenous factors. Magnetic field (MF) is one of these exogenous factors. When repair mechanisms are not sufficient, mainly because of imbalance in damage or mistakes in repair mechanisms, methylation of DNA results in polymorphism-related abnormalities. In this study, low intensity static magnetic field-induced DNA damage and methylation in wheat calli were investigated by using Random Amplified Polymorphic DNA and Coupled Restriction Enzyme Digestion-Random Amplification techniques. Calli were derived from mature embryos of wheat. Both 7- and 14-day-old wheat calli were exposed to 7 mT (millitesla) static MF for 24, 48, 72, 96, or 120 h of incubation period. The highest change in polymorphism rate was obtained in calli exposed to 7 mT MF for 120 h in both 7- and 14-day-old calli. Increase in MF duration caused DNA hypermethylation in both 7- and 14-day-old calli. Polymorphism and DNA methylation ratio were higher in 7-day-old calli. The highest methylation level with a value of 25.1% was found in 7-day-old calli exposed to MF for 120 h. Results suggested that low intensity static magnetic field may trigger genomic instability and DNA methylation. Bioelectromagnetics. 37:504-511, 2016. © 2016 Wiley Periodicals, Inc.

EGTA, a calcium chelator, affects cell cycle and increases DNA methylation in root tips of Triticum aestivum L.

In this study, when germinated &lt;em&gt;Triticum aestivum&lt;/em&gt; L. seeds were treated with 0, 2, 4 and 6 mM ethyl glycol tetraacetic acid (EGTA), root growth was suppressed and the mitotic index decreased. These inhibitory effects were positively correlated with EGTA concentration. RT-PCR analysis revealed that the expression of several gene markers related to the G1/S transition of the cell cycle were significantly downregulated. Confocal microscopy of Fluo-3/AM-stained roots showed chelation of nearly all of the Ca&lt;sup&gt;2+&lt;/sup&gt; within the root meristematic regions. Both random amplified polymorphic DNA (RAPD) and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques showed significant increases in the levels of genomic DNA polymorphisms and degree of DNA methylation. The study provides information concerning the impact of Ca&lt;sup&gt;2+&lt;/sup&gt; chelator, EGTA, on the growth, expression of cell cycle transition marker genes, and changes in DNA structure and methylation in the wheat roots.

Cytogenetic and epigenetic alterations by cobalt and nickel on Zea mays L

Changes in DNA methylation in Zea mays plantlets in response to cobalt and nickel (5, 10, 20, and 40 mmol L−1) evaluated by the coupled restriction enzyme digestion-random amplification technique revealed dose-related increases in hypermethylation. The effects on mitotic index and phytohormone levels were also determined. Co and Ni caused a decrease in mitotic index and an attenuation in the growth-promoting hormones gibberellic acid, zeatin, and indole acetic acid, while abscisic acid levels rose. These findings have shown that the increase in abscisic acid levels and DNA methylation depend on the concentrations of Co and Ni.