Mutagenic effects of sodium azide on in vitro mutagenesis, polymorphism and genomic instability in wheat (Triticum aestivum L.).

INTRODUCTION: Breeding studies are commonly conducted to develop new cultivars with high yield levels and improved quality traits. Chemically-induced mutations are used to create genetic variations in wheat genomes. Various physical and chemical mutagens are used to increase frequency of mutations and facilitate the selection processes. Sodium azide (SA) is largely employed to induce mutations of the genes regulating essential traits. Such mutations may also elucidate gene functions of the mutant phenotypes. Present experiments were conducted to investigate potential use of conventional chemical mutagenesis technique through SA for mature embryo culture in wheat. METHODS AND RESULTS: Sodium azide mutagenesis was experimented with 4 treatment durations (1, 2, 3 and 4 h) and 5 treatment concentrations (0, 1, 2, 3 and 4 mM). Mature embryos were subjected to experimental treatments to detect optimum doses of mutagenesis and to estimate polymorphism and genomic instability. Primarily, 50% reduction in number of regenerated plants as compared to the control (LD50) was adopted as the optimum dose. Based on LD50 criterion, the optimum value was achieved at 1 h duration of 4 mM SA concentration. Afterwards, inter-primer binding site markers were applied to investigate polymorphism and genomic instability in the regenerated plants. CONCLUSIONS: Present findings revealed that efficiency of chemical mutagenesis could be improved through the use of molecular technology and such mutations may assist plant breeders in developing high-yield cultivars.

Phylogenetic relationship among taxa in the genus Adonis L. collected from Türkiye based on nrDNA internal transcribed spacer (ITS) markers.

BACKGROUND: Genus Adonis L. contain approximately 40 annual and perennial species, which are widely distributed in the temperate zones of Asia and Europe, and less frequently in southwestern Asia, northern Africa and the Mediterranean region. The aim of the study was to evaluate the phylogenetic relationship among Adonis taxa collected from Türkiye based on nrDNA Internal transcribed spacer (ITS) markers. METHODS: Samples of 64 individual genotypes from 21 populations of 10 Adonis taxa were collected from different regions of the country during vegetation period between 2014 and 2018. ITS1, ITS4, P16 and P25 primers within ITS technique was used to genotype the plant materials. Then, genotypic data was used to estimate magnitude and organization of infraspecific variation in different populations of Adonis. RESULTS: About 600 bp DNA sequences were obtained from each 64 Adonis genotypes belonging to 21 different populations. The dendrogram obtained from Adonis taxa and out-group sequences had two large main groups. While the out-group species were placed in the first large main group, the sect. Consiligo (perennial) and sect. Adonis (annuals) were placed in different sub-groups of the second large main group. Genetic similarity among Adonis taxa varied between A. microcarpa and A. dentata (98.46%). Principal component analysis indicated that two important components in Adonis taxa genotypes. The expected heterozygosity ranged from 0.0252 (sub-population A) to 0.3460 (sub-population C), with an average of 0.1154. In addition, population differentiation measurements (Fst) ranged from 0.0025 (sub-population C) to 0.9016 (sub-population A) with a relatively high average 0.6601. CONCLUSIONS: Present analyses revealed that phylogenetic classification (grouping) of Adonis taxa largely depended on morphological structure and present ITS primers were quite efficient in putting forth the genetic diversity of such species. The results of this study suggested that ITS markers could be used in the identification of genetic diversity among the Adonis taxa. The results obtained from molecular data can be used to explore the genetic variation pattern, population structure, and the evolutionary history of genus Adonis in the future.

Exploring genetic diversity and Population structure of five Aegilops species with inter-primer binding site (iPBS) markers.

BACKGROUND: Turkey is not only a center of origin for wheat, but also contains wild forms of various cereals. Turkey, located in the Fertile Crescent, has conserved its genetic richness to the present day. The aim of the study was to investigate the genetic diversity of 70 wild wheat species, to evaluate the structure of diversity in germplasm and to generate useful data for further breeding programs. METHODS AND RESULTS: Genetic diversity and population structure of 70 wild wheat species (Ae. cylindrica, Ae. geniculata, Ae. triuncialis, T. dicocoides, Ae. columnaris) collected from Eastern and Southeastern Anatolia regions of Turkey were investigated in this study with the use of inter-primer binding site (iPBS) markers. Of 35 iPBS primers used, 11 yielded a total of 61 alleles. Number of alleles per marker varied between 2 (iPBS-2085) and 9 (iPBS-2394) with an average value of 5.55. Polymorphic information content (PIC) values varied between 0.22 and 0.47, with an average value of 0.35. Average number of effective alleles (Ne) was identified as 1.9488, Nei's genetic diversity (H) as 0,4861 and Shannon's information index (I) as 0.6791. Cluster analysis through unweighted pair-group mean average (UPGMA) method revealed that 70 wild wheats were divided into three main clusters. Genetic similarity between the genotypes, calculated with the use of NTSYS-pc software, varied between 19% (YB2 and YB70) and 98% (YB66 and YB67). Principal coordinate analysis (PCoA) revealed that three principal coordinates explained 62.33% of total variation. Moreover, population structure analysis showed that all genotypes formed three sub-populations. Expected heterozygosity values varied between 0.2666 (the first sub-population) and 0.2330 (third sub-population), with an average value of 0.2500. Average population differentiation measurement (Fst) was identified as 0.3716 for the first sub-population, 0.3930 for the second subpopulation and 0.4804 for the third sub-population. CONCLUSIONS: Based on present findings population structure of 70 wild wheat genotypes collected from Eastern and Southeastern Anatolia regions of Turkey were successfully characterized with the use of iPBS markers. Present findings suggested that iPBS-retrotransposon markers could reliably be used to elucidate genetic diversity of Aegilops genotypes.

Improvement of Drought Tolerance by Exogenous Spermidine in Germinating Wheat (Triticum aestivum L.) Plants Is Accompanied with Changes in Metabolite Composition.

Drought is one of the most important environmental factors reducing the yield and production of crops, including wheat. Polyamines are closely associated with plant stress tolerance. The present study investigated the mechanisms through seed germination with spermidine protecting wheat varieties from drought stress. In the first experiment, the effects of spermidine on the germination of wheat varieties, namely Rakhshan, Mihan, Sirvan and Pishgam, were investigated in three drought levels, namely 0, -2, and -4 MPa induced by polyethylene glycol 6000. Analysis of variance indicated that spermidine, drought stress and interaction between varieties and drought stress were significant for all traits, and with severity of stress, all traits significantly decreased. In the second experiment, detailed gene expression and non-targeted metabolomics analyses were carried out using the Rakhshan and Mihan varieties after germination, with or without spermidine treatment and/or drought stress. According to the biomass parameters, the Mihan variety showed relatively better growth compared to the other variety, but the Rakhshan one showed more pronounced responses at gene expression level to exogenous spermidine than the Mihan variety. Overall, these results showed that spermidine increased the drought tolerance of wheat at the germination stage, due to specific role of polyamine metabolism in the development of effective responses under drought stress.

Effects of mammalian sex hormones on in vitro organogenesis of common bean (Phaseolus vulgaris L.).

Beans are an important plant species and are one of the most consumed legumes in human nutrition, especially as a protein, vitamin, mineral, and fiber source. Common bean (Phaseolus vulgaris L.) is a plant that also has an important role in natural nitrogen fixation. Currently, in vitro regeneration and micropropagation applications are limited in relation to genetic factors in bean Accordingly, there is great need to optimize micropropagation and tissue culture methods of the bean plant. To date, the effect of mammalian sex hormones (MSH) on in vitro conditions in P. vulgaris L. is poorly understood. This study examined the effects of different types of explants (embryo, hypocotyl, plumule, and radicle), MSH type (progesterone, 17 ß-estradiol, estrone, and testosterone), and MSH concentration (10-4, 10-6, 10-8 and 10-10 mmol L-1) on the responding explants induction rate (REI), viability of plantlets rate (VPR), shoot proliferation rate (SPR), root proliferation rate (RPR), and callus induction rate (CIR). The effects of mammalian sex hormones, concentrations, explant type, and their interactions were statistically significant (p ≤ 0.01) in all examined parameters. The best explants were embryo and plumule. Our results showed that the highest REI rate (100%) was recorded when 10-10 mmol L-1 of all MSH was applied to MS medium using the plumule explant. The highest VPR (100%) was obtained when 10-10 mmol L-1 of all MSH was applied to MS medium using the plumule explant. The highest root proliferation rates (77.5%) were recorded in MS medium supplemented with 10-8 mmol L-1 17ß-estradiol using embryo explant. The highest percentage of shoot-forming explants (100%) generally was obtained from embryo and plumule cultured in the MS culture medium with low MSH concentration. In addition, the highest CIR (100%) was obtained from embryo and plumule explant cultured in MS medium containing 10-10 mmol L-1 of all MSH types. In conclusion, we observed that mammalian sex hormones may be used in bean in vitro culture.

Genetic Diversity and Population Structure in Türkiye Bread Wheat Genotypes Revealed by Simple Sequence Repeats (SSR) Markers.

Wheat genotypes should be improved through available germplasm genetic diversity to ensure food security. This study investigated the molecular diversity and population structure of a set of Türkiye bread wheat genotypes using 120 microsatellite markers. Based on the results, 651 polymorphic alleles were evaluated to determine genetic diversity and population structure. The number of alleles ranged from 2 to 19, with an average of 5.44 alleles per locus. Polymorphic information content (PIC) ranged from 0.031 to 0.915 with a mean of 0.43. In addition, the gene diversity index ranged from 0.03 to 0.92 with an average of 0.46. The expected heterozygosity ranged from 0.00 to 0.359 with a mean of 0.124. The unbiased expected heterozygosity ranged from 0.00 to 0.319 with an average of 0.112. The mean values of the number of effective alleles (Ne), genetic diversity of Nei (H) and Shannon's information index (I) were estimated at 1.190, 1.049 and 0.168, respectively. The highest genetic diversity (GD) was estimated between genotypes G1 and G27. In the UPGMA dendrogram, the 63 genotypes were grouped into three clusters. The three main coordinates were able to explain 12.64, 6.38 and 4.90% of genetic diversity, respectively. AMOVA revealed diversity within populations at 78% and between populations at 22%. The current populations were found to be highly structured. Model-based cluster analyses classified the 63 genotypes studied into three subpopulations. The values of F-statistic (Fst) for the identified subpopulations were 0.253, 0.330 and 0.244, respectively. In addition, the expected values of heterozygosity (He) for these sub-populations were recorded as 0.45, 0.46 and 0.44, respectively. Therefore, SSR markers can be useful not only in genetic diversity and association analysis of wheat but also in its germplasm for various agronomic traits or mechanisms of tolerance to environmental stresses.

Identification of Novel QTLs Associated with Frost Tolerance in Winter Wheat (Triticum aestivum L.).

Low temperature (cold) and freezing stress is a major problem during winter wheat growth. Low temperature tolerance (LT) is an important agronomic trait in winter wheat and determines the plants' ability to cope with below-freezing temperatures; thus, the development of cold-tolerant cultivars has become a major goal of breeding in various regions of the world. In this study, we sought to identify quantitative trait loci (QTL) using molecular markers related to freezing tolerance in winter. Thirty-four polymorphic markers among 425 SSR markers were obtained for the population, including 180 inbred lines of F12 generation wheat, derived from crosses (Norstar × Zagros) after testing with parents. LT50 is used as an effective selection criterion for identifying frost-tolerance genotypes. The progeny of individual F12 plants were used to evaluate LT50. Several QTLs related to wheat yield, including heading time period, 1000-seed weight, and number of surviving plants after overwintering, were identified. Single-marker analysis illustrated that four SSR markers with a total of 25% phenotypic variance determination were linked to LT50. Related QTLs were located on chromosomes 4A, 2B, and 3B. Common QTLs identified in two cropping seasons based on agronomical traits were two QTLs for heading time period, one QTL for 1000-seed weight, and six QTLs for number of surviving plants after overwintering. The four markers identified linked to LT50 significantly affected both LT50 and yield-related traits simultaneously. This is the first report to identify a major-effect QTL related to frost tolerance on chromosome 4A by the marker XGWM160. It is possible that some QTLs are closely related to pleiotropic effects that control two or more traits simultaneously, and this feature can be used as a factor to select frost-resistant lines in plant breeding programs.

Investigation of the Influence of Polyamines on Mature Embryo Culture and DNA Methylation of Wheat (Triticum aestivum L.) Using the Machine Learning Algorithm Method.

Numerous factors can impact the efficiency of callus formation and in vitro regeneration in wheat cultures through the introduction of exogenous polyamines (PAs). The present study aimed to investigate in vitro plant regeneration and DNA methylation patterns utilizing the inter-primer binding site (iPBS) retrotransposon and coupled restriction enzyme digestion-iPBS (CRED-iPBS) methods in wheat. This investigation involved the application of distinct types of PAs (Put: putrescine, Spd: spermidine, and Spm: spermine) at varying concentrations (0, 0.5, 1, and 1.5 mM). The subsequent outcomes were subjected to predictive modeling using diverse machine learning (ML) algorithms. Based on the specific polyamine type and concentration utilized, the results indicated that 1 mM Put and Spd were the most favorable PAs for supporting endosperm-associated mature embryos. Employing an epigenetic approach, Put at concentrations of 0.5 and 1.5 mM exhibited the highest levels of genomic template stability (GTS) (73.9%). Elevated Spd levels correlated with DNA hypermethylation while reduced Spm levels were linked to DNA hypomethylation. The in vitro and epigenetic characteristics were predicted using ML techniques such as the support vector machine (SVM), extreme gradient boosting (XGBoost), and random forest (RF) models. These models were employed to establish relationships between input variables (PAs, concentration, GTS rates, Msp I polymorphism, and Hpa II polymorphism) and output parameters (in vitro measurements). This comparative analysis aimed to evaluate the performance of the models and interpret the generated data. The outcomes demonstrated that the XGBoost method exhibited the highest performance scores for callus induction (CI%), regeneration efficiency (RE), and the number of plantlets (NP), with R2 scores explaining 38.3%, 73.8%, and 85.3% of the variances, respectively. Additionally, the RF algorithm explained 41.5% of the total variance and showcased superior efficacy in terms of embryogenic callus induction (ECI%). Furthermore, the SVM model, which provided the most robust statistics for responding embryogenic calluses (RECs%), yielded an R2 value of 84.1%, signifying its ability to account for a substantial portion of the total variance present in the data. In summary, this study exemplifies the application of diverse ML models to the cultivation of mature wheat embryos in the presence of various exogenous PAs and concentrations. Additionally, it explores the impact of polymorphic variations in the CRED-iPBS profile and DNA methylation on epigenetic changes, thereby contributing to a comprehensive understanding of these regulatory mechanisms.

Machine Learning Analysis of the Impact of Silver Nitrate and Silver Nanoparticles on Wheat (Triticum aestivum L.): Callus Induction, Plant Regeneration, and DNA Methylation.

The objective of this study was to comprehend the efficiency of wheat regeneration, callus induction, and DNA methylation through the application of mathematical frameworks and artificial intelligence (AI)-based models. This research aimed to explore the impact of treatments with AgNO3 and Ag-NPs on various parameters. The study specifically concentrated on analyzing RAPD profiles and modeling regeneration parameters. The treatments and molecular findings served as input variables in the modeling process. It included the use of AgNO3 and Ag-NPs at different concentrations (0, 2, 4, 6, and 8 mg L-1). The in vitro and epigenetic characteristics were analyzed using several machine learning (ML) methods, including support vector machine (SVM), random forest (RF), extreme gradient boosting (XGBoost), k-nearest neighbor classifier (KNN), and Gaussian processes classifier (GP) methods. This study's results revealed that the highest values for callus induction (CI%) and embryogenic callus induction (EC%) occurred at a concentration of 2 mg L-1 of Ag-NPs. Additionally, the regeneration efficiency (RE) parameter reached its peak at a concentration of 8 mg L-1 of AgNO3. Taking an epigenetic approach, AgNO3 at a concentration of 2 mg L-1 demonstrated the highest levels of genomic template stability (GTS), at 79.3%. There was a positive correlation seen between increased levels of AgNO3 and DNA hypermethylation. Conversely, elevated levels of Ag-NPs were associated with DNA hypomethylation. The models were used to estimate the relationships between the input elements, including treatments, concentration, GTS rates, and Msp I and Hpa II polymorphism, and the in vitro output parameters. The findings suggested that the XGBoost model exhibited superior performance scores for callus induction (CI), as evidenced by an R2 score of 51.5%, which explained the variances. Additionally, the RF model explained 71.9% of the total variance and showed superior efficacy in terms of EC%. Furthermore, the GP model, which provided the most robust statistics for RE, yielded an R2 value of 52.5%, signifying its ability to account for a substantial portion of the total variance present in the data. This study exemplifies the application of various machine learning models in the cultivation of mature wheat embryos under the influence of treatments and concentrations involving AgNO3 and Ag-NPs.

SSR-Based Molecular Identification and Population Structure Analysis for Forage Pea (Pisum sativum var. arvense L.) Landraces.

Plant genetic diversity has a significant role in providing traits that can help meet future challenges, such as the need to adapt crops to changing climatic conditions or outbreaks of disease. Our aim in this study was to evaluate the diversity of 61 forage pea specimens (P. sativum ssp. arvense L.) collected from the northeastern Anatolia region of Turkey using 28 simple sequence repeat (SSR) markers. These primers generated a total of 82 polymorphic bands. The number of observed alleles (Na) per primer varied from 2 to 4 with a mean of 2.89 alleles/locus. The mean value of expected heterozygosity (Exp-Het = 0.50) was higher than the mean value of observed heterozygosity (Obs-Het = 0.22). The mean of polymorphic information content (PIC) was 0.41 with a range of 0.03-0.70. The mean number of effective alleles (Ne) was found to be 2.15, Nei's expected heterozygosity (H) 0.49, and Shannon's information index (I) 0.81. Cluster analysis through the unweighted pair-group mean average (UPGMA) method revealed that 61 forage pea landraces were divided into three main clusters. Genetic dissimilarity between the genotypes, calculated with the use of NTSYS-pc software, varied between 0.10 (G30 and G34) and 0.66 (G1 and G32). Principal coordinate analysis (PCoA) revealed that three principal coordinates explained 51.54% of the total variation. Moreover, population structure analysis showed that all genotypes formed three sub-populations. Expected heterozygosity values varied between 0.2669 (the first sub-population) and 0.3223 (third sub-population), with an average value of 0.2924. Average population differentiation measurement (Fst) was identified as 0.2351 for the first sub-population, 0.3838 for the second sub-population, and 0.2506 for the third sub-population. In general, current results suggest that SSR markers could be constantly used to illuminate the genetic diversity of forage pea landraces and can potentially be incorporated into future studies that examine the diversity within a larger collection of forage pea genotypes from diverse regions.

iPBS-Retrotransposon Markers in the Analysis of Genetic Diversity among Common Bean (Phaseolus vulgaris L.) Germplasm from Türkiye.

Beans are legumes that play extremely important roles in human nutrition, serving as good sources of protein, vitamins, minerals, and antioxidants. In this study, we tried to elucidate the genetic diversity and population structure of 40 Turkish bean (Phaseolus vulgaris L.) local varieties and 5 commercial cultivars collected from 8 different locations in Erzurum-Ispir by using inter-primary binding site (iPBS) retrotransposon markers. For molecular characterization, the 26 most polymorphic iPBS primers were used; 52 bands per primer and 1350 bands in total were recorded. The mean polymorphism information content was 0.331. Various diversity indices, such as the mean effective allele number (0.706), mean Shannon's information index (0.546), and gene diversity (0.361) revealed the presence of sufficient genetic diversity in the germplasm examined. Molecular analysis of variance (AMOVA) revealed that 67% of variation in bean germplasm was due to differences within populations. In addition, population structure analysis exposed all local and commercial bean varieties from five sub-populations. Expected heterozygosity values ranged between 0.1567 (the fourth sub-population) and 0.3210 (first sub-population), with an average value of 0.2103. In contrary, population differentiation measurement (Fst) was identified as 0.0062 for the first sub-population, 0.6372 for the fourth subpopulations. This is the first study to investigate the genetic diversity and population structure of bean germplasm in Erzurum-Ispir region using the iPBS-retrotransposon marker system. Overall, the current results showed that iPBS markers could be used consistently to elucidate the genetic diversity of local and commercial bean varieties and potentially be included in future studies examining diversity in a larger collection of local and commercial bean varieties from different regions.

Determining Genetic Diversity and Population Structure of Common Bean (Phaseolus vulgaris L.) Landraces from Türkiye Using SSR Markers.

Assessment of genetic diversity among different varieties helps to improve desired characteristics of crops, including disease resistance, early maturity, high yield, and resistance to drought. Molecular markers are one of the most effective tools for discovering genetic diversity that can increase reproductive efficiency. Simple sequence repeats (SSRs), which are codominant markers, are preferred for the determination of genetic diversity because they are highly polymorphic, multi-allelic, highly reproducible, and have good genome coverage. This study aimed to determine the genetic diversity of 40 common bean (Phaseolus vulgaris L.) landraces collected from the Ispir district located in the Northeast Anatolia region of Türkiye and five commercial varieties using SSR markers. The Twenty-seven SSR markers produced a total of 142 polymorphic bands, ranging from 2 (GATS91 and PVTT001) to 12 (BM153) alleles per marker, with an average number of 5.26 alleles. The gene diversity per marker varied between 0.37 and 0.87 for BM053 and BM153 markers, respectively. When heterozygous individuals are calculated proportional to the population, the heterozygosity ranged from 0.00 to 1.00, with an average of 0.30. The expected heterozygosity of the SSR locus ranged from 0.37 (BM053) to 0.88 (BM153), with an average of 0.69. Nei's gene diversity scored an average of 0.69. The polymorphic information content (PIC) values of SSR markers varied from 0.33 (BM053) to 0.86 (BM153), with an average of 0.63 per locus. The greatest genetic distance (0.83) was between lines 49, 50, 53, and cultivar Karacasehir-90, while the shortest (0.08) was between lines 6 and 26. In cluster analysis using Nei's genetic distance, 45 common bean genotypes were divided into three groups and very little relationship was found between the genotypes and the geographical distances. In genetic structure analysis, three subgroups were formed, including local landraces and commercial varieties. The result confirmed that the rich diversity existing in Ispir bean landraces could be used as a genetic resource in designing breeding programs and may also contribute to Türkiye bean breeding programs.

The Effect of Mammalian Sex Hormones on Polymorphism and Genomic Instability in the Common Bean (Phaseolus vulgaris L.).

Mammalian sex hormones are steroid-structured compounds that support the growth and development of plants at low concentrations. Since they affect the physiological processes in plants, it has been thought that mammalian sex hormones may cause modifications to plant genomes and epigenetics. This study aims to determine whether different mammalian sex hormones (17 ß-estradiol, estrogen, progesterone, and testosterone) in several concentrations (0, 10-4, 10-6, and 10-8 mM) affect genetic or epigenetic levels in bean plants, using in vitro tissue cultures from plumule explants. We investigated levels of DNA damage, changes in DNA methylation and DNA stability in common bean exposed to mammalian sex hormones (MSH) using inter-primer binding site (iPBS) and Coupled Restriction Enzyme Digestion-iPBS (CRED-iPBS) assays, respectively. The highest rate of polymorphism in iPBS profiles was observed when 10-4 mM of estrogen (52.2%) hormone was administered. This finding indicates that genetic stability is reduced. In the CRED-iPBS profile, which reveals the methylation level associated with the DNA cytosine nucleotide, 10-4 mM of estrogen hormone exhibited the highest hypermethylation value. Polymorphism was observed in all hormone administrations compared to the control (without hormone), and it was determined that genomic stability was decreased at high concentrations. Taken together, the results indicate that 17 ß-estradiol, estrogen, progesterone, and testosterone in bean plants affect genomic instability and cause epigenetic modifications, which is an important control mechanism in gene expression.

Effects of Zinc, Copper and Iron Oxide Nanoparticles on Induced DNA Methylation, Genomic Instability and LTR Retrotransposon Polymorphism in Wheat (Triticum aestivum L.).

Nanomaterials with unique and diverse physico-chemical properties are used in plant science since they improve plant growth and development and offer protection against biotic and abiotic stressors. Previous studies have explored the effects of such nanomaterials on different plant mechanisms, but information about the effects of nanomaterials on induced DNA methylation, genomic instability and LTR retrotransposon polymorphism in wheat is lacking. Therefore, the present study highlights the key role of nanoparticles in DNA methylation and polymorphism in wheat by investigating the effects of ZnO, CuO and γ-Fe3O4 nanoparticles (NPs) on mature embryo cultures of wheat (Triticum aestivum L.). Nanoparticles were supplemented with Murashige and Skoog (MS) basal medium at normal (1X), double (2X) and triple (3X) concentrations. The findings revealed different responses to the polymorphism rate depending on the nanoparticle type and concentration. Genomic template stability (GTS) values were used to compare the changes encountered in iPBS profiles. ZnO, CuO and γ-Fe3O4 NPs increased the polymorphism rate and cytosine methylation compared to the positive control while reducing GTS values. Moreover, non-γ-Fe3O4 NPs treatments and 2X ZnO and CuO NP treatments yielded higher polymorphism percentages in both MspI- and HpaII-digested CRED-iPBS assays and were thus classified as hypermethylation when the average polymorphism percentage for MspI digestion was considered. On the other hand, the 3X concentrations of all nanoparticles decreased HpaII and MspI polymorphism percentages and were thus classified as hypomethylation. The findings revealed that MS medium supplemented with nanoparticles had epigenetic and genotoxic effects.