Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 75-81, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-138 Citation: I. Petrescu, I. Sarac, E. Bon- ciu, E. Madosa, C.A. Rosculete, M. Butnariu (2020) Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Oci- mum basilicum L.). Caryologia 73(1): 75-81. doi: 10.13128/caryologia-138 Received: January 9, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 I. Petrescu, I. Sarac, E. Bonciu, E. Madosa, C.A. Rosculete, M. Butnariu. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) Irina Petrescu1, Ioan Sarac1, Elena Bonciu2, Emilian Madosa1, Cata- lin Aurelian Rosculete2,*, Monica Butnariu1 1 Banat’s University of Agricultural Science and Veterinary Medicine “Regele Mihai I al României” Timisoara, Romania 2 University of Craiova, Faculty of Agronomy, Craiova, Romania *Corresponding author. E-mail: catalin_rosculete@yahoo.com Abstract. The cytogenetic study on the meristematic tissues of basil (Ocimum basilicum L.) aimed to evaluate some cytotoxic effects induced by two heavy metals (cadmium - Cd and zinc - Zn) applied in three different concentrations: 10, 50 and 100 ppm. Cytogenetic tests reveal a decrease of the mitotic index and the occurrence of various chromosomal aberrations following heavy metal treatments. The cell division was significantly affected, especially in the case of Cd treatment, which showed the highest degree of toxicity in all variants compared to control variant. Instead, Zn has a lower degree of toxicity but only at concentrations of 50 ppm and 100 ppm. Types of chromosomal aberrations were rela- tively varied, being randomly distributed and concentration dependent, for both Cd and Zn. Were observed cells with large nucleus and disorganized-looking; interphases with pyknotic nucleus; cells with laggard chromosomes, pyknotic and sticky chromosomes, as well as cells with telophase bridge. The results reveal that Cd (at all tested concentrations) and Zn in concentrations higher than 10 ppm exhibit significant cytotoxic potential to Ocimum basilicum L. as a result of the effects reported in cell divisions of the meris- tematic tissues. We can also appreciate that the Ocimum basilicum L. species could be used as a test plant to determine the degree of soil pollution with heavy metals. Keywords. Basil, cadmium, chromosomal aberrations, mitodepresive, zinc. INTRODUCTION Basil (Ocimum basilicum L.) is an herbaceous, annual and aromatic plant belonging to the Lamiaceae family. Basil is currently grown in many other parts of the world. Due to medical and culinary properties, as well as the spiritual and symbolic connotations that have been in the culture of the Romanian people since ancient times, basil is one of the most appreciated aromatic plants in Romania, along with rosemary, mint and sage. The basil is used as a seasonal functional food, being used both in tea and fresh salads, due to its health benefits. Functional foods have an important contribution to improving the quality of life (Butnariu and Caunii, 2013). 76 Irina Petrescu et al. Basil is a plant used not only in the food industry but also in the pharmaceutical and perfume industry. For example, from a pharmaceutical point of view, the basil may be used as a nutritional supplement or thera- peutic drug to protect against aspirin-induced gastric ulcers, a common problem resulting from the use of aspirin (Abd El-Ghffar et al., 2018). Heavy metals are identifiable components in the environment, occurring in significant concentrations and under natural conditions. In the 21st century, the metaliferic loading of air, water, soils, and consequent- ly of plants, animals and the human body became an urgent concern for nature pollution. The aim of this study was to determine how the basil (Ocimum basilicum L.) responded to increasing Cd and Zn concentrations in terms of changes in cellular activity and especially in chromosomes structure. The vegetal meristematic tissues that are used for testing the effects of chemicals on chromosomes should be easy to obtain and less expensive and from this point of view, the basil can be suitable. MATERIALS AND METHODS Plant material Dry seeds of Ocimum basilicum L. belong to the Genovese variety was placed in glass Petri dishes on fil- ter paper. Three treatment variants with 4 replicates were performed for each of the heavy metals experienced (Cd and Zn). Solutions for the treatment of seeds have been obtained by dissolving the respective amounts of heavy metals in distilled water. Equal volumes of the different concentrations of cadmium nitrate Cd(NO3)2 and zinc nitrate Zn(NO3)2 solutions (10, 50 and 100 ppm), respec- tively were administered while the control was treated with distilled water. These concentrations have been established taking into account that basil is an aromatic and medicinal herbaceous plant that reacts easily to any stressful environmental factor, being easily contaminat- ed with heavy metals during growth. The seeds (in the amount of 100 seeds per every variant) were germinat- ed in climatic chamber (model Binder KBF 720, Binder manufacturer, USA), at 22°C. After 72 hours, the basil roots that grew to a length of 1-1.5 cm were cut and pro- cessed for microscopic preparation. Microscopic preparations The biological material were fixed with a mixture of absolute ethyl alcohol and glacial acetic acid in a vol- ume ratio of 3:1 for 24 hours at 6°C in the refrigerator, followed by hydrolysis with 1 N hydrochloric acid for 5 minutes at room temperature. The stage of the meris- tematic roots staining was performed using the Feulgen- Rossenbeck method (Baik et al. 2017; Rosculete CA et al. 2019). Colouring was achieved in a basic fuchsine solu- tion, in concentration of 10%. The microscopic slides were prepared using the squash technique (Asita Okorie et al. 2017). Five slides for each variant were analysed for calcu- lating the mitotic index and the chromosomal aberration frequency. The same slides used to calculate the mitotic index were studied to identify the chromosomal aberra- tion. All slides were examined using a Kruss microscope with digital camera (Kruss manufacturer Hamburg, Germany). Statistical analyses Statistical analysis was done using MS Excel 2007. The data obtained were analysed to determine the effects of Cd and Zn treatments on the mitotic activity to Oci- mum basilicum L. The mean and standard error (SE) were calculated for the mitotic index (MI) and differenc- es between treatment means were compared using the LSD-test at probability level of 0.05% (Botu and Botu, 1997) after ANOVA analysis. The mitotic index (MI) was calculated according to Balog (1982): Total number of cells in division MI (%) = × 100 Total number of analysed cells The index of the chromosomal aberrations (CA) and the percentage of germination (G) were also calculated: Total number of aberrant cells CA (%) = × 100 Total number of cells in division Germinated seed G (%) = × 100 Total seed RESULTS The heavy metals have differently influenced seed germination and root length to Ocimum basilicum L. as can be seen in Table 1. The inhibitory effect on germi- nation is evident to the highest concentration of heavy 77Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) metals (V4/100 ppm) at which the germination percent- age was 23.33% for Cd and 70.00% for Zn. Also the con- centration of 50 ppm, both to Cd and Zn, inhibited the germination of basil seeds in the proportion of 56.66% (Cd) and 74.11% respectively (Zn). The highest germination percentage was recorded to V2/Zn/10 ppm variant (93.33%, percentage equal to that of the untreated control). As for the increase in length of the roots, the highest values were recorded in the variants with the lowest con- centrations of heavy metals: V2/Zn/10 ppm (1.28 ± 0.13 cm) respectively V2/Cd/10 ppm (1.16 ± 0.19 cm). The most powerful inhibitory effect was found in the V4/Cd/100 ppm variant, where the average length of the roots was 0.21 ± 0.04 cm, compared to the control (2.26 ± 0.37). Table 2 presents the results of the effects of Cd and Zn on the mitotic index and the cell division phases to Ocimum basilicum L. A significant reduction (p=0.05) of the mitotic index compared to the control was observed in all treatment variants. Mitotic index value decreased with the increase con- centration of heavy metal solutions. Thus, the intensity of mitotic activity was decreasing in order of treatment with Cd to Zn treatment. The higher mitodepresive effect was found in the treatment of Cd at the concentra- tion of 100 ppm, when MI was 9.23%, i.e. 79.8% lower mitotic activity compared to control variant. However, in all variants treated with Cd, there was a significant decrease in the mitotic index compared to the control (10.26% - V3/50 ppm and 12.64% - V2/10 ppm). In case of the Zn-treated variants, the decrease in the mitotic index was also correlated with the increase in the concentration of heavy metal, but the strongest mitodepresive effect compared to the control was found only at the concentration of 100 ppm (V4 - 17.29% and 50 ppm (V3 - 29.14%). In low concentrations (10 ppm), Zn did not negatively influence the values of the mitotic index as compared to control variant. From point of view of the cell distribution on mitot- ic phases, the highest percentage was registered by pro- phase, followed by telophase, metaphase and anaphases in all the analysed variants, including the control. Frequency of cells in prophase ranged from 73.47- 85.61% for Cd-treated variants and 75.54-84.11% for Zn-treated variants. The frequency of cells in metaphase ranged from 7.33% (V4/Zn/100 ppm) to 10.29% (V2/ Cd/10 ppm). On the other hand, the frequency of cells in anaphase stage ranged from 1.58% (V4/Cd/100 ppm) to 4.82% (V3/Zn/50 ppm). The smallest values of the mitot- ic index of telophase compared to control were record- ed at the highest concentrations of heavy metals: 3.70% (V4/Cd/100 ppm) and 6.54% (V4/Zn/100 ppm) respec- tively. Heav y metals tested induced a high number of mitotic aberrations when compared with control. The increase of mitotic aberrations was dependent on the increasing treatment concentrations (Table 3). The types of chromosomal aberrations identified in meristematic Table 1. Influence of cadmium and zinc on the seeds germination and root length to Ocimum basilicum L. Variants Germination (%) Root length (X ± SE) (cm) V1 (Control) 93.33 2.26±0.37 V2/Cd/10 ppm 80.00 1.16±0.19 V3/Cd/50 ppm 56.66 0.26±0.04 V4/Cd/100 ppm 23.31 0.21±0.04 V2/Zn/10 ppm 93.33 1.28±0.13 V3/Zn/50 ppm 74.11 1.18±0.19 V4/Zn/100 ppm 70.00 0.65±0.09 Table 2. Mitotic index (%) and the cell division phases (%) to Ocimum basilicum L. treated with different concentrations of cadmium and zinc nitrate. Variants TCN MI ± SE % MIP % MIM % MIA % MIT % V1 (Control) 500 45.82±0.68 75.35 6.35 1.93 16.37 V2/Cd/10 ppm 500 12.64±0.42* 73.47 10.29 1.86 14.38 V3/Cd/50 ppm 500 10.26±0.35* 78.36 9.74 1.64 10.26 V4/Cd/100 ppm 500 9.23±0.34* 85.61 9.11 1.58 3.70 V2/Zn/10 ppm 500 43.61±0.63 76.28 7.46 2.01 14.25 V3/Zn/50 ppm 500 29.14±0.60* 75.54 9.22 4.82 10.42 V4/Zn/100 ppm 500 17.29±0.58* 84.11 7.33 2.02 6.54 TCN = Total cells number; MI = Mitotic index; MIP = Mitotic index of Prophase; MIM = Mitotic index of Metaphase; MIA = Mitotic index of Anaphase; MIT = Mitotic index of Telophase; SE = Standard error; * Significant at level 5% (p=0.05). 78 Irina Petrescu et al. cells of Ocimum basilicum L. were interphases with pyk- notic nucleus; pyknotic and sticky chromosomes, cells with laggard chromosomes, as well as cells with telo- phase bridge. The most common types of chromosomal aberra- tions were stickiness and pyknosis while the least fre- quent were bridges. Compared with the control variant, total chromosomal aberration rate recorded insignificant values for all variant exposed to Zn, from 4.58% (V2/ Zn/10 ppm) to 14.51% (V4/Zn/100 ppm). On the other hand, in all variants exposed to Cd treatment total chro- mosomal aberration recorded significantly positive val- ues from 19.51% (V2/Cd/10 ppm) to 39.70% (V4/Cd/100 ppm) respectively. DISCUSSION The contamination of soil and water by heavy met- als is a major environmental problem. In this regard it presents an ecotoxicology risk for food chains because of strongly toxic properties of these elements for all human beings (Lassoued et al. 2014; Bonciu et al. 2018; Coroian et al. 2017; Puia et al. 2019). Understanding the phenom- enon of bioaccumulation of heavy metals in living sub- stance is of extremely complex. This contamination can have very long-term effects (Bilal et al. 2014; Lassoued et al. 2014; Bonciu et al. 2018). Heavy metal pollution is one of the most serious problems of industrialization, who affects significantly soil and biodiversity and its impact continues to increase (Bae et al. 2016), due to these metals’ non-biodegrada- bility and high toxicity (Chul Kong, 2013). Generally, heavy metals are dangerous because they tend to bioac- cumulate and can cause altered physiological and meta- bolic processes to plants or disturbing the metabolism of essential elements (Petrescu et al. 2015; Sarac et al. 2015; Wójcik and Tukiendorf 2014; Butnariu 2012; Mohanpu- ria et al. 2007; Dong et al. 2006). In our experiment, the seed germination of Ocimum basilicum L. was heavily affected by the concentration of heavy metals, especially by Cd, which exhibited the highest degree of toxicity. Instead, zinc recorded a lower degree of toxicity and only at concentrations of 50 ppm and 100 ppm. The inhibition of seed germination with increasing concentration of Cd has been found also in other plants: Vigna radiata (Maheswari et al. 2017); Trit- icum aestivum (Guilherme et al. 2015); Suaeda salsa (Liu et al. 2012); Spartiana alterniflora (Mrozek and Funicelli, 1982), etc. We can appreciate that the highest toxicity in basil seed germination as well as the increase in length of meristematic roots was induced by Cd at the concentra- tion of 100 ppm. In other authors’ opinion, at low con- centrations Cd is not toxic to plants, but at higher con- centrations it is toxic and preferentially accumulates in the meristematic and elongation root zones (Xu et al. 2009; Karcz and Kurtyka, 2007). The heavy metals can disturb the nucleolar cycle. Indirect immunof luores- cence detects nucleolar material and their movement into the cytoplasm following heavy metal stress (Liu et al. 2016). The length roots of Ocimum basilicum L. was influ- enced differently from one heavy metal to another and from one concentration to the other, the most power- Table 3. Type and percentage of mitotic aberrations induced by cadmium and zinc on the meristematic roots to Ocimum basilicum L. Variants Mitotic aberrations (%) Total aberrations (%)PN PC L S B V1 (Control) 0 0 0 1.05 0 1.05 V2/Cd/10 ppm 4.03 5.27 3.89 5.09 1.23 19.51* V3/Cd/50 ppm 6.21 8.63 4.03 7.26 1.58 27.71* V4/Cd/100 ppm 8.15 12.86 6.23 10.04 2.42 39.70* V2/Zn/10 ppm 0 1.04 1.53 2.01 0 4.58 V3/Zn/50 ppm 3.05 1.01 2.03 2.34 0.89 9.32 V4/Zn/100 ppm 2.84 2.63 3.82 4.01 1.21 14.51 PN = Pyknotic Nucleus; PC = Pyknotic Chromosomes; L = Lag- gards; S = Stickiness; B = Bridges; * Significant at level 5% (p=0.05). A B C D Figure 1. Some chromosomal aberrations identified in meristematic cells of Ocimum basilicum L. exposed to Cd and Zn: pyknosis (A); sticky metaphase whit laggards chromosomes (B); bridges (C); dis- turbed telophase whit pyknotic chromosomes (D). 79Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) ful inhibitory effect being found at the highest Cd con- centrations. Results suggest that Cd is highly toxic and can affect the metabolism of meristematic roots. Similar results have been reported by Gharebaghi et al. (2017) to two basil species (Ocimum basilicum L. and Ocimum basilicum var. Purpurescens). Cytogenetic tests on Ocimum basilicum L. show a decrease of the mitotic index following heavy metal treatments. The mitodepresive effect of cadmium was obvious even at the lowest concentration (10 ppm). The other authors results showed that Cd causes irregulari- ties in mitotic activity to Pisum sativum (Fusconi et al. 2007) and Allium sativum (Xu et al. 2009) and can induce increased frequency of the chromosomal aber- rations to Allium cepa (1.5 times more than in control group), while mitotic index was significantly decreased (Evseeva et al. 2001). To Allium cepa, Cd affected the spindle and decreased anaphase and telophase stages while the metaphase stage was increased. In the presence of certain external stimuli, the cel- lular progress can be blocked in one of the phases of the cell cycle or cell division, and their action is called mitoinhibition. Mitogens act to overcome intracellular braking mechanisms that block cell cycle progression, and their action is called mitostimulatory. Any devia- tion from the orderly and directed progression of the cell cycle, and respectively, of mitosis and cytokinesis, is reflected in a state of cytotoxicity and genotoxicity (Bon- ciu et al. 2018; Rosculete E et al. 2019) and some chro- mosomes variation (Bouziane et al. 2019). Some research shows that the electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) are good methods for identifying sites of localiza- tion of heavy metals at the sub-cellular level in cell orga- nelles, cytoplasm or cell walls and clarifying the process involved in their uptake, transport and deposition or detoxification in plant cells (Liu and Kottke 2003, 2004). The results of previous investigations indicate that heavy metals including Cd and Zn at excessive concen- tration can disturb cell division process and induce CA comprising c-mitosis and lagging chromosomes, ana- phase bridges, and chromosome stickiness in the root tips of A. cepa (Liu et al. 1995). During mitosis, metal ions can interfere with the proper positioning of nucleo- lar organizing regions on chromosomes. Under metal stress, an obviously toxic phenomenon appears in nucle- oli of root tips of A. cepa (Bonciu et al. 2018). The results of this study highlight the strong cyto- toxic effect of Cd to Ocimum basilicum L. even at low concentrations of 10 and 50 ppm. The most common types of chromosomal aberrations were stickiness; sticky chromosomes can lead, in opinion of some authors, to cell death (Singh, 2015; Karaismailoğlu, 2017). In most cases the percentages of abnormal mitotic phases were seen to increase with increasing concentration, this result being recorded in other studies also (Samanta and Bandyopadhyay, 2012; Verma et al. 2016; Șuțan et al. 2018). The cytotoxicity effect of Zn occurred only at con- centrations higher than 10 ppm. In low concentrations, Zn did not negatively influence the values of the mitotic index, the percentage of chromosome aberrations being insignificant. Of the two heav y metals tested, Cd showed the highest degree of cy totoxicity and inhibits normal growth to Ocimum basilicum L. The results also suggest that basil may be used for bio-greening of soil, since it absorbs the heavy metals and synthesizes them in the cells. Besides, the decontamination of soils polluted with heavy metals through phytoremediation is one of the cheapest and simplest methods, and from this point of view, the cultivation of basil involves very low costs. REFERENCES Abd El-Ghffar EA,  Al-Sayed E, Shehata SM, Eldahshan OA,  Efferth T. 2018. The protective role of  Ocimum basilicum  L. (Basil) against aspirin-induced gastric ulcer in mice: Impact on oxidative stress, inflamma- tion, motor deficits and anxiety-like behaviour. Food Funct. 9(8):4457-4468. Asita Okorie A, Moramang S, Rants’o T, Magama S. 2017. Modulation of mutagen-induced genotoxicity by vita- min C and medicinal plants in Allium cepa L. Caryo- logia. 70(2):151-165. Bae J, Benoit DL, Watson AK. 2016. Effect of heavy met- als on seed germination and seedling growth of com- mon ragweed and roadside ground cover legumes. Environ Pollut. 213(1):112-118. Baik N, Maamri F, Bandou H. 2017. Karyological study and meiotic analysis of four species of Aegilops (Poaceae) in Algeria. Caryologia. 70(4):324-337 Balog C. 1982. The mitotic index in diploid and triploid Allium roots. Cytologia. 47(3/4):689-697. Bilal E, Guenolé–Bilal I, Bounahkla M, Iatan LE, Macha- do de Mello F, Doumas M, Tahri M, Gallice F, Grail- lot D, Piegay H, Lassoued N. 2014. The freshwater mussels are a good bioindicator for monitoring the water pollution by heavy metal (The Saint– Victor– sur–Loire Lake, France). Banat¢s Journal of Biotech- nology. V(10):5-11. Bonciu E, Firbas P, Fontanetti CS, Wusheng J, Karaismailoğlu MC, Liu D, Menicucci F, Pesnya DS, 80 Irina Petrescu et al. Popescu A, Romanovsky AV, Schiff S, Ślusarczyk J, De Souza CP, Srivastava A, Sutan A  & Papini A. 2018. An evaluation for the standardization of the  Allium cepa  test as cytotoxicity and genotoxicity assay. Caryologia. 71(3):191-209. Botu I, Botu M. 1997. Statistical parameters used in the evaluation of the research of fruit plants. In: Methods and techniques of research in pomiculture. Rm. Val- cea (RO): Conphys Publishing. p. 213-236. Bouziane Z, Issolah R, Tahar A. 2019. Analysis of the chromosome variation within some natural popula- tions of subterranean clover (Trifolium subterraneum L., Fabaceae) in Algeria. Caryologia. 72(4):93-104. Butnariu M. 2012. Vegetable Bioindicators of Heavy Met- al Pollution. J Ecosys Ecograph. 2(5):e114. Butnariu M, Caunii A. 2013. Design management of functional foods for quality of life improvement. Annals of agricultural and environmental medicine. 20(4):736-741. Chul Kong, I. 2013. Joint effects of heavy metal bina- ry mixtures on seed germination, root and shoot growth, bacterial bioluminescence, and gene muta- tion. J Environ Sci. 25(5):889-894. Coroian A, Miresan V, Cocan D, Raducu C, Longodor AL, Pop A, Feher G, Andronie L, Marchis Z. 2017. Physical-chemical parameters and the level of heavy metals in cow milk in the Baia Mare area. Banat¢s Journal of Biotechnology. VIII(16):69-74. Dong J,  Wu FB,  Zhang GP, 2006. Influence of cadmium on antioxidant capacity and four microelement con- centrations in tomato seedlings (Lycopersicon esculen- tum). Chemosphere. 64(10):1659-1666. Evseeva TI,  Geras’kin SA,  Khramova ES. 2001. Cytoge- netic effects of separate and combined action of 232Th and Cd nitrates on Allium cepa root meristem cells. Tsitologiia. 43(8):803-808. Fusconi A, Gallo C, Camusso W. 2007. Effects of cad- mium on root apical meristems of Pisum sativum L.: cell viability, cell proliferation and microtubule pat- tern as suitable markers for assessment of stress pol- lution. Mutat Res. 632 (1-2):9-19. Gharebaghi A, Alborzi Haghighi MH, Arouiee H. 2017. Effect of Cadmium on seed germination and earlier basil (Ocimum basilicum L. and Ocimum basilicum var. Purpurescens) seedling growth. Trakia J Sci. 15(1):1-4. Guilherme de Souza MF, Habyhabanne de Oliveira M, Da Silva E. 2015. Cadmium toxicity on seed germi- nation and seedling growth of wheat Triticum aesti- vum. Acta Sci. 37(4):499-504. Karaismailoğlu MC. 2017. Assessments on the potential genotoxic effects of Fipronil insecticide on Allium cepa somatic cells. Caryologia. 70(4):378–384. Karcz W, Kurtyka R. 2007. Effect of cadmium on growth, proton extrusion and membrane potential in maize coleoptile segments. Biol Plant. 51(4):713-719. Lassoued N, Khelil MN, Rejeb S, Bilal E, Chaouachi M, Rejeb MN, Guenolé–Bilal I. 2014. Transfert heavy metal sewage sludge as fertilizer from soil to ray grass. Banat¢s Journal of Biotechnology. V(9):86-95. Liu D, Jiang W, Wang W, Zhai L. 1995. Evaluation of metal ion toxicity on root tip cells by the Allium test. Israel J Plant Sci. 43:125-133. Liu D, Kottke I. 2003. Subcellular localization of chromi- um and nickel in root cells of Allium cepa by EELS and ESI. Cell Biol Toxicol. 19:299-311. Liu DH, Kottke I. 2004. Subcellular localization of cad- mium in the root cells of Allium cepa by electron energy loss spectroscopy and cytochemistry. J Biosci. 29:329-335. Liu S, Yang C, Xie W, Xia C, Fan P. 2012. The Effects of Cadmium on Germination and Seedling Growth of Suaeda salsa. Procedia Environ Sci 16(0):293-298. Liu X, Shi Q, Zou J, Wang J, Wu H, Wang J, Jiang W, Liu D. 2016. Chromosome and nucleolus morphological characteristics in root tip cells of plants under metal stress. Fresenius Environ Bull. 25:2419–2426. Maheswari UL, Subramani A. 2017. Inhibitory effects of cadmium on seed germination characteristics of Vigna radiata (L.) Wilczek. Int J of Current Research. 9(03):48551-48554. Mohanpuria P,  Rana NK,  Yadav SK. 2007. Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis (L.) O. Kuntze. Environ Toxicol. 22(4):368-374. Mrozek E, Funicelli NA. 1982. Effect of lead and on ger- mination of Spartiana alterniflora seeds at various salinities. Env Exp Bot. 22 (1):23-32. Petrescu I., Sarac I, Chereches B, Madosa E. 2015. Effect of lead on the growth of Coriandrum sativum L. J of Hortic Forest and Biotechnology 19(4):93-95. Puia M, Mihon B, Ionescu B, Butucel E, Chicinas A, Far- cas E, Puskas H, Raducu C, Longodor AL, Coroian A. 2019. Assessing the level of heavy metals in differ- ent geographical areas in Romania. Banat¢s Journal of Biotechnology. X(19):5-10. Rosculete CA, Bonciu E, Rosculete E, Olaru LA. 2019. Determination of the environmental pollution poten- tial of some herbicides by the assessment of cytotoxic and genotoxic effects on Allium cepa. Int J Environ Res Public Health. 16(1):75. Rosculete E, Olaru AL, Rosculete CA, Bonciu E. 2019. Assessment of Cytological Effects of Food Preserva- tive Potassium Metabisulphite to Allium cepa. Ameri- can Journal of Plant Sciences. 11(1):11-23. 81Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) Samanta A, Bandyopadhyay B. 2012. Prevention of Cad- mium induced genotoxicity with Emblica officinalis L. (Amla) in Allium Test.. RJPBCS. 3(2):890-897. Sarac I, Petrescu I, Madosa E, Antonie IF. 2015, Copper effects at seed germination of Secale cereale L. J of Hortic Forest and Biotechnology 19(4):84-88. Singh P. 2015. Toxic effect of chromium on genotoxic- ity and cytotoxicity by use of Allium cepa L. IJREAS. 5(10):1-10. Şuţan NA, Uţă G, Bărbuceanu D. 2018. Oxidative stress and cytogenetic effects in root tip cells of Allium cepa L. induced by alcoholic extracts of Leptinotarsa decemlineata (Say). Caryologia. 71(4):405-413. Verma S, Arora K, Srivastava A. 2016. Monitoring of gen- otoxic risks of nitrogen fertilizers by Allium cepa L. mitosis bioassay. Caryologia. 69(4):343–350. Xu P, Liu D, Jiang W. 2009. Cadmium effects on the organization of microtubular cytoskeleton in inter- phase and mitotic cells of Allium sativum. Biol Plant. 53(2):387-390. Wójcik M,  Tukiendorf A. 2014. Phytochelatin synthesis and cadmium localization in wild type of  Arabidopsis thaliana. Plant Growth Regul. 44(1):71-80. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 1 - 2020 Firenze University Press Karyotypic investigation concerning five Bromus Species from several populations in Iran Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat High genetic diversity and presence of genetic structure characterise the endemics Ruta corsica and Ruta lamarmorae (Rutaceae) Marilena Meloni1, Caterina Angela Dettori2, Andrea Reid3, Gianluigi Bacchetta2,4,*, Laetitia Hugot5, Elena Conti1 Cytogenetic effects of C6H4 (CH3)2 (xylene) on meristematic cells of root tips of Vicia faba L. and mathematical analysis Cihangir Alaca1, Ali Özdemir1, Bahattın Bozdağ2, Canan Özdemir2,* Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. Sazada Siddiqui*, Sulaiman Al-Rumman Temporal Analysis of Al-Induced Programmed Cell Death in Barley (Hordeum vulgare L.) Roots Büşra Huri Gölge, Filiz Vardar* Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. Shahram Mehri*, Hassan Shirafkanajirlou, Iman Kolbadi A new diploid cytotype of Agrimonia pilosa (Rosaceae) Elizaveta Mitrenina1, Mikhail Skaptsov2, Maksim Kutsev2, Alexander Kuznetsov1, Hiroshi Ikeda3, Andrey Erst1,4,* Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) Irina Petrescu1, Ioan Sarac1, Elena Bonciu2, Emilian Madosa1, Catalin Aurelian Rosculete2,*, Monica Butnariu1 Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts Nicoleta Anca Şuţan1,*, Andreea Natalia Matei1, Eliza Oprea2, Victorița Tecuceanu3, Lavinia Diana Tătaru1, Sorin Georgian Moga1, Denisa Ştefania Manolescu1, Carmen Mihaela Topală1 Phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm Bombyx mori induced by microsporidian infection Hungund P. Shambhavi1, Pooja Makwana2, Basavaraju Surendranath3, Kangayam M Ponnuvel1, Rakesh K Mishra1, Appukuttan Nair R Pradeep1,* Electrophoretic study of seed storage proteins in the genus Hypericum L. in North of Iran Parisa Mahditabar Bahnamiri1, Arman Mahmoudi Otaghvari1,*, Najme Ahmadian chashmi1, Pirouz Azizi2 Melissa officinalis: A potent herb against EMS induced mutagenicity in mice Hilal Ahmad Ganaie1,2,*, Md. Niamat Ali1, Bashir A Ganai2 Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers Rayan Partovi1, Alireza Iaranbakhsh1,*, Masoud Sheidai2, Mostafa Ebadi3 In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) Saeed Tarkesh Esfahani1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2 Long-term Effect Different Concentrations of Zn (NO3)2 on the Development of Male and Female Gametophytes of Capsicum annuum L. var California Wonder Helal Nemat Farahzadi, Sedigheh Arbabian*, Ahamd Majd, Golnaz Tajadod A karyological study of some endemic Trigonella species (Fabaceae) in Iran Hamidreza Sharghi1,2, Majid Azizi1,*, Hamid Moazzeni2 Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Kishan Saha*, Rabindra Kumar Sinha, Sangram Sinha