Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 141-148, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1091 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Özgün Tuna-Gülören, Ferhan Korkmaz, Meltem Erdir, Ebru Ataşlar (2021) Cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some Gypsophila L. species using Allium cepa assay. Caryologia 74(2): 141-148. doi: 10.36253/caryolo- gia-1091 Received: September 22, 2020 Accepted: July 20, 2021 Published: October 08, 2021 Copyright: © 2021 Özgün Tuna-Gülören, Ferhan Korkmaz, Meltem Erdir, Ebru Ataşlar. 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, distribution, and reproduction in any medium, 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. Cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some Gypsophila L. species using Allium cepa assay Özgün Tuna-Gülören1, Ferhan Korkmaz2*, Meltem Erdir2, Ebru Ataşlar2 1 Eskişehir Osmangazi University, Graduate School of Natural and Applied Sciences, Department of Biology, 26040, Eskişehir, Turkey 2 Eskişehir Osmangazi University, Faculty of Science and Letters, Department of Biology, 26040, Eskişehir, Turkey *Corresponding author; e-mail ferhanatahan@gmail.com; ferhanka@ogu.edu.tr Abstract. In this study, the cytotoxic and genotoxic effects of Gypsophila perfoliata L. var. perfoliata, Gypsophila perfoliata L. var. araratica Kit Tan, Gypsophila pilosa Hud- son and Gypsophila osmangaziensis Ataşlar & Ocak plant extracts have been exam- ined using Allium assay. Methanol extracts of plants have been prepared in 4 differ- ent concentrations (0.625 mg/ml, 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml). After the onion roots were treated in these concentrations of plant extracts for 24 hours and 48 hours, mitosis slides were prepared from these root tips. With the data obtained by examining the slides, mitotic index (%) and chromosome aberration (%) values have been calculated. Distilled water has been used as the control group. It was found that mitotic index and chromosome aberration values of all species showed significant dif- ferences compared to the control group in the extract concentration range of 1.250– 5.000 mg/ml. It has been also determined that the most widely observed chromosome aberrations were disturbed metaphase, sticky metaphase, c-metaphase, disturbed ana- phase and anaphase bridge. Keywords: Gypsophila L., Caryophyllaceae, methanol extracts, Allium test, mitotic index, chromosome aberrations. INTRODUCTION Gypsophila L., is a genus of Caryophyllaceae family, which is found between 30° and 60° latitudes and is represented with nearly 150 species in this region (Antkowiak and Dyba 2004, Schweingruber 2007). Their under- ground parts, containing saponins (4-25%), were used for washing wool and silk, giving halva its fragility, and as fire extinguisher agents, while in medi- cine they were believed to have expectorant, laxative, and emetic properties (Kołodziej et al. 2018). Gypsophila which is the third largest genus of the Caryophyllaceae in Turkey, is represented by nearly 60 genus, 67% of which are endemic. Turkey 142 Özgün Tuna-Gülören et al. is located in Gypsophila species’ main centers of variation and is one of the most important locations for their gene diversity (Barkoudah 1962, Bittrich 1993, Davis 1967, Davis et al.1988, Güner et al. 2000, Güner et al. 2012). Studies on the saponin content of Gypsophila species have shown that the ratio of pure saponin can reach up to 25% in some species, for example G. bicolor (Freyn. & Sint.) Grossh. (Sezik 1982). Furthermore, one of the recent studies related to this subject was conducted by Kołodziej et al. (2018). In the study, 7 Gypsophila spe- cies with a potential use in the pharmaceutical industry for saponin production have been examined. The study shows that those species which were most abundant in saponins were G. acutifolia Steven ex Spreng., G. pacifica Kom., G. scorzonerifolia Ser. and G. zhegulensis Kras- nova. On the other hand, Kołodziej et al. (2019) studied antimicrobial and antioxidant activities of the G. panicu- lata L., G. pacifica, and G. scorzonerifolia. The results of this study showed that Gypsophila had valuable bioactive properties and the hexane extracts showed higher anti- fungal and antibiotic activity. Also, Kotelnaya et al. (2019) mentioned plant saponins exert cardiotonic, neurotrophic, hypotensive, tonic, hypocholesterolemic and antiscle- rotic, diuretic, corticotropic, adaptogenic, sedative, anti- ulcer genic and mild laxative effects on human subjects. Natural products are potential anticancer agent sources and these are considered as alternatives to synthetic anti- cancer drugs which have disadvantages such as toxicity, high costs and negative side effects. It is stated that nearly 3000 plant types produce metabolites that have antican- cer activity (Hartwell 1971). Furthermore, 350 plant types have been defined as potential source of agents against cancer (Graham et al. 2000). Due to the reason that mod- ern drugs are costly, the demand for natural plant prod- ucts has increased in clinical applications. Usage of plant extracts as traditional drugs in the health area is a prac- tice which is as old as the human history. Allium test is an important and well known test system which is used in determining safe concentrations in the therapeutic use of plant extracts. (Roy and Roy 2019). Besides, Allium test is recommended as the standard test, especially for cyto- genotoxicity in environmental monitoring (Fiskesjö 1985). This test has advantages such as being useful, having low costs, and showing good correlation with mammalian test systems. Results of Allium test are also compatible with test systems composed of prokaryotes and/or eukaryotes (Çelik and Aslantürk 2007). In this study, the cytotoxic and genotoxic effects of methanol extracts from four Gypsophila species, two of which are endemic, were investigated using Allium cepa root tip meristem cells. The species that were chosen for this study were: Gypsophila perfoliata L. var. perfoliata, Gypsophila perfoliata L. var. araratica Kit Tan (endemic), Gypsophila pilosa Hudson and Gypsophila osmangazien- sis Ataşlar & Ocak (endemic). There are some reports about cytotoxicity of different Gypsophila species such as Gypsophila bicolor (Freyn & Sint.) Grossh. and Gypsophi- la ruscifolia Boiss. (Rad et al. 2018). To the best of our knowledge this is the first report on the cy totoxicity and genotoxicity of methanolic extracts of vegetative parts ofall of the studied species and we hope our research will helpshed light on other studies about Gypsophlia species and provide data for future studies on medicinal plants. MATERIALS AND METHODS Plant samples In this study, four Gypsophila species were used. Plant samples were collected from two different loca- tions. Voucher specimens were deposited at the Herbar- ium of the Department of Biology, Eskişehir Osmangazi University (OUFE). G. perfoliata var. perfoliata and G. perfoliata var. araratica samples were collected from B3 Afyon: Emirdağ-Çifteler junction point, steppe, 1035 m, 39°22´34.27” N and 31°02´15.21” E, 23.08.2009 (Ataşlar, OUFE 15942, OUFE 15943). On the other hand, the samples of G. pilosa and G. osmangaziensis were col- lected from B3 Eskişehir: Eskişehir Osmangazi Uni- versity campus area, west sides, open stone areas, 810 m, 39°45´10.6164” N and 30°29´16.9620” E, 19.06.2009 (Ataşlar, OUFE 15944, OUFE 15945). These sites lie in the Central Anatolian Region which is characterized by its continental climate, extreme heat and virtually no rainfall in summers with winters recieving heavy, lasting snows. Long term avarage of annual temperature is around 10 °C while humidity is around 65% (Apaydin et al. 2011) Preparation of plant extracts The extraction procedure of the plant samples was performed according to Ataşlar et al. (2019). Briefly, fresh vegetative parts of the studied species were treated with 0.8% Tween 80, tap water and distilled water and then were dried at room temperature. The dried samples were grinded to obtain the powder form of the stud- ied Gypsophila species. Ten grams of powdered samples were extracted with 250 ml 80% petroleum ether to remove their oily constituents with soxhlet apparatus. The degreased plant materials were dried overnight and were extracted with 250 ml 70% methanol. At this step, the flasks were mixed for half an hour in the blender 143Cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some Gypsophila species using Allium cepa assay consecutively three times. All of the methanol extracts were combined and filtrated with Whatman 1 filter paper. The methanol in the total extract was removed by rotary evaporator. The obtained dry extracts (yield= 6.90%, 5.00%, 8.96% and 7.36%, respectively) were main- tained at 4 °C for future use in genotoxicity studies. Genotoxicity Test Genotoxicities of plant extracts used in the study have been determined with Allium Test. For this purpose, the methanol extracts of G. perfoliata var. perfoliata, G. perfoliata var. araratica, G. pilosa and G. osmangaziensis species were prepared in 4 different concentrations (0.625 mg/ml, 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml). Distilled water has been used as negative control. For each concentration, 6 onions rooted in distilled water for 24 hours. Onion roots were left to interact with extract concentrations at 25 ± 1 ºC for 24-48 hours. At the end of 24 and 48 hours, the root tips were cut and included in the Farmer fixative (3: 1 ethyl alcohol: glacial acetic acid) and stored at +4 ºC. Fixative residues that could be found on the root tips were removed by washing with distilled water. Afterwards, the root tips have been hydrolyzed for 12 minutes in 1 N HCl acid at 60 ºC water bath. After the hydrolysis, the roots were submerged in Feulgen dye and chromosomes were stained for 1 hour with the Schiff reaction. After this procedure, slides were prepared from the dyed root tips (1-2 mm) by crushing and spreading. (Fiskesjö 1985, Rank et al. 2002, Rank 2003). Slides were phtographed using a light microscope, Nikon Eclips 80i. Mitotic Index % (MI%). Mitotic Index (MI%) have been calculated with the following for- mula (Sehgal et al. 2006) and Chromosome Aberration % (CA%) have been calculated with the other formula (Ivanova et al. 2003). Mitotic Index (MI) (%)=(P+M+A+T)/(Total number of cells)×100 Chromosome Aberration (CA) (%)=(Number of abnor- mal cells)/(Number of cells in mitosis)×10 where (P+M+A+T) is the sum of all cells in phase as pro- phase, metaphase, anaphase and telophase, respectively. Statistical Analysis The results have been interpreted statistically, using independent sampling T test, one-way variance analysis (ANOVA) and Tukey test. RESULTS AND DISCUSSION The values of MI% (Mitotic Index %) and CA% (Chromosome Aberration %) of the methanol extracts of Gypsophila species used in the study are given in Table 1. In the 24-hour treatment, 5.000 mg / ml concentra- tionss significantly decreased MI% in G. osmangiensis and G. pilosa extracts compared to the control group (P <0.05). MI% values have statistically decreased to a sig- nificant extent compared to the control group, at 2.500 mg/ml and 5.000 mg/ml concentrations of G. perfoliata var. perfoliata extract and at 1.250 mg/ml, 2.500 mg/ ml and 5.000 mg/ml concentrationsof G. perfoliata var. araratica extract (P<0.05). As a result of treatment for 48 hours, none of the MI% values relating to G. osmanga- ziensis extract showed a significant difference compared to the control group. For G. pilosa extract, 0.625 mg/ml, 1.250 mg/ml concentrations have increased MI% com- pared to the control group, whereas 2.500 mg/ml has reduced it. When MI% values of G. perfoliata var. perfo- liata have been considered, it was determined that only 5.000 mg/ml has shown a significant decrease compared to the control group and for G. perfoliata var. araratica extract, it was determined that 2.500 mg/ml and 5.000 mg/ml concentrations have shown a significant decrease compared to the control group (P<0.05). The mitotic index is a cytogenetic parameter that helps measure the proliferation (M phase) of mitot- ic cells in the cell cycle, and inhibition of the mitot- ic index is considered as cell death. (Öney-Birol and Gündüz 2019). Taking this into consideration, the result of the treatment for 24 hours in the G. Osmangaziensis extract which had a concentration of 5.000 mg/ml, MI% decreased to a significant extent, which means that it showed a cytotoxic effect. However, no such effect could be observed from 48 hours treatment in the same con- centration for 48 hours. In the case of G. Pilosa, in the samples that were treated for 24 hours, the 5.000 mg/ ml concentration decreased MI% and in the samples that were treated for 48 hours, 2.500 mg/ml concentra- tion decreased MI%, meanwhile 1.250 mg/ml and 0.625 mg/ml concentrations increased MI%. When we look at the experiment data of the G. perfoliata var. perfoliata extract samples, it is seen that after 24 hours treatment the 2.500 mg/ml and the 5.000 mg/ml concentrations decreased MI% meanwhile in the samples that were treated for 48 hours only the 5.000 mg/ml concentra- tion decreased MI%. The treatment of samples in G. perfoliata var. araratica for 24 hours showed a decrease in MI% in 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml concentrations while treatment for 48 hours only showed a decrease in MI% in 2.500 mg/ml and 5.000 mg/ml 144 Özgün Tuna-Gülören et al. Table 1. Mitotic index and chromosome aberration types and their frequency induced by Gypsophila extracts in root tip cells of Allium cepa. D ur at io n Plant species Concentration of treatment (mg/ml) Total number of cells scored Number of dividing cells Number of abnormal cells Mitotic index (%)±SD Chromosome aberration types (%) The frequency of total chromosome aberration (CA %)±SD D is tu rb ed m et ap ha se St ic ky m et ap ha se c- m et ap ha se D is tu rb ed a na ph as e A na ph as e br id ge “2 4 ho ur s G. osmangaziensis Control 12959 740 21 5.76±2.69 43 48 0 9 0 2.65±1.05 0.625 13498 902 29 6.72±1.40 45 38 3 3 11 3.25±085 1.250 12704 966 53 7.69±1.88 43 30 4 17 7 5.50±1.41 b 2.500 12740 739 46 5.81±1.19 54 31 0 13 2 6.16±1.49 b 5.000 11916 364 30 2.90±1.25a 53 43 0 4 0 6.09±4.71 b G. pilosa Control 11094 588 0 5.41±1.02 0 0 0 0 0 0.00±0.00 0.625 12763 636 0 5.06±0.75 0 0 0 0 0 0.00±0.00 1.250 12698 648 0 5.18±0.81 0 0 0 0 0 0.00±0.00 2.500 11397 511 30 4.44±1.35 43 27 0 27 3 6.28±2.53 b 5.000 11827 231 28 2.02±1.29 a 39 29 0 32 0 14.15±10.97 b G. perfoliata var. perfoliata Control 13001 687 0 5.43±1.47 0 0 0 0 0 0.00±0.00 0.625 12756 905 0 7.11±1.24 0 0 0 0 0 0.00±0.00 1.250 12202 695 0 5.70±0.60 0 0 0 0 0 0.00±0.00 2.500 13271 389 19 2.86±1.22 a 63 27 0 5 5 5.51±5.52 b 5.000 10574 281 16 2.61±1.10 a 13 50 6 31 0 6.09±4.26 b G. perfoliata var. araratica Control 10753 849 0 7.83±1.39 0 0 0 0 0 0.00±0.00 0.625 11338 820 0 7.38±1.88 0 0 0 0 0 0.00±0.00 1.250 12230 689 13 5.85±1.68 a 38 31 8 23 0 2.05±1.64 b 2.500 12744 609 23 4.80±0.87 a 48 30 0 13 9 3.88±2.18 b 5.000 11810 337 37 2.92±1.02 a 54 16 3 27 0 10.61±5.39 b 48 h ou rs G. osmangaziensis Control 12243 550 1 4.29±2.82 100 0 0 0 0 1.19±2.92 0.625 12628 612 27 4.88±1.47 52 33 0 11 4 3.78±3.23 1.250 12149 520 45 4.28±1.07 20 31 0 49 0 8.60±7.62 b 2.500 12429 558 15 4.53±1.75 20 53 0 27 0 3.19±2.82 5.000 13159 502 13 3.84±1.39 31  69   0  0  0 2.44±2.07 G. pilosa Control 12904 661 0 5.22±0.77 0 0 0 0 0 0.00±0.00 0.625 11542 811 0 7.07±0.78 b 0 0 0 0 0 0.00±0.00 1.250 10919 587 0 5.28±1.25 b 0 0 0 0 0 0.00±0.00 2.500 10854 340 15 3.13±1.30 a 40 47 0 13 0 5.11±3.02 b 5.000 11708 238 12 1.97±1.23 61 23 8 8 0 4.48±2.68 b G. perfoliata var. perfoliata Control 14266 726 0 5.11±1.22 0 0 0 0 0 0,00±0,00 0.625 12969 827 0 6.39±1.00 0 0 0 0 0 0.00±0.00 1.250 11701 704 0 5.99±0.71 0 0 0 0 0 0.00±0.00 2.500 12947 520 33 4.10±1.99 49 42 0 9 0 5.43±3.70 b 5.000 11895 155 16 1.26±0.70 a 31 38 6 25 0 9.44±6.02 b G. perfoliata var. araratica Control 10731 675 0 6.15±1.85 0 0 0 0 0 0.00±000 0.625 12499 732 0 5.94±0.82 0 0 0 0 0 0.00±0.00 1.250 12960 793 22 6.11±0.69 18 59 0 23 0 2.76±1.50 b 2.500 12507 523 29 4.28±0.83 a 48 28 3 21 0 5.56±3.84 b 5.000 12121 362 33 2.94±1.03 a 52 36 0 12 0 10.95±6.51 b Means in a column followed by the same superscript letters are significantly different according to their control groups (P<0.05, one-way ANOVA, Tukey post hoc test; a: reduction of MI and CA, b: increase in MI and CA). 145Cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some Gypsophila species using Allium cepa assay concentrations. Thus, it can be said that at the end of 48 hours a decrease in the cytotoxicity of these plant extracts is observed. When reviewing the literature, it can be seen that Caryophyllaceae, which the studied plant species are included, constitute a wide family that has cytotoxic species. Cytotoxic activity of G. bicolor and G. ruscifo- lia’s methanol extracts on MCF-7 (human breast adeno- carcinoma), A-549 (non-small cell lung carcinoma) and AGO1522 (human fibroblast) cell lines were examined using MTT method and it was determined to be cyto- toxic for MCF-7 (human breast adenocarcinoma) cells Figure 1. Chromosome aberrations in Allium cepa root meristem cells after treatment with extracts of Gypsophila species. a: Disturbed met- aphase (G. perfoliata var. araratica-24 hours-5.000 mg/ml); b: Sticky metaphase (G. pilosa-24 hours-2.500 mg/ml); c: c-metaphase (G. per- foliata var. araratica-24 hour-5.000 mg/ml); d: Disturbed anaphase (G. perfoliata var. perfoliata-48 hours-5.000 mg/ml); e: Anaphase bridge (G.osmangaziensis-24 hours-2.500 mg/ml);f: normal metaphase and; g: normal anaphase. 146 Özgün Tuna-Gülören et al. (Rad et al. 2018). In another study, it was determined that Gypsophila saponins showed a synergistic cytotox- icity in macrophage-like (PMA-treated) U937 cells with type I RIPs saporin and his-tagged saporin (Weng et al. 2008). In a study conducted by Gevrenova et al. (2014), it was determined that saponins were plant glycosides hav- ing one or more sugar chains being covalently linked as a steroid or triterpenoid aglycon or aglycon and it was emphasized that Caryophyllaceae was an extremely rich source of triterpene saponin. In this study, it was shown for the first time that extracts of Caryophyllaceae species including Saponaria officinalis L., Gypsophila trichotoma Wend. and Dianthus sylvestris Wulffen, had impact on the vitality of mammalian monocytes/macrophage cell lines and that they induced apoptosis through caspase-3 activation (Gevrenova et al. 2014). In the literature research that has been made, no study was found using the Allium Test in determining the cytotoxicity of plant extracts belonging to Gypsophila species. Another parameter determined in this studyis geno- toxicity. For this purpose, CA% values were calculated. These values are shown in Table 1. In the chromosome analysis of the four Gypsophila species being studied, aberrations in the form of disturbed metaphase, sticky metaphase, c-metaphase, disturbed anaphase and ana- phase bridge were observed. These aberrations are shown in Figure 1. Similar results were observed in the literature. Ždralović and colleagues found that methanol extracts of the Plantago lanceolata L. plant also caused chromosome aberrations like sticky metaphase and ana- phase bridge in Allium cepa chromosomes (Ždralović et al. 2019). The deterioration of microtubules frequently causes mitotic aberrations like laggard chromosomes result- ed from disturbed anaphase-telophase (Amer and Ali, 1986). Various abnormalities like lagging chromosomes, vagrants, distrubed metaphases and anaphases, and chromosome stickiness can be induced by the inhibi- tion of proteins’ effect on the spindle function (Tkalec et al. 2009). And chromosome stickiness, usually of an irreversible type leading to cell death, is definite proof of genotoxicity (Khanna, N., & Sharma, S. (2013). Moreo- ver, vagrant chromosomes and c-metaphases increase the risk for aneuploidy, whereas chromosome bridges indicate the clastogenic effect caused by chromosome breaks (Leme and Marin-Morales 2009). When plant extracts’ genotoxicity was examined, distilled water was used as negative control. In the 24 hours treatment, it was seen that CA% values of G. osmangaziensis extract with concentrations of 1.250 mg/ml and 2.500 mg/ml 5.000 mg/ml increased sig- nificantly compared to the control group. For G. pilosa extract, concentrations of 2.500 mg/ml and 5.000 mg/ ml increased CA%. For G. perfoliata var. perfoliata, con- centrations of 2.500 mg/ml and 5.000 mg/ml increased CA% significantly compared to the control group and for G. perfoliata var. araratica, concentrations of 1.250 mg/ml, 2.500 mg/ml and 5.000 mg/ml increased CA% significantly compared to the control group (P<0.05). As a result of treatment for 48 hours, it was seen that for G. osmangaziensis extract, only the concentrationof 1.250 mg/ml increased CA% statistically and that for G. pilosa, concentrations of 2.500 mg/ml and 5.000 mg/ ml statistically increased the CA% value. For G. perfo- liata var. perfoliata, concentrations of 2.500 mg/ml and 5.000 mg/ml increased CA% values and for G. perfoliata var. araratica, concentrations of 1.250 mg/ml, 2.500 mg/ ml and 5.000 mg/ml increased CA values. According to these results; after 24 hour treatment, only the 1250 mg/ml, 2250 mg/ml and 5000 mg/ml concentrations of G. Osmangaziensis extract and after 48 hour treat- ment, only the 1250 mg/ml concentration of the same extract exhibited a significant increase. For the other 3 plant extracts; a significant difference in CA% increase between the 24 hour and 48 hour treatments could not be observed. Thus it can be said that treatment period does not effect genetoxicity to a great extent concentra- tion. In the literature review that has beenmade, no oth- er genotoxicity study was found on these plant extracts. The fact that G. Osmangaziensis is a new specie (Ataşlar and Ocak 2005), emphasizes the importance of data sub- mitted in this study. However, it is considered appropri- ate for in vitro tests such as this to be evaluated with other test systems. Our future studies will aimto support the data in this study related to the species investigated with other in vitro test systems. ACKNOWLEDGEMENTS Authors are thankful to Dr. Mustafa Yamaç for his contribution in writing and Mrs. Demet Büyükemir for her technical support during preparation. REFERENCES Amer, S. M., Ali, E. M. 1986. Cytological effects of pes- ticides XVII. Effect of the insecticide dichlorvos on root-mitosis of Vicia faba. Cytologia 51(1): 21-25. Apaydin, H., Anli, A. S., Ozturk, F., 2011. Evaluation of topographical and geographical effects on some cli- 147Cytotoxic and genotoxic effects of methanol extracts of vegetative parts of some Gypsophila species using Allium cepa assay matic parameters in the Central Anatolia Region of Turkey. Int J Climatol 31(9): 1264-1279. Ataşlar E, Ocak A, 2005. Gypsophila osmangaziensis (Car- yophyllaceae), a new species from Central Anatolia, Turkey. Ann Bot Fenn. 42: 57–60. Ataşlar E, Tuna-Gülören Ö, Filik-İşcen C, İlhan S. 2019. In vitro antimicrobial and antioxidant activities of four Gypsophila L. species plant extracts. Fresen Environ Bull. 28 (3): 1841–1851. Antkowiak W, Dyba S. 2004. Effect of differentiated NPK fertilization on saponin content in roots of tall and spread gypsophyll (Gypsophila paniculata L. and G. repens L.) in the second and third years of cultiva- tion. Prace z Zakresu Nauk Rolniczych. 97: 9–14. Barkoudah, YI. 1962. A revision of Gypsophila, Bolanthus, Ankyropetalum and Phryna. Wentia 9: 1–203. Bittrich V. 1993. Caryophyllaceae. In: Kubitzki, K., Rohw- er, J. G. and Bittrich, V. (eds.) The families and gen- era of vascular plants, Flowering plants, Dicotyle- dons, Magnoliid, Hamamelid and Caryophyllid fami- lies, Vol. 2. Springer-Verlag, Berlin. Çelik T, Aslantürk Ö. 2007. Cytotoxic and genotoxic effects of Lavandula stoechas aqueous extracts. Bio- logia. 62(3): 292–296. Davis PH. 1967. Flora of Turkey and the East Aegean Islands, Vol. 2. Edinburgh University Press, Edin- burgh. Davis PH, Mill RR, Tan K. 1988. Flora of Turkey and the East Aegean Islands, Vol. 10. Edinburgh University Press, Edinburgh. Fiskesjö G. 1985. The Allium test as a standard in envi- ronmental monitoring. Hereditas. 102(1): 99–112. Gevrenova R, Joubert O, Mandova T, Zaiou M, Chapleur Y, Henry M. 2014. Cytotoxic effects of four Caryo- phyllaceae species extracts on macrophage cell lines. Pharm Biol. 52(7): 919–925. Graham, J. G., Quinn, M. L., Fabricant, D. S. and Farns- worth, N. R. 2000. Plants used against cancer an extension of the work of Jonathan Hartwell. J. Eth- nopharmacol. 73: 347–377. Güner A, Özhatay N, Ekim T, Başer KHC. 2000. Flora of Turkey and the East Aegean Islands, Vol. 11. Edin- burgh University Press, Edinburgh. Güner A, Aslan S, Ekim T, Vural M, Babaç M (eds.). 2012. Turkey Plant List (Vascular Plants). Nezahat Gökyiğit Botanical Garden and Flora Research Asso- ciation Publication, İstanbul-Turkey. Hartwell, J. L. 1971. Plants used against cancer. A survey. Lloydia 34: 386–425. Ivanova, E., Staikova, T., Velcheva, I., & Kostadinovn, K. (2003). Somatostatic Effect of Heavy Metal Con- taminated Waters In The Region of The Town Of Panagjurishte, Bulgaria. Journal of Environmental Protection, 4(2): 284-287. Kamali M, Mosaddegh M, Delnavazi MR, Shahrestani R, Malek Mohammadi M, Hamzeloo-Moghadam M. 2020. Evaluation of the cytotoxicity activity of Gyp- sophila ruscifolia by bioassay-guided fractionation. Res J Pharmacogn. 7(4): 75–82. Khanna, N., Sharma, S. 2013. Allium cepa root chromosom- al aberration assay: a review. Indian Journal of Pharma- ceutical and Biological Research, 1(03): 105-119. Kołodziej B, Okoń S, Nucia A, Ociepa T, Luchowska K, Sugier D, Gevrenova R, Henry M. 2018. Morphologi- cal, chemical, and genetic diversity of Gypsophila L. (Caryophyllaceae) species and their potential use in the pharmaceutical industry. Turk J Bot. 42: 257–270. Kołodziej B, Sęczyk L, Sugier D, Kędzia B, Chernetskyy M, Gevrenova R, Henry M. 2019. Determination of the yield, saponin content and profile, antimicrobial and antioxidant activities of three  Gypsophila  species. Ind. Crops Prod. 138: Article 111422. Kotelnaya YI, Alekhina EA, Efremov AN, Bolotova YV, Guselnikova MV, Nikolaenko SA, Cezary Toma C. 2019. Notes on the saponins in the plants of the fam- ily Hydrocharitaceae. Bot Pac. 8(1): 57–61. Leme, D.M.; Marin-Morales, M.A. 2009. Allium cepa test in environmental monitoring: A review on its appli- cation. Mutat Res Rev Mutat Res, 682, 71–81. Öney-Birol S, Gündüz F. 2019. Assessment of cytotoxic and genotoxic effects of L-carnitine in Allium cepa root tip assay. KSÜ J Agr Natur. 22(4): 650–658. Rad AS, Esmaeili S, Motamed SM, Hamzeloo-Moghadam M. 2018. Cytotoxicity of two Gypsophila species to human breast adenocarcinoma (MCF-7). Int Pharm Acta. 1(1): 82–82. Rank J, Lopez LC, Nielsen MH, Moretton J. 2002. Geno- toxicity of maleic hydrazide, acridine and DEHP in Allium cepa root cells performed by two different lab- oratories. Hereditas. 136: 13–18. Rank J. 2003. The method of Allium anaphase-telophase chromosome aberration assay. Ekologija. 1: 38–42. Roy A, Roy S. 2019. Assessment of cytotoxic effects of aqueous and methanolic leaf extracts of Cleroden- drum inerme (L.) Gaertn. and C. viscosum Vent. using Allium Test. Cytologia. 84(1): 73–76. Schweingruber FH. 2007. Stem anatomy of Caryophyl- laceae. Flora. 202: 281–292. Sehgal, R., Roy, S., Kumar, V. L. 2006. Evaluation of cyto- toxic potential of latex of Calotropis procera and Podophyllotoxin in Allum cepa root model. Biocell, 30(1): 9. Sezik E. 1982. The origin and the quality of the Turkish soaproots. J Fac Pharm Ankara Univ. 12: 41–64. 148 Özgün Tuna-Gülören et al. Tkalec M., Malaric K., Pavlica M., Pevalek-Kozlina B., Vidakovic-Cifrek Z. 2009. Effects of radiofrequency electromagnetic fields on seed germination and root meristematic cells of Allium cepa L. Mutat Res, Genet Toxicol Environ Mutagen 672: 76-81. Weng A, Melzig MF, Bachran C, Fuchs H. 2008. Enhancement of saporin toxicity against U937 cells by Gypsophila saponins. J. Immunotoxicol. 5(3): 287– 292. Ždralović, A., Mesic, A., Eminović, I., Parić A. 2019. Cytotoxic and genotoxic activity of Plantago major L. extracts. Caryologia 72(3): 35-40. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 1 - 2021 Firenze University Press