Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 89-99, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1571 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Made Pharmawati, Ni Nyoman Wirasiti, Luh Putu Wrasiati (2022) Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate. Caryologia 75(2): 89-99. doi: 10.36253/caryologia-1571 Received: February 08, 2022 Accepted: March 24, 2022 Published: September 21, 2022 Copyright: © 2022 Made Pharmawati, Ni Nyoman Wirasiti, Luh Putu Wrasiati. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) 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. ORCID MP: 0000-0002-3064-4582 Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate Made Pharmawati1,*, Ni Nyoman Wirasiti1, Luh Putu Wrasiati2 1 Biology Study Program, Faculty of Mathematics and Natural Sciences, Universitas Udayana, Jalan Raya Kampus Unud, Jimbaran, Kecamatan Kuta Selatan, Kabupaten Badung, Bali 80361, Indonesia 2 Agroindustrial Technology Study Program, Faculty of Agricultural Technology, Universi- tas Udayana, Jalan Raya Kampus Unud, Jimbaran, Kecamatan Kuta Selatan, Kabupaten Badung, Bali 80361, Indonesia *Corresponding author. E-mail: made_pharmawati@unud.ac.id Abstract. Several environmental pollutants can cause damage to chromosomes, one of which is the heavy metal NiNO3. Some plant extracts have antigenotoxic proper- ties that result in a decrease in chromosomal damage. Member of flowering plants that need to be tested is seagrass. One seagrass species is Enhalus acoroides which was found to contain phytochemical compounds. This study aimed to analyse the geno- toxic effect and the potential of encapsulated E. acoroides leaf extract as antigenotoxic against nickel nitrate NiNO3. The extraction was conducted using a mixture of chloro- form and ethanol, and crude extract encapsulated using maltodextrin and tween 80. Chromosomal aberrations were evaluated using the squash technique of Allium cepa var. aggregatum root tips. Triphenyltetrazolium chloride and Evans Blue staining were used to observe mitochondrial and apoptotic activities. The results showed that at higher concentrations (250 ppm and 500 ppm), the encapsulated E. acoroides extract decreased mitotic indices; however, no chromosome aberration observed. NiNO3 itself induced a genotoxic effect as observed by low mitotic index and a high percentage of chromosome aberration. The modulation of NiNO3 effect by adding the encapsulated E. acoroides extract at low concentration (100 ppm) increased mitotic index compared to treatment with Ni alone, but did not reduce chromosome aberration. Simultaneous encapsulated E. acoroides extract and Ni treatment, significantly reduced nuclear frag- mentation and nuclear lesion. The encapsulated E. acoroides extract can repair several types of nuclear damage but cannot minimise chromosomal damage. Keywords: chromosome aberration, Enhalus acoroides, heavy metal, nuclear abnor- mality, seagrass. INTRODUCTION Heavy metals are hazardous inorganic environmental pollutants due to their toxicity. However, when present in low amounts, several heavy metals such as Cu, Fe, Mn, Co, Zn, and Ni are required for plants and animals as 90 Made Pharmawati, Ni Nyoman Wirasiti, Luh Putu Wrasiati micronutrients (Singh et al. 2020). Recently, due to high industrial activities and the extensive use of fertilizer and pesticides, heavy metals are present in enormous amounts in the environment, posing a serious global environmental threat. One of the most common heavy metal contaminants found in the environment is nickel (Ni), along with Arsenic (As), Cadmium (Cd), Chromium (Cr), Copper (Cu), Mercury (Hg), Lead (Pb), and Zinc (Zn) (Huang et al. 2020). Nickel is widely distributed in the environ- ment including air, water, soil, and biological materials. It is mainly derived from natural sources such as wind- blown dust, resulting from the weathering of rocks and soils, forest fires, and volcanic activity. Nickel is also present in the environment due to the combustion of coal, diesel oil, and fuel oil, as well as the incineration of trash and sewage (Cempel and Nikel 2006). In plants, high nickel concentrations can inhibit growth by caus- ing oxidative damage and disrupting nutrient uptake and translocation (Amjad et al. 2020). Nickel has also been reported to have cytotoxic and mutagenic effects in plants (Gantayat et al. 2018). Natural ingredients with bioactive compounds that can fight mutagenic and carcinogenic effects are now getting more and more attention. Compounds capable of reducing the mutagenicity of physical and chemi- cal mutagens are referred to as antimutagens. However, considering that all mutagens are genotoxic, then com- pounds that reduce DNA damage caused by genotoxic agents are also called antigenotoxic agents (López-Rome- ro et al. 2018). For example, aqueous extracts of medici- nal plants, Spondias mombin, Nymphea lotus and Luffa cylindrica reduced chromosomal and nuclear aberration induced by PbNO3 in A. cepa root tip cells (Oyeyemi and Bakare 2013). Butanol and ethyl acetate fractions of Par- kinsonia aculeata L. leaf extract demonstrated the most significant reduction in chromosomal abnormalities in A. cepa cells treated with maleic hydrazide, indicat- ing that they had chemo-preventive efficacy (Sharma et al. 2018). Previously, Sharma et al. (2012) reported that using the A. cepa root chromosomal aberration assay, the chloroform extract of Brasicca juncea seeds possesses antigenotoxic potential against mercury-induced geno- toxicity. Exploration of antitoxic properties from natural products has also been done at marine organisms such as Ulva fasciata (Rodeiro et al. 2015), Sargassum sp. (Kil- awati and Islamy 2019). However, limited studies were conducted in seagrass. Seagrass are flowering plants that grow in a marine environment. One of the seagrass spe- cies is Enhalus acoroides (L.f.) Royle. Enhalus acoroides is tropical seagrass, a member of the Family of Hydro- charitaceae, found throughout the Indo-Pacific region, including southern Japan, Southeast Asia, northern Australia, southern India, and Sri Lanka (Short and Waycott 2010). In Indonesia, this species is distributed widely in Papua, North Maluku, Ambon, Sulawesi, Bali, Java, Borneo, and Sumatra in Indonesia (Kiswara and Hutomo 1985). Extract of E. acoroides leaves contains phytochemical compounds such as phenols, flavonoids, and tannins as well as several pigments including chlo- rophyll, lutein, pheophytin, and b-carotene (Pharmawati and Wrasiati 2020). It has been known that flavonoids, phenolic compounds and pigments have antioxidant activity. To extend self-life and protect oxidative stabil- ity of plant extract, microencapsulation is often applied (Yusop et al. 2017) using colloidal particles such as maltodextrin, Arabic gum, or chitosan (Özkan and Bilek 2014, Šturm et al, 2019). The aim of this study was to analysed the genotoxic effect of encapsulated E. acoroides leaves extracts and its antigenotoxic potential against heavy metal nickel nitrate Ni(NO3)2.6H2O using Allium cepa var. aggrega- tum root tips assay. MATERIALS AND METHODS Sample Collection, Extraction and Encapsulation Leaves of E . acoroides were col lected f rom Semawang Beach, Denpasar, Bali, Indonesia. The meth- ods of den Hartog and Kuo (2006) and McKenzie and Yoshida (2009) were used to identify E. acoroides based on morphological traits. The voucher specimen was deposited in the Herbarium Biology Udayana (HBU- MP10), Biology Study Program, Universitas Udayana. Leaves were washed in running water, cut into 10 cm long, and air-dried for three days. The leaves were then further dried for one day using an oven at 50°C. Using a blender, the dried leaves were mashed and sieved through a 60-mesh sieve. Using 200 mL of chloroform: ethanol at a 9:1 (v/v) ratio, up to 20 g of dried leaves powder was extracted. The extraction was done using a Soxhlet extractor. The solvent was filtered using What- man filter paper, and the filtrate was vacuum evaporated using an IKA® RV10 rotary evaporator at 40°C and 100 mbar (Pharmawati and Wrasiati 2020) The encapsulation of crude extract was conducted using a 20% maltodextrin solution. As much as 10% extract of E. acoroides and 2% tween 80 were mixed with the encapsulated solution and homogenized at 6000 rpm for 30 minutes. After that, the mixture was dried up to 8% moisture content, mashed with a blender and then sieved through a 60 mesh sieve (Sulistyadewi et al. 2014). 91Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate Treatment of Allium cepa var. aggregatum root The base of A. cepa var. aggregatum bulbs were soaked in water to induce roots. When the length of the root was approximately 1 cm, bulbs were transferred to a glass jar containing treatment solutions. The treatments were 30 ppm Ni in the form of Ni(NO3)2.6H2O, encap- sulated E. acoroides extract at 100 ppm, 250 ppm, and 500 ppm, and combined 30 ppm Ni with each of 100 ppm, 250 ppm and 500 ppm of encapsulated E. acoroides extract. The treatments were given for 72 hrs. As con- trols, bulbs were soaked in H2O. Three bulbs were used in each treatment. Chromosome preparation Modified procedures were used to prepare mitotic squash (Sharma and Sharma 1994). Following treat- ments, roots were rinsed in distilled water and cut in the morning. Roots were then soaked in Farmer’s fixa- tive containing of ethanol and acetic acid (3:1) for 24 hrs in the refrigerator. For hydrolysis, root tips were cut 2 mm long and treated with 1N HCl for 15 minutes. The root tips were cleaned in distilled water before being col- oured with 2% acetoorcein for 20 min. Excess stain was absorbed using filter paper. Stained root tips were cov- ered with cover glass and then squashed. Slides were inspected for mitotic chromosomes and aberration using microscope binocular XSZ 107BN (Nanjing BW Optics and Instrument Co.) with 400x total magnification. The photographs were taken using the top mount camera Optilab Advance (Miconos). The data was collected from a total of six roots of three bulbs for each treatment and six fields chosen at random from each root. Metabolic Activity Following the treatment and control procedures, 5 root tips were removed and soaked in 0.5% 2,3,5-triphenyl tetrazolium chloride (TTC) for 15 minutes in the dark at 35°C. The root tips were then analysed qualitatively after being rinsed with distilled water. Furthermore, the roots were soaked in 95% ethanol to extract the colourful triph- enyl formazan complex. The absorbance was measured at 490 nm (Vazhangat and Thoppil 2017). Three replications were conducted in this experiment. Apoptotic Activity The Evans Blue staining method was used to investi- gate the loss of cell viability. After treatments, five roots with identical lengths were cut and dyed with a 0.25% (w/v) aqueous Evans Blue solution for 15 minutes before being rinsed with distilled water for 30 minutes. The experiment was conducted with three replications. The roots were then macro-imaged to determine cell death qualitatively. Roots then were soaked in 3 mL of N,N- dimethylformamide for 1 hour at room temperature for a quantitative estimation by measuring the absorbance of Evans Blue at 600 nm (Vazhangat and Thoppil 2017). Data Analyses The mitotic index (%) was computed as the number of dividing cells divided by the total number of cells ´ 100. The chromosomal aberrations were calculated by dividing the number of abnormal cells by the total num- ber of cells counted × 100 (Sarac et al. 2019). Phase index (%) was determined by calculating the number of divid- ing cells in phases by the total number of dividing cells ´ 100 (Kumar and Thonger 2016). The antigenotoxicity of encapsulated E. acoroides extract was determined by calculating the inhibitory activity of chromosomal aberration induced by Ni. The formula used was following Prajitha and Thoppil (2016). Inhibitory activity (%)= A−B: A−C × 100, where A: Number of aberrant cells induced by Ni, B: Number of aberrant cells induced by the mixture of Ni and encap- sulated E. acoroides extract, C: Number of aberrant cells induced in the control Statistical analyses were performed using Minitab 20, with randomised experimental design. The differ- ences between treatments were analysed using the Tukey test with a 95% confidence level. The data were presented as mean ± standard deviation, except for the data of the types of aberration. RESULTS Mitotic Index Using Allium cepa var. aggregatum root tips, the antigenotoxic potential of encapsulated extract of E. acoroides leaves was investigated. One of the metrics used to assess antigenotoxicity was mitotic activity as measured by the mitotic index. The mitotic indices were significantly affected by the treatments (p<0.01). Treat- ment of A. cepa root with Ni reduced mitotic index sig- nificantly. There are no differences between the mitotic index of control and treatment with 100 ppm encapsu- lated extract of E. acoroides leaves. The concentration of 250 ppm and 500 ppm encapsulated extract had a sig- 92 Made Pharmawati, Ni Nyoman Wirasiti, Luh Putu Wrasiati nificantly lower mitotic index than control, but signifi- cantly higher than nickel (Table 1). Treatment with nickel resulted in the lowest mitotic index indicating genotoxic activity of nickel. When nick- el and encapsulated extract of E. acoroides leaves were given simultaneously, the mitotic indices were higher than the mitotic index of Ni alone; however, statistical analysis showed that only the addition of 100 ppm encap- sulated extract had a significant increase of the mitotic index. Table 1 shows the mitotic indices of control, treat- ment with Ni, encapsulated E. acoroides extract, and combined treatment of Ni and encapsulated extract. Phase index The distribution of mitotic phases was shown in Table 2. The treatments significantly affected prophase, metaphase and telophase indices (p<0.05), while ana- phase index was not affected by treatments. The major- ity of chromosomes in all treatments and control were in metaphase. Treatment of Nickel resulted in the highest percentage of metaphase chromosomes, and Ni inhibited telophase as indicated by the significantly lowest index of telophase in Ni treatment. The addition of encapsulat- ed E. acoroides extract to the Ni treatment increased the percentage of telophase. Chromosomal Aberration and Nuclear Abnormality Statistical analysis shows that the treatments affect- ed chromosomal aberration (p<0.01) and nuclear abnor- mality (p<0.01). Mitotic chromosomal aberrations were detected in all treatments including control (Table 1) and control has a very low percentage of aberration. Treatment with Ni resulted in 1,677% of aberrant chro- mosomes. The percentage of the aberrant chromo- some at root tips treated with encapsulated E. acoroides extract at the concentration of 100 ppm, 250 ppm and 500 ppm had no significant difference to control, sug- gesting that the encapsulated extract had no or very low genotoxic effect. Simultaneous treatments of Ni and encapsulated E. acoroides extract at concentrations 100 ppm, 250 ppm, and 500 ppm showed a similar per- centage of chromosomal aberration to treatment with Ni alone. Modulation of Ni-induced genotoxicity with encapsulated E. acoroides extract showed no significant reduction of chromosomal aberration. The inhibitory activities of encapsulated extract to the genotoxic activ- ity of Ni were only 4.9%, 6.5%, and 14.4% with simul- taneous addition of 500 ppm, 250 ppm, and 100 ppm encapsulated extract. The types of chromosomal aberration at mitotic phases included prolonged prophase, stickiness, frag- ment, chromosoma l brea k /fragmentation at meta- phase, diagonal metaphase, diagonal telophase, chro- mosome bridge, star anaphase, fragment at anaphase, and vagrant telophase. Figure 1 shows normal mitotic phases, while Figure 2 shows types of aberrant chro- mosomes. Table 3 shows the percentage of each type of chromosomal aberration. Nuclear abnormalities were observed in a different set of fields of view than that of chromosomal aberra- tion. The nuclear abnormalities observed were micro- nuclei, nuclear fragments, and nuclear lesions (Figure Table 1. The Mitotic index and percentage of chromosomal aber- ration of A. cepa root tip cells induced by Ni, encapsulated E. acoroides leaves extract and mixture of Ni and encapsulated extract. Treatment (ppm) Mitotic index Chromosome aberration (%) Control 5.036± 0.497a 0.091±0.1b 30Ni 2.248±0.497c 1.677±0.487a 100Ea 5.048±0.864a 0.553±0.462b 250Ea 3.612±0.444b 0.542±0.227b 500Ea 3.383±0.418b 0.551±0.2097b 30Ni+100Ea 3.262±0.29b 1.453±0.343a 30Ni+250Ea 2.905±0.2176bc 1.575±0.1974a 30Ni+500Ea 2.779±0.489bc 1.601±0.289a Ni=nickel in the form of Ni(NO3)2.6H2O; Ea=encapsulated E. acoroides leaves extract. Means with same letters at the same column are not significantly different. Table 2. Phase index of mitosis of A. cepa root tip cells after treat- ment with Ni, encapsulated E. acoroides leaves extract and mixture of Ni and encapsulated extract. Treatment (ppm) Prophase Index Metaphase Index Anaphase Index Telophase Index Control 21.68±8.02a 27.8±5.38b 27.08±7.57a 23.44±5.08a 30 Ni 19.22±8.82a 59.17±18.87a 19.14±13.58a 2.74±3.83b 100 Ea 26.6±8.27a 32.96±4.89b 20.13±7.21a 18.31±7.08a 250 Ea 22.98±6.02a 37.88±5.54b 16.61±4.96a 24.22±3.21a 500 Ea 24.11±9.85a 37.72±13.86b 14.91±4.54a 21.57±3.49a 30 Ni+100 Ea 35.1±12.19a 29.42±11.86b 19.3±6.22a 16.17±5.75a 30 Ni+250 Ea 33.86±7.03a 36.21±4.82b 14.77±10.07a 15.16±5.46a 30 Ni+500 Ea 22.69±8.88a 43.34±15.44ab 17.97±2.83a 16±9.7a Ni=nickel in the form of Ni(NO3)2.6H2O; Ea=encapsulated E. acoroides leaves extract. Means with same letters at the same column are not significantly different. 93Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate 3, Table 4). Micronuclei were not detected in control and in treatment using 100 ppm, 250 ppm of encapsu- lated E. acoroides extract, while it was detected at 500 ppm encapsulated E. acoroides extract but not signifi- cantly different than control. Treatments with Ni alone and combined Ni and encapsulated E. acoroides extract resulted in the formation of micronuclei and statisti- cally, they were not significantly different, although the percentage of combined treatments was much lower than Ni alone. Fragmented nuclei were not observed in control and in treatment with 100 ppm, 250 ppm, and 500 ppm of encapsulated E. acoroides extract. The highest percentage of nuclear fragmentation was induced by Ni treatment only. The encapsulated E. acoroides extract also induced nuclear fragmentation in a significantly lower percentage than Ni treatment. The encapsulated E. acoroides extract given simultaneously with Ni, significantly reduced the percentage of fragmented nuclear (Table 4). Another nuclear abnormality observed was nuclear lesion and Ni treatment showed the highest percentage. The addition of encapsulated extract to Ni treatments significantly reduced the percentages of nuclear lesions compared to Ni treatment (Table 4). Metabolic activity The triphenyl tetrazolium chloride (TTC) stain- ing was used to examine the influence of Ni and encap- sulated E. acoroides extract on mitochondrial func- tion. Treatment of roots with Ni revealed a substantial decrease in mitochondrial activity. Visually, the encap- sulated E. acoroides extract as well as combined encap- sulated extract and Ni shows an increase in mitochon- drial activity (Figure 4). Based on the absorbance value of 490 nm, the encapsulated extract at concentrations of 100 ppm and 250 ppm has no effect, while 500 ppm extract reduced mitochondrial activity; however, the reduction was sig- nificantly less than treatment with Ni (Table 5). Simulta- neous treatment of encapsulated extract at 100 ppm and Ni showed improvement of mitochondrial activity com- pared to Ni alone. In comparison, the addition of 250 ppm and 500 ppm encapsulated extract to Ni treatment did not show improvement of mitochondrial activity. Apoptotic activity Evans blue stain was used to analyse in situ cell death by assessing the cell membrane’s integrity. Living cells keep the dye out due to the semipermeable nature of cell membranes. On the other hand, damaged cells are unable to remove the dye and are thus stained blue (Roy et al. 2019). Figure 5 shows the visualization of cell death using Evan’s blue staining. Evan’s Blue staining method for in situ cell death revealed that the encapsulated E. acoroides extract at con- centrations 100 ppm, 250 ppm, and 500 ppm showed less colour than treatment with Ni only. Simultaneous treat- ment of Ni and encapsulated E. acoroides extract also showed a reduction of blue colour indicating a reduc- tion of cell death (Figure 5). Quantitative analysis using a spectrophotometer is shown in Table 5. Statistical analy- sis revealed that 100 ppm and 250 ppm of encapsulated extract had similar effect as control. The 500ppm extract showed higher absorbance than control, but significantly lower than Ni alone. The data of in situ cell death is simi- lar to that of mitochondrial activity. The 100 ppm encap- Figure 1. Normal mitosis of A. cepa root tip cells. a. Prophase; b. Metaphase; c. Anaphase; d. Telophase. Scale bar=10μm. Figure 2. Types of chromosomal aberrations of A. cepa root tip cells after treatment with Ni, encapsulated E. acoroides leaves extract and combined Ni and encapsulated extract. a=prophase abnormality with fragments; b= sticky metaphase; c= fragment at metaphase; d=chain metaphase; e=vagrant telophase (circle); f=diagonal anaphase; g=chromosome bridge; h=diagonal meta- phase; i=fragment at anaphase; j=star anaphase; k=sticky anaphase. Scale bar=10μm. Arrows indicate abnormalities. 94 Made Pharmawati, Ni Nyoman Wirasiti, Luh Putu Wrasiati sulated extract when given simultaneously with Ni demon- strated significantly less cell death than treatment with Ni. DISCUSSION As a result of increased urbanization and industrial- isation, toxic metal poisoning has become a global issue. Moreover, accumulated heavy metal in plants has been known to induced chromosome abnormalities as shown by Sabeen et al. (2020). This present study confirmed that nickel has a geno- toxic effect by significantly decreasing mitotic index and inducing chromosomal aberration. At the concentration of 30 ppm Ni(NO3)2.6H2O the reduction of the mitotic index was 44.53% in comparison to control. The decline of the mitotic index below 50% has sub-lethal effects and is known as the limit value of cytotoxicity (Madike et al. 2019). The genotoxic effect of nickel has been studied using nickel chloride (NiCl2) (Ganesan and Panneersel- vam 2013), nickel sulfate (NiSO4.6H2O) (Pavlova 2017) and nickel nitrate Ni(NO3)2 (Sarac et al. 2019). The con- centration of 30 ppm Ni was used in this study, to evalu- ate a lower concentration than that used by Sarac et al. (2019) which was 50 ppm. Nickel at 30 ppm induced chromosomal aberration where chromosome stickiness and chromosome break Table 3. The percentage of each type of chromosomal aberration of A. cepa root tip cells after treatment with Ni, encapsulated E. acoroides leaves extract and combined Ni and encapsulated extract. Treatment (ppm) Frag.pro (%) Sty.meta (%) Frag.meta (%) Ch.meta (%) Diag.meta (%) Diag.ana (%) Star.ana (%) Sty.ana (%) Frag.ana (%) Bridge (%) Vr.telo (%) Control 0c 0.091b 0c 0a 0a 0a 0b 0a 0a 0a 0a 30Ni 0.256ab 0.509a 0.656ab 0.062a 0.062a 0.042a 0.1ab 0a 0.081a 0.023a 0a 100Ea 0.069b 0.180ab 0.071c 0.071a 0.032a 0a 0.039ab 0.026a 0.027a 0.039a 0a 250Ea 0.034bc 0.156ab 0.226c 0.026a 0.033a 0a 0b 0.033a 0.034a 0a 0a 500Ea 0.029bc 0.151ab 0.233c 0.032a 0a 0.037a 0.034ab 0a 0.036a 0a 0a 30Ni+100Ea 0.394ab 0.221ab 0.357bc 0.034a 0.069a 0.029a 0.225a 0.059a 0.064a 0a 0a 30Ni+250Ea 0.224ab 0.292ab 0.73ab 0.027a 0a 0a 0.086ab 0.1a 0.027a 0.027a 0.065a 30Ni+500Ea 0.229ab 0.181ab 0.758a 0.034a 0.029a 0.029a 0.13ab 0.068a 0.073a 0.073a 0a Ni=nickel in the form of Ni(NO3)2.6H2O; Ea=encapsulated E. acoroides leaves extract. Frag.pro=prophase abnormality with fragments; Sty.meta= sticky metaphase; Frag.meta=fragment at metaphase; Ch.meta=chain metaphase; Diag.meta=diagonal metaphase; Diag.ana=diagonal anaphase; Star.ana=star anaphase; Sty.ana=sticky anaphase, Frag.ana=fragment at ana- phase; Bridge=chromosome bridge; Vr.telo=vagrant telophase. Means with same letters at the same column are not significantly different. Table 4. The percentages of nuclear abnormalities of A. cepa root tip cells after treatment with Ni, encapsulated E. acoroides leaves extract and combined Ni and encapsulated extract. Treatment (ppm) Micronuclei (%) Nuclear lesion (%) Nuclear fragmentation (%) Control 0±0b 4.57±5.75d 0±0b 30 Ni 0.222±0.192a 90.67±3.58a 0.73±0.609a 100 Ea 0±0b 20,11±2.84c 0±0b 250 Ea 0±0b 20.03±3.78c 0±0b 500 Ea 0.020±0.063b 21.76±3.84c 0±0b 30 Ni+100 Ea 0.083±0.092ab 60.43±8.2b 0.083±0.091b 30 Ni+250 Ea 0.082±0.092ab 62.73±6.29b 0.079±0.087b 30 Ni+500 Ea 0.091±0.1ab 67.48±6.14b 0.076±0.084b Ni=nickel in the form of Ni(NO3)2.6H2O; Ea=encapsulated E. acoroides leaves extract. Means with same letters at the same column are not significantly different. Table 5. Metabolic and apoptotic activities of A. cepa root tips after treatment with Ni, encapsulated E. acoroides leaves extract and combined Ni and encapsulated extract. Treatment (ppm) Metabolic activity Apoptotic activity Control 0.482±0.034a 0.166±0.03c 30 Ni 0.157±0.006d 0.448±0.053a 100 Ea 0.486±0.05a 0.268±0.07bc 250 Ea 0.479±0.024a 0.286±0.071bc 500 Ea 0.334±0.026b 0.301±0.035b 30 Ni+100 Ea 0.277±0.022bc 0.304±0.015b 30 Ni+250 Ea 0.242±0.057bcd 0.320±0.011ab 30 Ni+500 Ea 0.222±0.024cd 0.344±0.053ab Ni=nickel in the form of Ni(NO3)2.6H2O; Ea=encapsulated E. acoroides leaves extract. Means with same letters at the same column are not significantly different. 95Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate were at a high percentage. This agrees with Sarac et al. (2019) and Kaur et al. (2019) who observed that chromo- some break and chromosome stickiness were the major types of chromosomal aberration found due to heavy metal treatments. Nickel promotes the generation of a large quantity of reactive oxygen species (ROS) which is a factor of nickel toxicity. Reactive oxygen species harm all cellular components in plants, including cell mem- branes, lipids, pigments, enzymes, chloroplasts, and nucleic acids (Gopal and Nautiyal 2012). Excessive ROS promotes DNA break which can be observed through the chromosomal break (Ganesan and Panneerselvam 2013). Chromosome stickiness is caused by chromo- some loss of physical identity due to physical attachment of chromatin material or inter-chromosomal connec- tions (Asita et al. 2017). Heavy metal complexes are very reactive, and their complexes interact directly or indi- rectly with DNA, histone, or non-histone proteins, caus- ing chromosomal surface properties to change, making them sticky (Kumar and Srivastava 2015). The cytotoxicity of encapsulated E. acoroides extract was tested by calculating the mitotic index. The mitotic index of encapsulated E. acoroides extract at a concen- tration of 100 ppm was similar to control. Higher con- centrations of encapsulated extract decreased mitotic index but were still higher than the mitotic index of Ni treatment. This suggests that at 250 ppm and 500 ppm, the encapsulated E. acoroides extract was less toxic than Ni. The reduction in the mitotic index shows that the encapsulated E. acoroides extract inhibits mitotic activity in A. cepa. The reduction in mitotic index is attributable to compounds in the aqueous extracts that have cytotox- ic effects, as the mitotic index is a quantitative measure of mitotic activity in an organism or a particular organ (Sreeranjini and Siril 2011). When Ni and encapsulated extract were given simultaneously, only 100 ppm encapsulated extract resulted in a significant increase of the mitotic index. This indicates that the encapsulated E. acoroides extract had the potential in modulated Ni-inhibited mitotic activity by increasing the proliferative activity of cells. The antigenotoxic activity of low concentrations of plant extract was also reported by Prajitha and Thoppil (2016). The lower concentration (5 ppm) of Amaranthus spino- sus employed in the antigenotoxicity experiment was beneficial in reversing the genotoxicity. Treatment with higher concentrations of encap- sulated E. acoroides extract combined with Ni did not significantly increase mitotic index. A study using Che- nopodium album extract found that at low concentra- tion, the extract reduced the genotoxic effect induced by EMS (ethylmethane sulfonate). At higher concentra- tions, C. album extract showed synergistic action with EMS, resulting in an increased genotoxic effect (Asita et al. 2015). In the present study, encapsulated E. acoroides extract did not have a synergetic effect with Ni since the addition of encapsulated extract together with Ni, had no significantly different with Ni alone on the chromo- somal aberration. Based on analysis of phase index, Ni treatment had a significantly higher metaphase index, while the ana- phase indices were not significantly different between treatments. This means that in Ni treatment the ana- phase index was low. The telophase index of Ni treat- ment was significantly lower than other treatments. Figure 5. Analysis of apoptotic activity using Evans Blue stain- ing of root of A. cepa. a=control, b=30 ppm Ni, c=100 ppm Ea, d=100 ppm Ea, d= 250 ppm Ea, e=500 ppm Ea, f=30ppmNi+100 ppm Ea, g=30 ppm Ni+250 ppm Ea, h=30 ppm Ni+500 ppm Ea. Ea=encapsulated E. acoroides leaves extract. Figure 4. Analysis of metabolic activity using TTC staining of root of A. cepa. a=control, b=30 ppm Ni, c=100 ppm Ea, d=100 ppm Ea, d= 250 ppm Ea, e=500 ppm Ea, f=30ppmNi+100 ppm Ea, g=30 ppm Ni+250 ppm Ea, h=30 ppm Ni+500 ppm Ea. Ea=encapsulated E. acoroides leaves extract. Figure 3. Types of nuclear abnormalities of A. cepa root tip cells after treatment with Ni, encapsulated E. acoroides leaves extract and combined Ni and encapsulated extract. a. nuclear fragmentation; b. micronucleus; c. nuclear lesion. Scale bar=10μm. 96 Made Pharmawati, Ni Nyoman Wirasiti, Luh Putu Wrasiati According to Asita et al. (2017), a decrease in the pro- portion of dividing cells in A + T indicates that the chromosome spindles were poisoned, resulting in metaphase arrest. Low anaphase and telophase indices can cause daughter cells to be damaged, limiting plant growth. The simultaneous addition of Ni and encapsu- lated E. acoroides extracts at all concentrations signifi- cantly increased the telophase indices. The percentage of chromosome abnormality between control and treatment with all concentrations of encap- sulated E. acoroides extract were not significantly differ- ent, indicating the possibility of the non-toxic effect of encapsulated E. acoroides extract. This result is impor- tant since the encapsulated E. acoroides extract had an antiproliferative effect by reducing the mitotic index. Therefore, it can be further explored in anticancer research. Chromosome aberrations were detected in the com- bined treatment of Ni and encapsulated E. acoroides extract at all concentrations tested. Although there were decreases in percentages of chromosome aberration in combined Ni and encapsulated extract treatments, the percentages were not significantly different from that of Ni treatment alone. This result indicates that the concen- trations of encapsulated E. acoroides extract used unable to effectively suppress chromosome aberration induced by Ni. However, it is worth noting that there was 14.4% inhibitory activity of Ni when 100 ppm encapsulates E. acoroides extract was given simultaneously with 30 ppm Ni. This suggests that the mixtures were less genotoxic than Ni alone. Nickel induced the formation of micronuclei and nuclear fragmentation at low levels but formed nuclear lesions in extremely high percentages. Nuclear lesions provide cytological evidence of DNA biosynthesis inhi- bition (Sajitha and Thoppil 2018). At all concentrations tested, the encapsulated E. acoroides extract did not induce micronuclei and chromosome fragmentation. However, it induced nuclear lesions at low percentag- es, significantly lower than induced by Ni. The control group had a low level of nuclear lesion, which could be due to unintentional DNA changes. According to Nefic et al. (2013), root tip cells show a very low frequency of spontaneous abnormalities. In TTC analysis, the roots treated with Ni were unable to convert TTC to red coloured TF, indicating a significantly lower activity of the mitochondrial res- piratory chain compared to control. The roots treated with encapsulated E. acoroides extract at 250 ppm and 500 ppm demonstrated no effect on mitochondrial activity. The addition of lower concentration of encap- sulated E. acoroides extract (100 ppm) to 30 ppm Ni increased mitochondrial activity compared to treat- ment with Ni alone. Apoptotic activity was highly induced in Ni treated root, while encapsulated E. acoroides extract showed lower apoptotic activity than Ni, but higher activity than control. This suggests that encapsulated E. acoroides extract was less toxic than Ni. Supplementation of encapsulated E. acoroides extracts to Ni, visually result- ed in lower apoptotic activity than Ni alone as observed as less blue colour. However, when measured using a spectrophotometer, there were no differences between the apoptotic activities at Ni treatment and Ni sup- plemented with 250 ppm and 500 ppm encapsulated E. acoroides extract. Lower concentration of encapsulated E. acoroides extract (100 ppm) when given together with 30 ppm Ni, induced reduction of apoptotic activity. The effects of simultaneous addition of Ni and encapsulated E. acoroides extract at a lower concentra- tion to metabolic activity and apoptotic activity agreed with their effect on the mitotic index. According to Pra- jitha and Thoppil (2016), a higher concentration of an extract can have mutagenic effect and a lower concen- tration can have an antimutagenic effect or vice versa. In mice, lower levels of b-carotene increased the anti- clastogenic activity of cyclophosphamide-induced clas- togenicity, but there was no protective impact at higher concentrations. This finding implies distinct processes of b-carotene modulation and a probable shift in the bal- ance of the promutagen activation/detoxification mecha- nism (Salvadori et al. 1992). Similar reasoning may apply to the effect of simultaneous addition of Ni and low con- centration encapsulated E. acoroides extract on increas- ing mitotic index and metabolic activity and reducing apoptotic activity. Enhalus acoroides leaves extract contained phyto- chemical compounds, including phenols, tannins, and flavonoids. The FTIR analysis confirmed the presence of flavonoid and polyphenols as a high C-H out-of-plane bending (oop bend) vibration for the substituted ben- zene ring was identified in the extract (Pharmawati and Wrasiati 2020). These phytochemical components in the plant extracts may be responsible for the reduced mitotic index in A. cepa root meristematic cells when roots were treated with encapsulated E. acoroides leaves extract. On the other hand, these phytochemical compounds may contribute to the increasing mitotic index, lowering nuclear abnormalities when the encapsulated extract is present together in the Ni treatment. This kind of result where plant extract showed the opposite effect was also observed by Prajitha and Thoppil (2016) in Amaranthus spinosus extract. The encapsulated E. acoroides leaves extract also contained pigments such as chlorophyll b, 97Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate ethyl-chlorophyllide a, Mg-free chlorophyll b, lutein, Mg free chlorophyll a, pheophytin, and β-carotene (Phar- mawati and Wrasiati 2020). It is well known that phe- nolic compounds, tannins, flavonoids, chlorophyll, and carotenoids have antioxidant properties (Aryal et al. 2019). Antioxidants containing phenolics can prevent the generation of free radicals and/or stop the spread of autoxidation. At the same plant pigments can chelate metals and transfer hydrogen to oxygen radicals, delay- ing oxidation (Brewer 2011). In the present investigation, the encapsulated E. acoroides extract was found to have preventive activity, as evidenced by the reduction and reversion of nuclear damages (nuclear lesions and nuclear fragmentations) caused by Ni. However, the encapsulated extract can- not reduce chromosomal aberration. Preincubation with the encapsulation extract before Ni treatment needs to be evaluated to test the ability of encapsulated extract to suppress chromosomal abnormalities. Further study is also needed to test the protective activity of the encapsu- lated extract on animal cells. ACKNOWLEDGMENT The authors thank Universitas Udayana for sup- porting this study through the Study Program Flagship Research Scheme No. B/773/UN14.2.8.II/PT.01.03/2021 FUNDING Universitas Udayana through the Study Program Flagship Research Scheme No. B/773/UN14.2.8.II/ PT.01.03/2021 REFERENCES Amjad M, Raza H, Murtaza B, Abbas G, Imran M, Sha- hid M, Naeem MA, Zakir A, Iqbal MM. 2020. Nick- el toxicity induced changes in nutrient dynamics and antioxidant profiling in two maize (Zea mays L.) Hybrids. Plants. 9(1):5. https://doi.org/10.3390/ plants9010005 Aryal S, Baniya MK, Danekhu K, Kunwar P, Gurung R, Koirala N. 2019. Total phenolic content, flavonoid content and antioxidant potential of wild vegetables from Western Nepal. Plants. 8(4):96. doi:10.3390/ plants8040096 Asita AO, Heisi DH, Tjale T. 2015. Modulation of muta- gen-induced genotoxicity by two Lesotho medicinal plants in Allium cepa L. Environ Nat Resour Res. 5(3):37-55 Asita OA, Moramang S, Rants’o T, Magama S. 2017. Mod- ulation of mutagen-induced genotoxicity by vitamin C and medicinal plants in Allium cepa L. Caryologia. 70: 51-165. doi: 10.1080/00087114.2017.1311166 Brewer MS. 2011. Natural antioxidants: sources, com- pounds, mechanisms of action, and potential appli- cations. Compr Rev Food Sci Food Saf. 10:221-247. doi: 10.1111/j.1541-4337.2011.00156.x Cempel M, Nikel G. 2006. Nickel: A Review of Its Sourc- es and Environmental Toxicology. Polish J Environ Stud. 15(3):375-382 den Hartog C, Kuo J. 2006. Taxonomy and biography of seagrasses. In: Larkum T, Orth RJ, Duarte CM, edi- tors. Seagrasses: Biology, ecology and conservation. The Netherlands: Springer: p. 1-23. Ganesan A, Panneerselvam N. 2013. Analysis of Ni induced genotoxicity in root meristem of Allium cepa. Inter. J. Biological Technol. 4:19-22 Gantayat S, Mania S, Pradhan C, Das AB. 2018. Ionic stress induced cytotoxic effect of cadmium and nickel ions on roots of Allium cepa L. Cytologia 83(2):143– 148 Gopal R, Nautiyal N. 2012. Growth, antioxidant enzymes activities, and proline accumulation in mustard due to nickel. Int. J. Veg. Sci . 18:223–234. Huang L, Rad S, Xu L, Gui L, Song X, Li Y, Wu Z, Chen Z. 2020. Heavy metals distribution, sources, and eco- logical risk assessment in Huixian Wetland, South China. Water.12(2):431. https://doi.org/10.3390/ w12020431 Kaur M, Sharma A, Soodan RK, Chahal V, Kumar V, Kat- noria JK, Nagpal AK. 2019. Allium cepa root chro- mosomal aberration assay: A tool to assess genotox- icity of environmental contaminants. Environ Con- tam Nat Prod. 2019:65-93 Kilawati Y, Islamy RA. 2019. The antigenotoxic activity of brown seaweed (Sargassum sp.) Extract Against Total Erythrocyte and Micronuclei of Tilapia (Oreochromis niloticus) Exposed by Methomyl-Base Pesticide. J Exp Life Sci. 9:205-210 Kiswara W, Hutomo M. 1985. Habitat Dan Sebaran Geo- grafik Lamun. Oseana. XII(1): 21-30. Kumar G, Srivastava A. 2015. Comparative genotoxicity of herbicide ingredients glyphosate and atrazine on root meristem of buckwheat (Fagopyrum esculentum Moench). Jordan J Biol Sci. 8(3):221-226 Kumar S, Thonger T. 2016. Study on 24 Hour Root Tip Cell Division Mitotic and Mitotic Phase Index of Allium chinense. American-Eurasian J. Agric. Envi- ron. Sci. 16(1):172-183 98 Made Pharmawati, Ni Nyoman Wirasiti, Luh Putu Wrasiati López-Romero D, Izquierdo-Vega JA, Morales-González JA, Madrigal-Bujaidar E, Chamorro-Cevallos G, Sánchez-Gutiérrez M, Betanzos-Cabrera G, Alvarez- Gonzalez I, Morales-González Á, Madrigal-Santillán E. 2018. Evidence of some natural products with antigenotoxic effects. Part 2: Plants, Vegetables, and Natural Resin. Nutrients, 10(12):1954. https://doi. org/10.3390/nu10121954 Madike LN, Takaidza S, Ssemakalu C, Pillay M. 2019. Genotoxicity of aqueous extracts of Tulbaghia violacea as determined through an Allium cepa assay. S Afr J Sci. 115(1/2), Art. #4391, https://doi. org/10.17159/sajs.2019/4391 McKenzie LJ, Yoshida RL. 2009. Seagrass-watch. Proceed- ing of a workshop for monitoring seagrass habitats in Indonesia. The Nature Conservancy, Coral Triangle Center, Sanur, Bali: pp. 29-32 Nefic H, Musanovic J, Metovic A, Kurteshi K. 2013. Chromosomal and nuclear alterations in root tip cells of Allium cepa L. induced by alprazolam. Med Arch. (Sarajevo, Bosnia and Herzegovina) 67(6):388–392. doi.org/10.5455/medarh.2013.67.388-392 Oyeyemi IT, Bakare AA. 2013. Genotoxic and antigeno- toxic effect of aqueous extracts of Spondias mombin L., Nymphea lotus L. and Luffa cylindrica L. on Alli- um cepa root tip cells. Caryologia. 66(4): 360-367. https://doi.org/10.1080/00087114.2013.857829 Özkan G, Bilek SE. 2014. Microencapsulation of natural food colourants. Intl J Nutr Food Sci. 3(3):145–56. doi:10.11648/j.ijnfs.20140303.13 Pavlova D. 2017. Nickel effect on root-meristem cell divi- sion in Plantago lanceolata (Plantaginaceae) seed- lings. Aust J Bot. 65:446-452. https://doi.org/10.1071/ BT17054 Pharmawati M, Wrasiati LP. 2020. Phytochemical screening and FTIR spectroscopy on crude extract from Enhalus acoroides leaves. Malays J Anal Sci. 24(1):70-77 Prajitha V, Thoppil JE. 2016. Genotoxic and antigenotoxic potential of the aqueous leaf extracts of Amaranthus spinosus Linn. using Allium cepa assay. S Afr J Bot. 102:18-25 https://doi.org/10.1016/j.sajb.2015.06.018. Rodeiro I, Olguín S, Santes R, Herrera JA, Pérez CL, Mangas R., Hernández Y, Fernández G, Hernández I, Hernández-Ojeda S, Camacho-Carranza R, Valencia- Olvera A, Espinosa-Aguirre JJ. 2015. Gas chroma- tography-mass spectrometry analysis of Ulva fasciata (green seaweed) extract and evaluation of its cytopro- tective and antigenotoxic effects. Evid Base Comple- mentary Altern Med. 2015. Article ID 520598. http:// dx.doi.org/10.1155/2015/520598 Roy B, Krishnan SP, Chandrasekaran N, Mukherjee A. 2019. Toxic effects of engineered nanoparticles (met- al/metal oxides) on plants using Allium cepa as a model system. In: Verma SK, Das AK, (editors). Com- prehensive Analytical Chemistry, Elsevier: p. 125-143 Sabeen M, Mahmood Q, Ahmad Bhatti, Z., Faridullah, Irshad M, Bilal M, Hayat MT, Irshad U, Ali Akbar T, Arslan M, Shahid N. 2020. Allium cepa assay based comparative study of selected vegetables and the chromosomal aberrations due to heavy metal accu- mulation. Saudi J Biol Sci. 27(5):1368–1374. https:// doi.org/10.1016/j.sjbs.2019.12.011 Sajitha MK, Thoppil JE. 2018. Screening of cytotoxicity, metabolic inhibition and possible apoptotic cell death induced by Gomphostemma heyneanum Wall. ex Benth. var. heyneanum using Allium cepa root tips. Int J Pharm Biol Sci. 8(2):56-64 Salvadori DMF, Ribeiro LR, Oliveira MDM, Pereira CAB, Beçak W. 1992. The protective effect of β-carotene on genotoxicity induced by cyclophosphamide. Mutat Res. 265(2):237–244 Sarac I, Bonciu E, Butnariu M, Petrescu I, Madosa E. 2019. Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test. Caryologia. 72:37-43. Sharma AK, Sharma A. 1994. Chromosome Technique A Manual. CRC Press, Taylor & Francis Group, Boca Raton, Florida, USA. p. 1-32 Sharma S, Nagpal A, Vig AP. 2012. Genoprotective potential of Brassica juncea (L.) Czern. Against mer- cury-induced genotoxicity in Allium cepa L. Turk J Biol. 36:622-629 doi:10.3906/biy-1110-18 Sharma S, Sharma S, Vig AP. 2018. Antigenotoxic potential of plant leaf extracts of Parkinsonia aculeata L. using Allium cepa assay. Plant Physiol Biochem. 130:314-323. https://doi.org/10.1016/j.plaphy.2018.07.017 Short, F.T. & Waycott, M. 2010. Enhalus acoroides. The IUCN Red List of Threatened Species 2010: e.T173331A6992567. https://dx.doi.org/10.2305/IUCN. UK.2010-3.RLTS.T173331A6992567.en. Accessed on 31 January 2022. Singh SB, Singh K, Butola SS, Rawat S, Arunachalam K. 2020. Determination of macronutrients, micronutri- ents and heavy metals present in Spilanthes acmella Hutch and Dalz: possible health effects. Nat Prod Sci. 26(1):50-58. 10.20307/nps.2020.26.1.50. Sreeranjini S, Siril EA. 2011. Evaluation of anti-genotox- icity of the leaf extracts of Morinda citrifolia Linn. Plant Soil Environ. 57:222–227 Šturm L, Črnivec IGO, Istenič K, Ota A, Megušar P, Slu- kan A, Humar M, Levic S, Nedović V, Kopinč R, Deželak M, Gonzales AP, Ulrih NP. 2019. Encapsula- tion of non-dewaxed propolis by freeze-drying and spray-drying using gum Arabic, maltodextrin and 99Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate inulin as coating materials. Food Bioprod Process. 116:196-211, Sulistyadewi ENP, Wrasiati LP, Wartini NM. 2014. Peru- bahan kadar MDA, SOD, dan kapasitas antioksidan hati tikus 13prague dawley pada pemberian ekstrak bubuk daun cemcem (Spondias Pinnata (L.f ) Kurz). Media Ilmiah Teknol Pangan. 1(1):71-80. Vazhangat P, Thoppil JE. 2016. Apoptotic induction via membrane/DNA damage and metabolic inactivation by synthetic food colorants in Allium cepa root mer- istem. Turk J Biol. 40: 922-933 Yusop FHM, Manaf SFA, Hamzah F. 2017. Preserva- tion of bioactive compound via microencapsulation. Chem Eng Res Bull. 19:50-56 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 2 - 2022 Firenze University Press Cytogenetic Studies of Six Species in Family Araceae from Thailand Piyaporn Saensouk1, Surapon Saensouk2,*, Rattanavalee Senavongse2 Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana Sherzad R. Abdull, Sahar H. Rashid*, Bakhtiar S. Ghafoor, Barzan S. Khdhir Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified polymorphism Wei Cao1, Xiao Chen2,*, Zhiwei Cao3 Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae) Xiaoju Zhang1, Li Bai2,*, Somayeh Esfandani-Bozchaloyi3 SCoT molecular markers are efficient in genetic fingerprinting of pomegranate (Punica granatum L.) cultivars Shiva Shahsavari1, Zahra Noormohammadi1,*, Masoud Sheidai2,*, Farah Farahani3, Mohammad Reza Vazifeshenas4 First record of nucleus migration in premeiotic antherial cells of Saccharum spontaneum L. (Poaceae) Chandra Bhanu Singh1, Vijay Kumar Singhal2, Manish Kapoor2,* Genetic Characterization of Salicornia persica Akhani (Chenopodiaceae) Assessed Using Random Amplified Polymorphic DNA Zhu Lin1,*, Hamed Khodayari2 Comparative chromosome mapping of repetitive DNA in four minnow fishes (Cyprinidae, Cypriniformes) Surachest Aiumsumang1, Patcharaporn Chaiyasan2, Kan Khoomsab3, Weerayuth Supiwong4, Alongklod Tanomtong2 Sumalee Phimphan1,* Classical chromosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand Weera Thongnetr1, Surachest Aiumsumang2, Alongklod Tanomtong3, Sumalee Phimphan2,* Genotoxic and antigenotoxic potential of encapsulated Enhalus acoroides (L. f.) Royle leaves extract against nickel nitrate Made Pharmawati1,*, Ni Nyoman Wirasiti1, Luh Putu Wrasiati2 Chromosomal description of three Dixonius (Squamata, Gekkonidae) from Thailand Isara Patawang1, Suphat Prasopsin2, Chatmongkon Suwannapoom3, Alongklod Tanomtong4, Puntivar Keawmad5, Weera Thongnetr6,* First Report on Classical and Molecular Cytogenetics of Doi Inthanon Bent-toed Gecko, Cyrtodactylus inthanon Kunya et al., 2015 (Squamata: Gekkonidae) in Thailand Suphat Prasopsin1, Nawarat Muanglen2, Sukhonthip Ditcharoen3, Chatmongkon Suwannapoom4, Alongklod Tanomtong5, Weera Thongnetr6,* Evaluation of genetic diversity and Gene-Pool of Pistacia khinjuk Stocks Based On Retrotransposon-Based Markers Qin Zhao1,*, Zitong Guo1, Minxing Gao1, Wenbo Wang1, Lingling Dou1, Sahar H. Rashid2 A statistical overview to the chromosome characteristics of some Centaurea L. taxa distributed in the Eastern Anatolia (Turkey) Mikail Açar1,*, Neslihan Taşar2 Cytotoxicity of Sunset Yellow and Brilliant Blue food dyes in a plant test system Elena Bonciu1, Mirela Paraschivu1,*, Nicoleta Anca Șuțan2, Aurel Liviu Olaru1