Cytological assessments and a methodological approach to observe the apoptotic effect of nickel sulfate (NiSO4) on Allium cepa L. root germination using EB/AO fluorescence staining


  • Fulya D. GÖKALP Trakya University, Science Faculty, Biology Department, 22030 Edirne (TR)
  • Didar GÜZEY Trakya University, Science Faculty, Biology Department, 22030 Edirne (TR)
  • Mihriban ABIS Trakya University, Science Faculty, Biology Department, 22030 Edirne (TR)
  • Özgür MERT Trakya University, Science Faculty, Biology Department, 22030 Edirne (TR)



acridine orange, Allium cepa, apoptosis, ethidyum bromide, nickel, nuclear abnormalities


Ethidium bromide–acridine orange (EB/AO) is one of the fastest, most economically valid methods for separating living and dead cells in plant root tips. The present study aimed to investigate the apoptotic effect, nuclear abnormalities and cell division index using an Allium cepa test assay with EB/AO staining and an ImageJ program. Nickel sulphate (NiSO4) concentrations (1.75, 3.5, 7 and 14 ppm) were exposed to the root germination of A. cepa for 48 and 72 h to observe mitotic abnormalities, cytotoxic effects and 5-day exposure to apoptotic effects. It was found that amorphous nuclei, vacuolisation and C-mitosis were the most observed abnormalities, which increased at 14 ppm NiSO4 exposure after 48 and 72 h. The total number of nuclear abnormalities significantly increased at all concentrations and exposure periods. The ImageJ program was used to determine apoptosis rates. The data obtained showed that high concentrations of NiSO4 caused significantly more cell death in root tips than in the control group, resulting in root growth inhibition and increased apoptosis with increased concentration and exposure period. NiSO4 caused toxic activity on root growth, identified as apoptosis, especially at the cortex and vascular region in the root tips. The study found affected damaged and apoptotic areas on the root tips using an EB/AO staining method, which can be used as a marker of damaged tissue areas.


Metrics Loading ...


Aggarwal N, Laura JS, Sheoran IS (1990). Effect of cadmium and nickel on germination, early seedling growth and photosynthesis of wheat and pigeon pea. International Journal of Tropical Agriculture 8(2):141-147.

Ahmad MSA, Ashraf M (2011). Essential roles and hazardous effects of nickel in plants. Reviews of Environmental Contamination and Toxicology 214:125-167.

Akbas H, Dane F, Meric C (2016). A comparative study on the significance of lead, nickel and cadmium on root growth, the kinetics of metal ion transport and pH changes in onion (Allium cepa) root. Fresenius Environmental Bulletin 25(5):1623-1629.

AlKahtani A, Alkahtany SM, Mahmood A, Elsafadi MA, Aldahmash AM, Anil S (2014). Cytotoxicity of QMix (TM) endodontic irrigating solution on human bone marrow mesenchymal stem cells. BMC Oral Health 14.

Bishnoi NR, Sheoran IS, Singh R (1993). Effect of cadmium and nickel on mobilization of food reserves and activities of hydrolytic enzymes in germinating pigeon pea-seeds. Biologia Plantarum 35(4):583-589.

Bozali K, Guler EM, Celikten M (2022). Investigation of the effect of curcumin on cytotoxicity, genotoxicity, and apoptosis on breast cancer cells. World Cancer Research Journal 9:1-8.

Brooks RR, Shaw S, Marfil AA (1981). The chemical form and physiological-function of nickel in some Iberian alyssum species. Physiologia Plantarum 51(2):167-170.

Bugs M, Cornelio M (2001). Analysis of the ethidium bromide bound to DNA by photoacoustic and FTIR spectroscopy. Photochemistry and Photobiology 74(4):512-520.

Byczkowska A, Kunikowska A, Kaźmierczak A (2013). Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining. Protoplasma 250(1):121-128.

Foy CD, Chaney RT, White MC (1978). The physiology of metal toxicity in plants. Annual Review of Plant Physiology 29(1):511-566.

Caicedo M, Jacobs JJ, Reddy A, Hallab NJ (2008). Analysis of metal ion-induced DNA damage, apoptosis, and necrosis in human (Jurkat) T-cells demonstrates Ni2+ and V3+ are more toxic than other metals: Al3+, Be2+, Co2+, Cr3+, Cu2+, Fe3+, Mo5+, Nb5+, Zr2+. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 86(4):905-913.

Cameron KS, Buchner V, Tchounwou PB (2011). Exploring the molecular mechanisms of nickel-induced genotoxicity and carcinogenicity: a literature review. Review of Environmental Health 26(2):81-92.

Ciniglia C, Pinto G, Sansone C, Pollio A (2010). Acridine orange/Ethidium bromide double staining test: A simple In-vitro assay to detect apoptosis induced by phenolic compounds in plant cells. Allelopathy Journal 26(2):301-308.

Cortes-Eslava J, Gomez-Arroyo S, Risueno MC, Testillano PS (2018). The effects of organophosphorus insecticides and heavy metals on DNA damage and programmed cell death in two plant models. Environmental Pollution 240:77-86.

Demchenko NP, Kalimova IB, Demchenko KN (2005). Effect of nickel on growth, proliferation, and differentiation of root cells in Triticum aestivum seedlings. Russian Journal of Plant Physiology 52(2):220-228.

Doreswamy K, Shrilatha B, Rajeshkumar T, Muralidhara (2004). Nickel-induced oxidative stress in testis of mice: evidence of DNA damage and genotoxic effects. Journal of Andrology 25(6):996-1003.

Eskew DL, Welch RM, Cary EE (1983). Nickel - an essential micronutrient for legumes and possibly all higher-plants. Science 222(4624):621-623.

Espen L PL, Cocucci SM (1997). Effects of Ni2+ during the early phases of radish (Raphanus sativus) seed germination. Environmental and Experimental Botany 38(2): 187-197.

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.

Jia J, Chen J (2008). Chronic nickel-induced DNA damage and cell death: the protection role of ascorbic acid. Environmental Toxicology 23(3):401-406.

Kang JH, Zhang DW, Chen J, Liu Q, Lin CJ (2005). Antioxidants and trichostatin A synergistically protect against in vitro cytotoxicity of Ni2+ in human hepatoma cells. Toxicology in Vitro 19(2):173-182.

Kargacin B, Klein CB, Costa M (1993). Mutagenic responses of nickel oxides and nickel sulfides in Chinese-Hamster V79 cell-lines at the xanthine guanine phosphoribosyl transferase locus. Mutation Research 300(1):63-72.

Kazmierczak A (2010). Endoreplication in Anemia phyllitidis coincides with the development of gametophytes and male sex. Physiologia Plantarum 138(3):321-328.

Kaźmierczak A (2008). Cell number, cell growth, antheridiogenesis, and callose amount is reduced and atrophy induced by deoxyglucose in Anemia phyllitidis gametophytes. Plant Cell Reports 27(5):813-821.

Khursheed A, Jain V (2021). Oxidative stress-mediated caspase-dependent intrinsic apoptosis and proautophagic therapeutic potential of Salvia moorcroftiana Wall. ex Benth. growing in the Kashmir Valley, India. Biomedical Research and Therapy 8(11).

Klaunig JE, Kamendulis LM (2004). The role of oxidative stress in carcinogenesis. Annual Review in Pharmacological Toxicology 44:239-267.

Kopittke PM, Asher CJ, Menzies NW (2007). Toxic effects of Ni2+ on growth of cowpea (Vigna unguiculata). Plant and Soil 292(1-2):283-289.

Kunikowska A, Byczkowska A, Kazmierczak A (2013). Kinetin induces cell death in root cortex cells of Vicia faba ssp minor seedlings. Protoplasma 250(4):851-861.

Leon V, Rabier J, Notonier R, Barthelemy R, Moreau X, Bouraima-Madjebi S, Viano J, Pineau R (2005). Effects of three nickel salts on germinating seeds of Grevillea exul var. rubiginosa, an endemic serpentine Proteaceae. Annals of Botany 95(4):609-618.

LePecq J-B, Paoletti C (1967). A fluorescent complex between ethidium bromide and nucleic acids: physical—chemical characterization. Journal of Molecular Biology 27(1):87-106.

Liu DH, Jiang WH, Guo L, Hao YQ, Lu C, Zhao FM (1994). Effects of nickel sulfate on root-growth and nucleoli in root-tip cells of Allium cepa. Israel Journal of Plant Sciences 42(2):143-148.

Liu DH, Jiang WS, Wang W, Zhai L (1995). Evaluation of metal-ion toxicity on root-tip cells by the Allium test. Israel Journal of Plant Sciences 43(2):125-133.

Maehle L, Metcalf RA, Ryberg D, Bennett WP, Harris CC, Haugen A (1992). Altered p53 gene structure and expression in human epithelial cells after exposure to nickel. Cancer Research 52(1):218-221.

Manosroi A, Akazawa H, Akihisa T, Jantrawut P, Kitdamrongtham W, Manosroi W, Manosroi J (2015). In vitro anti-proliferative activity on colon cancer cell line (HT-29) of Thai medicinal plants selected from Thai/Lanna medicinal plant recipe database "MANOSROI III". Journal of Ethnopharmacology 161:11-17.

Mcilveen WD, Negusanti JJ (1994). Nickel in the terrestrial environment. Science of the Total Environment 148(2-3):109-138.

Murch SJ, Haq K, Rupasinghe HPV, Saxena PK (2003). Nickel contamination affects growth and secondary metabolite composition of St. John's wort (Hypericum perforatum L.). Environmental and Experimental Botany 49(3):251-257.

O’brien IE, Baguley BC, Murray BG, Morris BA, Ferguson IB (1998). Early stages of the apoptotic pathway in plant cells are reversible. The Plant Journal 13(6):803-814.

Page V, Feller U (2005). Selective transport of zinc, manganese, nickel, cobalt and cadmium in the root system and transfer to the leaves in young wheat plants. Annals of Botany 96(3):425-434.

Petriccione M, Forte V, Valente D, Ciniglia C (2013). DNA integrity of onion root cells under catechol influence. Environmental Science and Pollution Research 20(7):4859-4871.

Rahman H, Sabreen S, Alam S, Kawai S (2005). Effects of nickel on growth and composition of metal micronutrients in barley plants grown in nutrient solution. Journal of Plant Nutrition 28(3):393-404.

Riesen O, Feller U (2005). Redistribution of nickel, cobalt, manganese, zinc, and cadmium via the phloem in young and maturing wheat. Journal of Plant Nutrition 28(3):421-430.

Samadi L, Behboodi BS (2005). Detection of apoptotic bodies in nickel-treated root apical cells of Allium cepa. Russian Journal of Plant Physiology 52(1):131-133.

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(2):37-43.

Seregin IV, Kozhevnikova AD (2006). Physiological role of nickel and its toxic effects on higher plants. Russian Journal of Plant Physiology 53(2): 257-277.

Sresty TVS, Rao KVM (1999). Ultrastructural alterations in response to zinc and nickel stress in the root cells of pigeonpea. Environmental and Experimental Botany 41(1):3-13.

Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006). Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 160(1):1-40.

Vanyushin BF, Bakeeva LE, Zamyatnina VA, Aleksandrushkina NI (2004). Apoptosis in plants: Specific features of plant apoptotic cells and effect of various factors and agents. International Review of Cytology - a Survey of Cell Biology, 233:135-179.

Wang L, Lu L (2007). Pathway-specific effect of caffeine on protection against UV irradiation-induced apoptosis in corneal epithelial cells. Investigative Ophthalmology & Visual Science 48(2):652-660.

Wang YF, Shyu HW, Chang YC, Tseng WC, Huang YL, Lin KH, Chou MC, Liu HL, Chen CY (2012). Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway. Toxicology and Applied Pharmacology 259(2):177-186.

Wong MH, Bradshaw AD (1982). A comparison of the toxicity of heavy-metals, using root elongation of rye grass, Lolium perenne. New Phytologist 91(2): 255-261.

Yan XL, Liu M, Zhong JQ, Guo JT, Wu W (2018). How human activities affect heavy metal contamination of soil and sediment in a long-term reclaimed area of the Liaohe River Delta, North China. Sustainability 10(2).

Yassin AM, El-Deeb NM, Elsaid FG, Shati AA, Cioca G, Tit DM, Bungau S, Popa A, Hafez EE (2019). Lectin from Pisum fulvum seeds as in vitro anticancer and apoptotic gene regulator. Revista De Chimie 70(4):1490-1495.




How to Cite

GÖKALP, F. D., GÜZEY, D., ABIS, M., & MERT, Özgür. (2024). Cytological assessments and a methodological approach to observe the apoptotic effect of nickel sulfate (NiSO4) on Allium cepa L. root germination using EB/AO fluorescence staining. Notulae Scientia Biologicae, 16(1), 11676.



Research articles
DOI: 10.55779/nsb16111676