Abstract
Cytotoxicity of different compounds are commonly evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay is mainly used to study cell viability in cell lines (Carmichael et al., 1987). In this study, the protocol is being used to determine the cell viability of plant roots, treated with different stress inducing agents. The basis of the assay is that the dye enters the living cell’s mitochondrion where it is reduced to insoluble formazan, which is solubilised by directly treating the cells with organic solvent (DMSO). Intensity of colour is directly proportional to the amount of formazan produced.In the present study, plants were treated for 16 h, with several phytotoxic agents, then the roots were incubated in MTT solution for 4 h. To solubilise the formazan, roots were excised. 2 N potassium hydroxide (KOH) along with DMSO was used to solubilize the cell wall components and thereby liberating the formazan granules in the DMSO solution. The rate of the cell viability was measured by measuring the colour intensity of the formazan.
Keywords: Cell viability, Cytotoxicity, MTT assay, Plant tissue, Rice seedling
Background
This protocol is designed to determine plant cell viability directly from root tissue. Till date, MTT assay has been profusely used for mammalian cell proliferation and viability assay. In case of plants usually the 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay (Kaundal et al., 2012) is preferably used. Cost effective MTT assay can also be used for plant tissue viability assay instead of XTT assay. This protocol can be used to determine the cytotoxicity of different stress inducing agents and their IC50 doses (the concentration of phytotoxic agent at which 50% cell death is obtained). Nicotinamide adenine dinucleotide phosphate (NAD(P)H) dependent dehydrogenase enzyme present in mitochondrion of living cell is capable of reducing tetrazolium dye MTT 3-(4,5-dimethylthia zol-2-yl)-2,5-diphenyltetrazolium bromide to an insoluble purple coloured formazan, which is measured spectrophotometrically. This reduction takes place when the mitochondrial enzymes are active and hence the degree of reduction can be directly correlated with the number of viable cells. The metabolically inactive cells will not show this property.
Materials and Reagents
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Acknowledgments
Authors wish to acknowledge the Department of Botany, Centre of Advanced Studies, Phase-VII, University of Calcutta for the instrumentation facilities. SM and TG wish to acknowledge DBT, GOI and CSIR for the financial assistance. The authors declare the absence of any conflict of interests.
References
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