According to the present study application of metribuzin had significant toxic effect to plant growth and development. Toxic effects of metribuzin are manifested by retarded growth and damaged roots due to altered metabolism associated with degradation of plant food reserve and absorption of minerals and water by roots (Tudi et al., 2021). Similar findings of dose dependent increase in morphotoxicity of metribuzin + 2,4-D were also reported in wheat varieties Choudhary et al. (2016). As per our experiment this toxicity can be overcome to some extend by the external application of SA (1mM) as the RN and RL increased in plants treated with SA along with the metribuzin when compared to plant stressed only with metribuzin. The ameliorating effect of SA under exposure of different fungicides, pesticides insecticides along with various abiotic stresses has been thoroughly discussed. In this context, it was also reported that SA improves morphological growth, physiological attributes and enhanced antioxidant machinery of Vigna radiata exposed to three different pesticides Metribuzin, Mancozeb and Chlorpyrifos (Fatma et al., 2018b).
A reliable parameter to identify cytotoxicity is mitotic index (MI) as it reflects the rate of the cell division in root meristematic tissues (Bianchi et al. 2016). According to our experiment MI decreased significantly (p ≤ 0.05) and progressively as the concentration of metribuzin was increased as compared to control. The reduction of mitotic activity seems to be a common effect of herbicide tested for their action on mitosis (Mesi and Kopliku 2013). This reduction in mitotic activity may be due to blocking of mitotic cell cycle during interphase (Fioresi et al., 2020), inhibition of nuclear-proteins synthesis essential for normal mitotic sequence (Venturelli 2015), suppression of DNA synthesis (Küçük et al. 2018) or change in the relative duration of the mitotic stages (Mekki et al., 2016).
In our experiment we tried to overcome the reduction in MI by the ameliorative effect of SA. It is evident from present study that SA treatment significantly improved the MI in root meristematic cells of A. cepa at different concentrations of metribuzin. Thus, from the data of the present study, it is apparent that SA is also effective at higher concentrations of metribuzin used in study. Present result was very much correlated to the result of Singh et al. (2013), investigating the possible mitigating role of salicylic acid (SA) in protecting plant (Vicia faba) from cytotoxicity of insecticides by observing significantly increased MI. Thus, the ameliorative response of SA which provides a basis to control herbicide contamination in crop production is by regulating plant response to organic contaminants (Varhney et al. 2015). Further Singh et al. (2012) reported that combined treatments of pendimethalin with SA increased MI when compared with pendimethalin treatments.
The experimental result of the current study showed that metribuzin is able to induce significant chromosomal abnormalities in the root meristematic cells of A. cepa as compared to control, which indicate the genotoxic potential of metribuzin. The various abnormalities recorded were stickiness, fragmentation, clumping disorientation, C-metaphase, bridge at anaphase, laggard and precocious movement of chromosomes (Özkara et al., 2015). SA treatment resulted in significant decrease in the frequency of cells with chromosomal abnormalities which implies that SA tries to ameliorate genotoxicity stress created by metribuzin. The current findings are in agreement with the earlier reports signifying SA-induced response to DNA damaging agents by inducing expression of defence genes (Ahmad et al., 2020). Among these abnormalities stickiness was the major abnormality noted which is considered to be a chromatid type physiological aberration (Ping et al. 2012) and attributed to the effect of environmental pollutants on degradation or depolymerization of chromosomal DNA (Kushwah et al., 2017), DNA condensation (Mahanty et al., 2017) and exchanges among chromosomes (Goujon et al. 2015). There was a significant increase in stickiness which was dose dependent and is considered as common sign of highly toxic effects on chromosomes and irreversible type that probably leading to cell death (Turkoglu 2012). Stickiness can be caused by loss of chromosome movement thus preventing them from reaching final destination or sub-chromatid linkage between chromosomes. This can also be explained as physical adhesion of the chromosome proteins (Goujon et al. 2015).
After stickiness, disorientation of chromosomes at meta and anaphase was seen increasing significantly, which may be due to spindle dissolution. This dissolution could be due to alteration of the gene which controls the spindle mechanism. Singh et al. (2021) when investigating the effect of pesticides on Vigna species found that disoriented metaphase induced due to the disturbance of spindle apparatus. In addition to this, bridges formation due to chromatid breakage and fusion during the translocation of unequal chromatids or due to dicentric chromosome presence thus, bridges resulting in structural chromosome mutations (Liman et al. 2015). On the other hand, C-metaphase and laggard chromosome are due to spindle failure (Mesi and Kopliku 2013) which was recorded at higher concentrations of metribuzin. Inhibition of spindle organization by various chemicals, can lead to conditions of scattering of chromosomes, also known as C-metaphase. Laggard chromosomes might be due to the hindrance of pro-metaphase movement of chromosomes accompanied by adhesion of centromere to the nuclear membrane (Kuchy et al., 2016) and as a result they increase the risk for aneuploidy. Similar finding was reported by Karaismailoglu (2016) while investigating the toxic effect of herbicide on A. cepa. In our study, SA application were found effective in inducing the adaptive response to genotoxic stress since it significantly reduces the chromosomal abnormalities in the root meristematic cells of A. cepa induced by herbicide metribuzin. The underlying mechanisms is not well known, but it might be due to activation of mitogen activate kinase protein and up regulation of other stress gene(s)