C 2000 The Japan Mendel Society Cytologia 65: 383-388, 2000 Cytological Effect of the Herbicide 2, 4-D isooctylester 48% on Root Mitosis of Allium cepa Aye Nihal Gomiirgen Biology Department, Science Faculty, Hacettepe University, Beytepe, Ankara, Turkey Accepted August 9, 2000 Summary The effect of the herbicide 2,4-D isooctylester 48% (Esteran 48) has been studied on root mitosis of Allium cepa. Root tips of Allium cepa were treated with a series of concentrations, ranging from 50 ppm to 50,000 ppm for 3, 6, 12, 24 h. Examinations of roots were done in permanent root tip squash preparations stained by the Feulgen technique. Esteran 48 effects the relative duration of each mitotic stage as compared with the control. It also caused reduction in the mitotic index, in- dicating mitotic inhibition, and increased in the frequency of abnormal mitosis. The type of the abnormalities induced: chromosome stickiness, C-metaphase, tetraploid cells, bridges, laggards, tripolar anaphases-telophases and micronuclei. The effect of Esteran 48 on root mitosis simulates that of colchicine in the type of abnormal metaphase (C-mitosis) and induction of polyploidy cells as well as the accumulation of metaphases. Key words 2,4-D isooctylester, Chromosome abnormalities. It is now widely known that herbicides are capable of inducing genetic effects on plants. Many studies have demonstrated the mutagenic action of herbicidal solutions on seeds, seedlings and growing plants. Plant assay systems have been found useful for the detection of environmental mu- tagens (De Serres 1978). It is ironical that although herbicides are used in modern technology of crop production, they are sources of potentially hazardous substances in the human environment. Many cytological studies have been carried out to detect the harmful effect of different pesticides and herbicides on different plants (Soriano 1984, Njagi and Goplan 1981, Mousa 1982, Amer and Farah 1987, El-Khodary et al. 1989). The use of chromosomal aberrations, induced by pesticides in crop plants, is there fore, being accepted as indicators of genetic damage (Ma 1982). The herbicide 2,4-D isooctylester 48% is also known as Esteran 48 and it is actively control of many broad leaf weeds. The present investigation was carried out to study the cytological effect of the herbicide Esteran 48 on the process of mitosis in the root tips of Allium cepa. Material and methods The bulbs of Allium cepa was used as plant test material. Roots while intact, were treated with different concentrations of the herbicide, Esteran 48 ranging from 50 ppm to 50,000 ppm for 3, 6, 12 and 24 h. Roots were fixed in acetic alcohol (1 : 3) for 24 h. Examination of the roots was done in permanent root tip squash preparations stained by the Feulgen technique. Three replicates were performed for each treatment and the control, and scoring was made from the 3 roots of each replicate. The mitotic index was calculated for each treatment as a number of dividing cells/100 cells. Results and discussion The used concentrations of Esteran 48 caused distinct decrease in the mitotic index when com- 384 Aye Nihal GOrniirgen Cytologia 65 pared with the control (Table 1). In all the treatments the decrease in MI values in the root meris- tems of Allium cepa with increasing concentrations is attribute to mitotic inhibitions. A drop in mi- totic activity was clearly observed when the roots were treated with high concentrations. Such a drop in the mitotic index indicates that Esteran 48 interferes in the normal sequence of mitosis thus preventing a number of cells entering the prophase, state at interphase. Such reduction in the mitot- ic activity could be due to the inhibition of DNA synthesis (Schneiderman et al. 1971). Beau et al. (1976) also showed that exposing the root tips of Vicia faba to high concentrations of the herbicide (Paraquate), led to inhibition of DNA synthesis. This may suggest that Esteran 48 could have the same effect. The reduction of mitotic activity seems to be common effect of most herbicides tested for their action on mitosis (Amer and Farah 1980, 1983, Tomkins and Grant 1972, Badr 1983, Badr and Ibrahim 1987). Mitotic inhibition by herbicides has been attributed to blocking of mitotic cycle during interphase which may result from prolonged G2 period or to the inhibition of DNA synthesis (Chand and Roy 1981). The degree of cytological aberrations either in mitosis or meiosis is regarded as one of the de- pendable criteria for estimating the effect of mutagen (Reddi and Reddi 1985). Esteran 48 exerted a marked mitodepressive action on mitosis and induced a number of chromosomal aberrations. These abnormalities are shown in Table 2. The colchicine C-metaphase (Fig. 1) configurations as the major mitotic abnormality produced in roots treated with high concentrations and/or prolonged periods of treatments. C-mitosis is one of the consequences of inactivation of spindle apparatus connected with the delay in the division of centromere. Deysson (1968) suggested that this might lead to the polyploid cells thus formed de- generate without further division. Esteran 48 also induced sticky metaphase (Fig. 2). Other mitotic abnormalities induced by this herbicide are poliploidy (Fig. 3), tripolar anaphases (Fig. 4) and tripo- lar telophases (Fig. 5), bridges (Fig. 6) and lagging chromosomes (Fig. 7) and micronuclei (Fig. 8). Aberrations involving the chromosomal structure as elegant indicators of chromosomal mutations. In the present study a remarkable correlation exists between stickness and bridges produced. These suports the hypothesis that bridges induced by Esteran 48 is due to the stickness of the chro- mosomes. Stickiness is regarded as physiological effect exerted by herbicides in plants, which has been considered of the chromosomes. On the other hand stickiness has been attributed to the im- proper folding of chromosome fibers, which makes the chromatids connected by means of subchro- matid bridges (McGill et al. 1974, Klasterska et al. 1976). The phenomena of chromosome sticki- ness dominant indicate that the toxicity of Esteran 48 and its low mutagenic potential. This herbicide tested on this study produced anaphase bridges (Table 2). Bridges were found to be the result of stickiness of the chromosomes. The irregular spreading of chromosomes may be at- tributed to the disturbance of spindle apparatus (Amer and Ali 1974). It is well known that frag- ments and bridges lead to structural changes in the chromosomes. Breaking up chromosomes fol- lowed by proximal chromatid reunion evidently results in dicentric chromosomes which form char- acteristic anaphase bridges (Tomkins and Grant 1972, Grant 1978), or these anaphase bridges can be attributed to the general stickiness of chromosomes (Abraham and Koshy 1979). Lagging chromosomes induced this herbicide are acting only on spindle apparatus resulting in scattered anaphases and laggard chromosomes. Tripolar and trinuclaeted cells are indicating the in- hibition of cytokinesis (Somashekar and Godwa 1984). The presence of micronuclei observed in the present study is not a deviant phenomenon (Amer and Ali 1974, Amer and Farah 1980, 1983), these are evidently the resultants of the chromosome fragments of the cells concerned. The formation of micronuclei, may be the result of lagging chro- mosome or chromosome segment produced during a preceding division. 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