Effects of Some Growth Substances on Mitosis

Transcription

1 Cytologia 42: , 1977 Effects of Some Growth Substances on Mitosis Ganesh1 Prasad and K. Das2 Department of Genetics and Plant Breeding, Banaras Hindu University, Varanasi , India Received November 20, Radiomimetic action of growth substances, also called hormones, has been demonstrated by several workers (Crocker 1954, Gori and Maugini 1955, Kato, 1958, Sawamura 1964, Sharma and Mookerjea 1954, Unrau and Larter 1952). Some of the growth substances are used as growth promotors while others are used as herbicides for weed control. As they prove toxic beyond certain concentrations, it is fruitful to study their effects at different concentrations. This paper describes the results of an experiment involving different concentrations of three growth sub stances on root tip mitosis in Vicia faba when treated for varying durations. Materials and methods Three growth substances namely, Gibberellic acid-ga (a naturally occurring phytohormone), Indole-butyric acid-iba (a synthetic hormone) and 2, 4-Dichioro phenoxyacetic acid-2, 4-D (a synthetic hormonic herbicide) were taken for this study. The seeds of Vicia faba were grown in petri dishes on blotting papers. When the roots attained the length of about 2cm, treatments were given by the aqueous solu tions of the chemicals, the concentrations being 10 and 50ppm. The durations of treatments were 3, 6 and 12 hours with a recovery period of 12 hours for each treat ment. Root tips were collected in 3: 1 alcohol-acetic acid solution and stored in 70 per cent alcohol. Slides were prepared using 0.5 per cent haematoxylin stain and examined under microscope. Results The cell metabolism was strongly disturbed by 2, 4-D. The protoplasm became gelated with higher concentration and the cell contents disappeared. IBA also caused some toxic effects like 2, 4-D while GA had no such effect. Data relating to comparative effects of these chemicals on cell division are shown in Table 1 and graphically presented in Fig. 1. GA caused increase in mitotic index with increasing duration at 10 ppm concentration. Frequency of dividing cells was 25.3 per cent after 12 hours treatment in contrast to 14.1 per cent in control. At 50ppm concentration, mitotic index decreased with increasing durations, however, 1 Present address: Department of Agricultural Botany, S. D. J. Post Graduate College, Chandesar, Azamgarh, U. P., India. 2 Present address: Emeritus Progessor, Department of Genetics and Plant Breeding, Assam Agricultural University, Jorhat, Assam, India.

2 4 G. Prasad and K. Das Cytologia 42 Table 1. Mitotic index and the frequencies of cells in different stages of mitosis after treatment with the three chemicals

3 1977 Effects of Some Growth Substances on Mitosis 325 the percentage of dividing cells with 3 hours duration was more than that in control. IBA and 2, 4-D inhibited the cell division and their inhibitory action increased with increasing concentration and duration. It is observed in Table 1 that the frequency of prophase cells was more in treated material than in control. On the other hand, metaphase and anaphase cells were less frequent as compared to control. Percent age of telophase cells in GA-treated material was more than that in control. Simi lar effect was also observed with 2, 4-D and IBA at some treatments (Table 1). Fig. 1. Frequency of cells in mitosis after treatment at 10ppm and 50ppm concentra tions with varying durations. Fig. 2. Frequency of abnormal cells induced by the three chemicals at 10ppm and 50ppm concentrations with varying durations. All the three chemicals induced aberrations in dividing cells. Different types of abnormalities were divided into four groups and their frequencies are shown in Table 2. Relevant data on the frequency of abnormal cells are also portrayed graphically in Fig. 2. a) Chromosome breakage Chromosome fragments were found at all the treatments. They were observed at metaphase (Fig. 3C) and anaphase (Fig. 3A, D). In general, the frequencies of cells containing fragments increased with increasing concentrations and durations. Highest frequency of fragments was observed with 2, 4-D. Large fragments were induced by GA and small fragments by 2, 4-D while IBA induced both types of fragments. These fragments were lying in the cytoplasm and sometimes caused inhibition of cell plate formation leading to binucleate cells. 2, 4-D and IBA caused chromosome erosion also. b) Bridge formation Bridges were induced by all the three chemicals. Some of the bridges persisted in telophase also (Fig. 3B). Single bridges were observed with GA. 2, 4-D induced single and double bridges while IBA induced upto 4 bridges. They were broken

4 Prasad and K. Das Cytologia 42 Table 2. Frequencies of different types of abnormalities induced by the three chemicals

5 1977 Effects of Some Growth Substances on Mitosis 327 by stretching or by cell plate formation. c) Spindle suppression Polyploid cells (Fig. 3F) were observed in varying frequencies. C-mitotic ac tivity was more frequent with 2, 4-D. Fragments were also observed in polyploid cells. Fig. 3. Some aberrations induced by the chemicals. A, fragments at anaphase. B, bridge per sisting in telophase. C, chromosome breakage at metaphase. D, fragments at anaphase. E, micronucleus. F, polyploid cell. d) Other abnormalities One of the abnormalities was the presence of micronuclei (Fig. 3E) which were formed by chromosome fragments lying outside the nucleus. IBA and 2, 4-D caused

6 328 G. Prasad and K. Das Cytologia 42 stickiness where outline of chromosomes became indistinct and the chromosomes tended to clump. 2, 4-D caused chromosome condensation where the chromosomes became thicker and shorter than the usual metaphase chromosomes. Discussion GA enhanced cell division at lower concentration but inhibited at higher con centration while IBA and 2, 4-D strongly inhibited mitosis at all the treatments. There might be two types of inhibition; the preprophase inhibition where the cells were prevented from entering into prophase and the prophase poisoning where cells entered into mitosis but they were arrested in the prophase resulting in high frequency of prophase cells. Regarding the cytotoxic effects of GA, the results agree with those obtained by Kato (1958). The results of 2, 4-D treatment are similar to those observed by Amer and Ali (1974), Crocker (1954), Gori and Maugini (1955) and Sawamura (1964). Stickiness was not observed with GA and it is probable that the bridges induced by this chemical were dicentric chromatid bridges due to rejoining of broken ends of chromosomes. On the other hand, bridges caused by 2, 4-D and IBA might have been formal due to stickiness. C-mitotic activity of 2, 4-D was observed by several workers (D'Amato 1950, Crocker 1954, Gori and Maugini 1955). D'Amato (1950) suggested that there are two types of mechanisms which produce C-mitosis. One is characterized by the action of colchicine with no delay in mitosis and other by the action of the chemicals where preprophase inhibition occurs. Chromosome condensation with 2, 4-D was also reported by Crocker (1954) in Allium ceps, Similar type of action was ob served by Amer and Farah (1974) in Gossypium barbadense treated with the insec ticide "Rogor". In connection with erosion, Sharma and Sharma (1960) indicated that certain chromosome sites become devoid of nucleic acids and appear as thinner areas com pared to the rest of the chromosome segments. Different chemicals act in different ways according to their structure. Some chemicals may cause disturbance in oxi dation reduction system within the nucleus, some may affect the sulfhydryl group (-SH) of protein and others may act through their influence on hydrogen bonds of nucleic acids (Auerbach 1952, Buttler 1954, Dustin 1949, Levan and Tjio 1948). Sharma and Sharma (1962) found that a disturbance in RNA metabolism leading to DNA protein deficiency was responsible for chromosome breakage. Ahnstrom and Natarajan (1966) also found that chromosome breakage was caused by inhibition of DNA synthesis. It may, therefore, be concluded that the chemicals act through different means all of which finally disturb the nucleic acid metabolism causing hazards in protein synthesis which results in breakage of chromosomes. Summary Cytological effects of three growth substances namely, Gibberellic acid (GA), Indole-butyric acid (IBA), and 2, 4-Dichlorophenoxyacetic acid (2, 4-D) were studied on root tip mitosis of Vicia faba treated with 10 and 50ppm concentrations and 3,

7 1977 Effects of Some Growth Substances on Mitosis and 12 hours durations. GA enhanced cell division while 2, 4-D and IBA inhibit ed it and were more toxic than GA. Main effects at chromosomal level were chro mosome breakage, bridge formation, C-mitosis, micronuclei formation, stickiness and chromosome condensation. Bibliography Auerbach, C Sensitivity of Drosophila germ cells to mutagens. Symposium on chromosome breakage. Heredity 6 Suppl: Ahnstrom, G. and Natarajan, A. T Mechanism of chromosome breakage a new theory. Hereditas 54: Amer S. M. and Ali, E. M Cytological effects of pesticides V. Effects of some herbicides on Vicia faba. Cytologia 39: and Farah, O. R Cytological effects of pesticides VI. Effect of the insecticide "Rogor" on the mitosis of Vicia faba and Gossypium barbadense. Cytologia 39: Buttler, J. A. V The action of carcinogenic agents and radiations-some implications of new ideas of structure of deoxyribonucleic acid. Acta 10: Crocker, H. B Effects of 2, 4-Dichlorophenoxyacetic acid and 2, 4-Trichlorophenoxyacetic acid on mitosis in Allium cepa. Bot. Gaz. 114: D'Amato, F The quantitative study of mitotic poisons by Allium cepa test, data and prob lems. Protoplasma 34: Dustin, P Mitotic poisoning at metaphase and -SH proteins. Proc. 7th Intern. Congr. Exptl. Cytol. (1949). Exptl. Cell Research (Suppl.) 1: Gori, C. and Maugini, E Cytological and root forming effects of some growth substances. Caryologia 7: Kato, Y Effect of gibberellin and fusarinic acid on mitosis. Senshokutai (Chromosome) 34: Levan, A. and Tjio, J. H Chromosome fragmentation induced by phenols. Hereditas 34: Sawamura, S Cytological studies on effect of herbicides on plant cell in vivo I. Hormonic herbicides. Cytologia 29: Sharma, A. K. and Sharma A Spontaneous and chemically induced chromosome breaks. Intern. Rev. Cytol and A study of the importance of nucleic acids in controlling chromosome breaks induced by different compounds. Nucleus 5: and Mookerjea, A Possibilities of the use of hormone in chromosome analysis. Caryo logia 6: Unrau, J. and Larter, E. N Cytological responses of cereals to 2, 4-D as a study of meiosis of plants treated at various stages of plant growth. Canad. J. Bot. 30: