77
Mutation Research, 263 (1991) 77-81 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 016579929100441 MUTLET 0493
2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells Mirjana Pavlica, Dra~ena Pape~ and Biserka Nagy Department of Molecular Biology, Faculty of Science, Universityof Zagreb, YU-41001Zagreb (Yugoslavia) (Received 4 January 1991) (Revision received 16 January 1991) (Accepted 17 January 1991)
Keywords: 2,4-Dichlorophenoxyaceticacid; Chromosome aberrations; Cytotoxicity; Mutagenicity
Summary The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 #g/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture ( > 5 #g/ml).
It is known that chromosome mutations as well as changes in chromosome number and structure, and also changes during the cell cycle are frequently observed in in vitro systems of plant and animal origin (Sunderland, 1973; D'Amato, 1978; Bayliss, 1980; Evans et al., 1980; Constantin, 1981; Pape~ et al., 1983). Plants regenerated from callus or cell suspension showed genetic and phenotypic variability (Larkin and Scowcroft, 1981), chromosome structure instability, and also polyploidy and aneu-
Correspondence: Dr. M. Pavlica, Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, P.O. Box 933, Yu-41001 Zagreb (Yugoslavia).
ploidy (Bennici and D'Amato, 1978; D'Amato, 1978). Nutrient medium composition, growth regulator concentrations and culture conditions (temperature, photoperiod and light) could be the main sources of these changes. Synthetic growth regulators are very frequently used in plant-cell and tissue culture due to their physiological activity similar to plant hormones. 2,4-Dichlorophenoxyacetic acid (2,4-D; CAS No. 94-75-7) is a synthetic auxin widely used in plant-cell and tissue culture. 2,4-D can induce mitotic and meiotic irregularities in plant cells in vivo and in vitro (Bayliss, 1973, 1977; Khalatkar and Bhargava, 1982). Bayliss (1980) reported that
78
concentrations higher than 5 mg/l of 2,4-D could induce different kinds of chromosome aberrations like spindle failure and chromosome mutations. The most frequent types of aberrations induced by 2,4-D were fragments, bridges, laggards and also polyploidy and aneuploidy (Mohandas and Grant, 1972; Fiskesj6 et al., 1981). 2,4-D can also induce chromatin and chromosome fragmentation and erosion (Mohandas and Grant, 1972). In the present study the chromatin and chromosome changes induced by 2,4-D in shallot root-tip cells and the genotoxic and mutagenic effects of V79 Chinese hamster fibroblast cells were examined and compared. Material and methods
Plant test material In our experiments equal-sized bulbs from a diploid (2n= 16) population of shallot Allium ascalonicum L. (syn. A. cepa var. aggregatum) were used. Bulbs were grown in water until the roots were about 1.5-2 cm long, and then roots were immersed in a 2,4-D (Sigma Chemical Co.) and water solution (45 #M and 450 #M). Normal tap water was used as a control (Fiskesj6, 1988). The roots were incubated in a growth room at 26 ___1°C, exposed to artificial light for 16 h per day at a light intensity of 17 W / m 2. For microscopic studies root tips were cut 2 and 24 h after treatment (recovery period was 24 h in tap water after each treatment). Material was fixed in ethanol:acetic
acid (3:1) for 24 h, and slides were prepared using the Feulgen squash technique (Sharma and Sharma, 1972). Permanent slides were prepared using liquid carbon dioxide and after 1 day drying they were mounted in Euparal. At least 2000 cells were scored (100 per slide) per treatment point and analyzed (Fiskesj6, 1985). Animal cells and culture conditions Chinese hamster V79 fibroblast cells were maintained as stock cultures in a-minimal essential medium (ot-MEM, Gibco) with 1007o fetal calf serum (Flow Laboratories) in a humidified atmosphere containing 5070 CO2 and 9507o air at 37°C. All experiments were performed in triplicate with exponentially growing cells. Survival studies Exponentially growing cultures of V79 cells were treated with various concentrations of 2,4-D (10, 25 and 100 #g/ml) for 60 min. 2,4-D water solution was autoclaved for 20 rain before use. After treatment all cultures were washed twice with PBS (8.1 mM Na2HPO4, 1.5 mM KHzPO4, 0.14 M NaCI, 2.6 mM KC1) and trypsinized. Cell survival was determined by plating appropriate numbers of cells to give about 200 colonies per dish, 6 dishes per experimental point (Nagy et al., 1986). Mutation assay for H G P R T mutants Cells were plated into 150-cm2 T-flasks at a cell density of 2 × 106 cells per flask. After 24 h of growth cells were treated with 2,4-D according to
TABLE 1 MITOTIC INDEX AND PERCENTAGE OF ABERRANT SHALLOT ROOT-TIP CELLS INDUCED BY 2,4-D Treatment time (h) 2
24 + 24 h of tap water *P
O 0
i
i
•
•
20
40
60
80
i
100
12o
2,4-D ~g/ml) Fig. 2. 6-Thioguanine-resistantmutant frequencyafter 1 h of treatment with 2,4-D.
2,4-D and the frequency of mutation colonies per survivor for all doses tested. Discussion The data obtained in this study prove that the synthetic auxin 2,4-D has a strong cytotoxic and mutagenic effect on plant and mammalian cells. Results on plant cells showed that 2,4-D affected cell division, and induced mitotic abnormalities in the form of chromosomal aberrations. The comparison of total chromosomal aberrations to mitotic activity showed that these two effects were parallel in both concentrations (Table 1). 2,4-D induced chromosome mutations such as ana- and telophase bridges, fragments and laggards, and micronuclei. Also, there was a strong effect on spindle function resulting in c-mitosis (Fiskesj6 et al., 1981) and disturbed anaphases. Both events could be a consequence of 2,4-D binding to protein which is subjected to spindle microtubule (Mohandas and Grant, 1972; Brinkley et al., 1985). Chromatin fragmentation and erosion in interphase nuclei (Grant, 1982b) which were noticed at a concentration of 450 ttM in the form of chromatin bodies have also been described in other plant species such as Allium cepa and Vicia faba (Mohandas and Grant, 1972). Chromosome stickiness appearing in meta-, aria- and telophase is probably a subchromatid aberration described by McGill et al. (1974) and Kla~tersk~t et al. (1976). 2,4-D stopped cell division in prophase which could be connected with the delay in spindle function. The same effect was noticed by Von D6bel (1983) who also mentioned that 2,4-D induced restitution nuclei, which we also observed in our experiments. Since it is known that 2,4-D integrates into chromosomes of root tips of Viciafaba and Allium cepa (Liao and Hamilton, 1966), and also forms complexes with proteins, a possible explanation for chromosomal aberration induction could be a direct effect of 2,4-D on nucleic acid. Data obtained on V79 cells showed that 2,4-D had cytotoxic and mutagenic effects in a concentration range higher than commonly used in
81
establishing plant tissue culture. Also, 2,4-D showed a genotoxic effect on bone marrow cells in vitro (Pilinskaya, cited by Grant, 1982a). However, there is clear evidence that 2,4-D produces similar effects on plant and mammalian cells. It induces chromosome aberrations in shallot root tips and H G P R T - mutants in V79 Chinese hamster fibroblast cells, suggesting that the plant cell system shows similarities and may replace mammalian cells in screening and testing mutagens in vitro (Clive and Spector, cited by Nilan et al., 1978; Ma, 1982). References Bayliss, M.W. (1973) Origin of chromosome number variation in cultured plant cells, Nature (London), 246, 529-530. Bayliss, M.W. (1977) Factors affecting the frequency of tetraploid cells in a predominantly diploid suspension culture, Protoplasma, 92, 109-115. Bayliss, M.W. (1980) Chromosomal variation in plant tissue in culture, Int. Rev. Cytol., Suppl. IlA. Bennici, A., and F. D'Amato (1978) In vitro regeneration of durum wheat plants, I. Chromosome numbers of regenerated plantlets, Z. Pflanzucht., 81,305-311. Brinkley, B.R., A. Tousson and M.M. Valdivia (1985) The kinetochore of mammalian chromosomes: structure and function in normal mitosis and aneuploidy, in: V.L. Dellarco, P.E. Voytek and A. Hollaender (Eds.), Aneuploidy, Etiology and Mechanisms, Plenum, New York, pp. 243-265. Constantin, M.J. (1981) Chromosome instability in cell and tissue cultures and regenerated plants, Environ. Exp. Bot., 21,359-368. D'Amato, F. (1978) Chromosome number variation in cultured cells and regenerated plants, in: T.A. Thorpe (Ed.), Frontiers of Plant Tissue Culture, University of Calgary Offset Printing Services, Calgary, pp. 287-295. Evans, H.J., M. Ishidate, M. Lung Jr., C.T. Miller, F. Mitelman and E. Vogel (1980) Cytogenetic damage as an end point in short term assay systems for detecting environmental carcinogens, in: R. Montesano et al. (Eds.), Molecular and Cellular Aspects of Carcinogens Screening Test, IARC Scientific Publications, Lyon, pp. 227-244. Fiskej6, G. (1985) The Allium-test as a standard in environmental monitoring, Hereditas, 102, 99-112. Fiskesj6, G. (1988) The Allium test - an alternative in environmental studies: the relative toxicology of metal ions, Mutation Res., 197, 243-291. Fiskesj6, G., C. Lassen and L. Renberg (1981) Chlorinated phenoxyacetic acids and chlorophenols in the modified Allium test, Chem.-Biol. Interact., 34, 333-344. Grant, W.F. (1982a) Chromosome aberration assays in Allium:
a report of the U.S. Environmental Protection Agency GeneTox Program, Mutation Res., 99, 273-291. Grant, W.F. (1982b) Cytogenetic studies of agricultural chemicals in plants, in: R.A. Fleck and A. Hollaender (Eds.), Genetic Toxicology: An Agricultural Perspective, Plenum, New York, pp. 353-378. Khalatkar, A.S., and Y.R. Bhargava (1982) 2,4-Dichlorophenoxyacetic acid - a new environmental mutagen, Mutation Res., 103, 111-114. Kla~tersk/t, A., T. Natarajan and C. Ramel (1976) An interpretation of the origin of subchromatid aberrations and chromosome stickiness as a category of chromatid aberrations, Hereditas, 83, 153-162. Larkin, P.J., and W.R. Scowcroft ( 1981) Somaclonal variation - a novel source of variability from cell cultures for plant improvement, TAG, 60, 197-214. Liao, S., and R.H. Hamilton (1966) Intracellular localization of growth hormones in plants, Science, 151,822-824. Ma, T.-H. (1982) Vicia cytogenetic tests for environmental mutagens: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 99, 257-271. McGill, M., S. Pathak and T.C. Hsu (1974) Effects of ethidium bromide on mitosis and chromosomes: a possible material basis for chromosome stickiness, Chromosoma, 47, 157-167. Mohandas, T., and W. Grant (1972) Cytogenetic effects of 2,4-D and amitrole in relation to nuclear volume and DNA content in some higher plants, Can. J. Genet. Cytol., 14, 773-783. Nagy, B., P.J. Dale and D. Grdina (1986) Protection against cis-diamminedichloroplatinum cytotoxicity and mutagenicity in V79 cells by 2-/(aminopropyl)amino/ethanethiol, Cancer Res., 46, 1132-1135. Nilan, R.A., J.L. Rosichan, P. Arenaz, A.L. Hodgon and A. Kleinhofs (1981) Pollen genetic markers for detection of mutagens in the environment, Environ. Health Perspect., 37, 19-25. Paper, D., V. Garaj-Vrhovac, S. Jelaska and B. KolevskaPletikapi~ (1983) Chromosome behaviour in cultured cell populations of higher plants, Kew Chromosome Conference II. Sharma, A.K., and A. Sharma (1972) Chromosome Techniques - Theory and Practice, University Park Press, Baltimore, MD, pp. 56. Sunderland, N. (1973) Nuclear cytology, in: H.E. Street (Ed.), Plant Tissue and Cell Culture, Blackwell, Oxford, pp. 161-190. Thacker, J., M.A. Stephens and A. Stretch (1976)Factors affecting the efficiency of purine analogues as selective agents for mutants of mammalian cells induced by ionising radiation, Mutation Res., 35,465-478. Von D6bel, P. (1983) Verringerung des Ploidiegrades und der Restitutionkernbildung in einer gegen hohe 2,4-DKonzentrationene Suspensionkultur yon Nigella damascena, Biol. Zbl., 102, 431-444. Communicated by M. Ala~evi6
Mutation Research, 263 (1991) 77-81 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 016579929100441 MUTLET 0493
2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells Mirjana Pavlica, Dra~ena Pape~ and Biserka Nagy Department of Molecular Biology, Faculty of Science, Universityof Zagreb, YU-41001Zagreb (Yugoslavia) (Received 4 January 1991) (Revision received 16 January 1991) (Accepted 17 January 1991)
Keywords: 2,4-Dichlorophenoxyaceticacid; Chromosome aberrations; Cytotoxicity; Mutagenicity
Summary The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 #g/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture ( > 5 #g/ml).
It is known that chromosome mutations as well as changes in chromosome number and structure, and also changes during the cell cycle are frequently observed in in vitro systems of plant and animal origin (Sunderland, 1973; D'Amato, 1978; Bayliss, 1980; Evans et al., 1980; Constantin, 1981; Pape~ et al., 1983). Plants regenerated from callus or cell suspension showed genetic and phenotypic variability (Larkin and Scowcroft, 1981), chromosome structure instability, and also polyploidy and aneu-
Correspondence: Dr. M. Pavlica, Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, P.O. Box 933, Yu-41001 Zagreb (Yugoslavia).
ploidy (Bennici and D'Amato, 1978; D'Amato, 1978). Nutrient medium composition, growth regulator concentrations and culture conditions (temperature, photoperiod and light) could be the main sources of these changes. Synthetic growth regulators are very frequently used in plant-cell and tissue culture due to their physiological activity similar to plant hormones. 2,4-Dichlorophenoxyacetic acid (2,4-D; CAS No. 94-75-7) is a synthetic auxin widely used in plant-cell and tissue culture. 2,4-D can induce mitotic and meiotic irregularities in plant cells in vivo and in vitro (Bayliss, 1973, 1977; Khalatkar and Bhargava, 1982). Bayliss (1980) reported that
78
concentrations higher than 5 mg/l of 2,4-D could induce different kinds of chromosome aberrations like spindle failure and chromosome mutations. The most frequent types of aberrations induced by 2,4-D were fragments, bridges, laggards and also polyploidy and aneuploidy (Mohandas and Grant, 1972; Fiskesj6 et al., 1981). 2,4-D can also induce chromatin and chromosome fragmentation and erosion (Mohandas and Grant, 1972). In the present study the chromatin and chromosome changes induced by 2,4-D in shallot root-tip cells and the genotoxic and mutagenic effects of V79 Chinese hamster fibroblast cells were examined and compared. Material and methods
Plant test material In our experiments equal-sized bulbs from a diploid (2n= 16) population of shallot Allium ascalonicum L. (syn. A. cepa var. aggregatum) were used. Bulbs were grown in water until the roots were about 1.5-2 cm long, and then roots were immersed in a 2,4-D (Sigma Chemical Co.) and water solution (45 #M and 450 #M). Normal tap water was used as a control (Fiskesj6, 1988). The roots were incubated in a growth room at 26 ___1°C, exposed to artificial light for 16 h per day at a light intensity of 17 W / m 2. For microscopic studies root tips were cut 2 and 24 h after treatment (recovery period was 24 h in tap water after each treatment). Material was fixed in ethanol:acetic
acid (3:1) for 24 h, and slides were prepared using the Feulgen squash technique (Sharma and Sharma, 1972). Permanent slides were prepared using liquid carbon dioxide and after 1 day drying they were mounted in Euparal. At least 2000 cells were scored (100 per slide) per treatment point and analyzed (Fiskesj6, 1985). Animal cells and culture conditions Chinese hamster V79 fibroblast cells were maintained as stock cultures in a-minimal essential medium (ot-MEM, Gibco) with 1007o fetal calf serum (Flow Laboratories) in a humidified atmosphere containing 5070 CO2 and 9507o air at 37°C. All experiments were performed in triplicate with exponentially growing cells. Survival studies Exponentially growing cultures of V79 cells were treated with various concentrations of 2,4-D (10, 25 and 100 #g/ml) for 60 min. 2,4-D water solution was autoclaved for 20 rain before use. After treatment all cultures were washed twice with PBS (8.1 mM Na2HPO4, 1.5 mM KHzPO4, 0.14 M NaCI, 2.6 mM KC1) and trypsinized. Cell survival was determined by plating appropriate numbers of cells to give about 200 colonies per dish, 6 dishes per experimental point (Nagy et al., 1986). Mutation assay for H G P R T mutants Cells were plated into 150-cm2 T-flasks at a cell density of 2 × 106 cells per flask. After 24 h of growth cells were treated with 2,4-D according to
TABLE 1 MITOTIC INDEX AND PERCENTAGE OF ABERRANT SHALLOT ROOT-TIP CELLS INDUCED BY 2,4-D Treatment time (h) 2
24 + 24 h of tap water *P
O 0
i
i
•
•
20
40
60
80
i
100
12o
2,4-D ~g/ml) Fig. 2. 6-Thioguanine-resistantmutant frequencyafter 1 h of treatment with 2,4-D.
2,4-D and the frequency of mutation colonies per survivor for all doses tested. Discussion The data obtained in this study prove that the synthetic auxin 2,4-D has a strong cytotoxic and mutagenic effect on plant and mammalian cells. Results on plant cells showed that 2,4-D affected cell division, and induced mitotic abnormalities in the form of chromosomal aberrations. The comparison of total chromosomal aberrations to mitotic activity showed that these two effects were parallel in both concentrations (Table 1). 2,4-D induced chromosome mutations such as ana- and telophase bridges, fragments and laggards, and micronuclei. Also, there was a strong effect on spindle function resulting in c-mitosis (Fiskesj6 et al., 1981) and disturbed anaphases. Both events could be a consequence of 2,4-D binding to protein which is subjected to spindle microtubule (Mohandas and Grant, 1972; Brinkley et al., 1985). Chromatin fragmentation and erosion in interphase nuclei (Grant, 1982b) which were noticed at a concentration of 450 ttM in the form of chromatin bodies have also been described in other plant species such as Allium cepa and Vicia faba (Mohandas and Grant, 1972). Chromosome stickiness appearing in meta-, aria- and telophase is probably a subchromatid aberration described by McGill et al. (1974) and Kla~tersk~t et al. (1976). 2,4-D stopped cell division in prophase which could be connected with the delay in spindle function. The same effect was noticed by Von D6bel (1983) who also mentioned that 2,4-D induced restitution nuclei, which we also observed in our experiments. Since it is known that 2,4-D integrates into chromosomes of root tips of Viciafaba and Allium cepa (Liao and Hamilton, 1966), and also forms complexes with proteins, a possible explanation for chromosomal aberration induction could be a direct effect of 2,4-D on nucleic acid. Data obtained on V79 cells showed that 2,4-D had cytotoxic and mutagenic effects in a concentration range higher than commonly used in
81
establishing plant tissue culture. Also, 2,4-D showed a genotoxic effect on bone marrow cells in vitro (Pilinskaya, cited by Grant, 1982a). However, there is clear evidence that 2,4-D produces similar effects on plant and mammalian cells. It induces chromosome aberrations in shallot root tips and H G P R T - mutants in V79 Chinese hamster fibroblast cells, suggesting that the plant cell system shows similarities and may replace mammalian cells in screening and testing mutagens in vitro (Clive and Spector, cited by Nilan et al., 1978; Ma, 1982). References Bayliss, M.W. (1973) Origin of chromosome number variation in cultured plant cells, Nature (London), 246, 529-530. Bayliss, M.W. (1977) Factors affecting the frequency of tetraploid cells in a predominantly diploid suspension culture, Protoplasma, 92, 109-115. Bayliss, M.W. (1980) Chromosomal variation in plant tissue in culture, Int. Rev. Cytol., Suppl. IlA. Bennici, A., and F. D'Amato (1978) In vitro regeneration of durum wheat plants, I. Chromosome numbers of regenerated plantlets, Z. Pflanzucht., 81,305-311. Brinkley, B.R., A. Tousson and M.M. Valdivia (1985) The kinetochore of mammalian chromosomes: structure and function in normal mitosis and aneuploidy, in: V.L. Dellarco, P.E. Voytek and A. Hollaender (Eds.), Aneuploidy, Etiology and Mechanisms, Plenum, New York, pp. 243-265. Constantin, M.J. (1981) Chromosome instability in cell and tissue cultures and regenerated plants, Environ. Exp. Bot., 21,359-368. D'Amato, F. (1978) Chromosome number variation in cultured cells and regenerated plants, in: T.A. Thorpe (Ed.), Frontiers of Plant Tissue Culture, University of Calgary Offset Printing Services, Calgary, pp. 287-295. Evans, H.J., M. Ishidate, M. Lung Jr., C.T. Miller, F. Mitelman and E. Vogel (1980) Cytogenetic damage as an end point in short term assay systems for detecting environmental carcinogens, in: R. Montesano et al. (Eds.), Molecular and Cellular Aspects of Carcinogens Screening Test, IARC Scientific Publications, Lyon, pp. 227-244. Fiskej6, G. (1985) The Allium-test as a standard in environmental monitoring, Hereditas, 102, 99-112. Fiskesj6, G. (1988) The Allium test - an alternative in environmental studies: the relative toxicology of metal ions, Mutation Res., 197, 243-291. Fiskesj6, G., C. Lassen and L. Renberg (1981) Chlorinated phenoxyacetic acids and chlorophenols in the modified Allium test, Chem.-Biol. Interact., 34, 333-344. Grant, W.F. (1982a) Chromosome aberration assays in Allium:
a report of the U.S. Environmental Protection Agency GeneTox Program, Mutation Res., 99, 273-291. Grant, W.F. (1982b) Cytogenetic studies of agricultural chemicals in plants, in: R.A. Fleck and A. Hollaender (Eds.), Genetic Toxicology: An Agricultural Perspective, Plenum, New York, pp. 353-378. Khalatkar, A.S., and Y.R. Bhargava (1982) 2,4-Dichlorophenoxyacetic acid - a new environmental mutagen, Mutation Res., 103, 111-114. Kla~tersk/t, A., T. Natarajan and C. Ramel (1976) An interpretation of the origin of subchromatid aberrations and chromosome stickiness as a category of chromatid aberrations, Hereditas, 83, 153-162. Larkin, P.J., and W.R. Scowcroft ( 1981) Somaclonal variation - a novel source of variability from cell cultures for plant improvement, TAG, 60, 197-214. Liao, S., and R.H. Hamilton (1966) Intracellular localization of growth hormones in plants, Science, 151,822-824. Ma, T.-H. (1982) Vicia cytogenetic tests for environmental mutagens: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 99, 257-271. McGill, M., S. Pathak and T.C. Hsu (1974) Effects of ethidium bromide on mitosis and chromosomes: a possible material basis for chromosome stickiness, Chromosoma, 47, 157-167. Mohandas, T., and W. Grant (1972) Cytogenetic effects of 2,4-D and amitrole in relation to nuclear volume and DNA content in some higher plants, Can. J. Genet. Cytol., 14, 773-783. Nagy, B., P.J. Dale and D. Grdina (1986) Protection against cis-diamminedichloroplatinum cytotoxicity and mutagenicity in V79 cells by 2-/(aminopropyl)amino/ethanethiol, Cancer Res., 46, 1132-1135. Nilan, R.A., J.L. Rosichan, P. Arenaz, A.L. Hodgon and A. Kleinhofs (1981) Pollen genetic markers for detection of mutagens in the environment, Environ. Health Perspect., 37, 19-25. Paper, D., V. Garaj-Vrhovac, S. Jelaska and B. KolevskaPletikapi~ (1983) Chromosome behaviour in cultured cell populations of higher plants, Kew Chromosome Conference II. Sharma, A.K., and A. Sharma (1972) Chromosome Techniques - Theory and Practice, University Park Press, Baltimore, MD, pp. 56. Sunderland, N. (1973) Nuclear cytology, in: H.E. Street (Ed.), Plant Tissue and Cell Culture, Blackwell, Oxford, pp. 161-190. Thacker, J., M.A. Stephens and A. Stretch (1976)Factors affecting the efficiency of purine analogues as selective agents for mutants of mammalian cells induced by ionising radiation, Mutation Res., 35,465-478. Von D6bel, P. (1983) Verringerung des Ploidiegrades und der Restitutionkernbildung in einer gegen hohe 2,4-DKonzentrationene Suspensionkultur yon Nigella damascena, Biol. Zbl., 102, 431-444. Communicated by M. Ala~evi6
