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Effect of Plant Tissue Culture Media on the Frequency of Somatic Mutations in Tradescantia Stamen Hairs
Effect of Plant Tissue Culture Media on the Frequency of Somatic Mutations in Tradescantia Stamen Hairs
J. DOLEZEL and F.]. NOVAK Czechoslovak Academy of Sciences, Institute of Experimental Botany, Sokolovska 6, CS-77200 Olomouc, Czechoslovakia Received October 31, 1983 . Accepted December 2, 1983
Summary The mutagenicity of the plant tissue culture medium and its hormonal composition was tested in the Tradescantia stamen hair system. Although this system is highly sensitive to chemical mutagens, no alteration was observed in the frequency of somatic mutations following direct application of the culture medium to Tradescantia inflorescences regardless of its hormonal composition. It is suggested that plant tissue culture media have no direct effect on the induction of mutations in cells cultured in vitro. Key words: Plant tissue culture, genetic variability, somatic mutations, growth regulators, Tradescantia stamen hairs.
Introduction Genetic instability of plant tissue cultures has been reported to be of very general and common occurrence (Bayliss, 1980; Chaleff, 1981; Constantin, 1981; D'Amato, 1977; Novak, 1978; Skirvin, 1978). Due to this fact plant tissue culture may be regarded as a novel source of genetic variability (Larkin and Scowcroft, 1981). For even more general use it would be desirable to know the mechanism and sources of plant tissue culture variability_ No adequate explanation has ever been found, however, for the occurrence of culture variation. Some authors (Ghosh and Gadgil, 1979; Partanen, 1963; Shamina, 1966) suggest that the physical and chemical composition of nutrient media, especially the presence of certain growth substances, may play an important role in the induction of genetic instability. Bayliss (1980) has recently proposed that it is disorganized growth that leads to karyological instability and not any direct effect of nutrient medium. The objective of the experiments presented here was to find out whether one of the plant tissue culture media (BDS; Dunstan and Short, 1977) with a varying content of cytokinin (kinetin, KIN) and auxins (J3-indoleacetic acid, IAA; 2,4-dichlorophenoxyacetic acid, 2,4-D) causes mutagenic activity in Tradescantia tested at the gene level.
Abbreviations: IAA indole-3-acetic acid; KIN kinetin; MNU N-methyl-N-nitrosourea.
Z. Pjlanzenphysiol. Ed. 114. S. 51-58. 1984.
52
J. DOLEZEL and F. J. NovAK
Materials and Methods The Tradescantia stamen hair system was initially developed for a study on mutagenic effects of ionizing radiation but it has recently been used as a detector of mutagenicity of chemical agents (Grant and Zura, 1982; Underbrink et aI., 1973). The system is based on the occurrence of somatic mutations in the stamen hairs of the clones heterozygous for flower colour (Underbrink et aI., 1973). In the heterozygous plants the phenotype is blue but the recessive pink colour can be expressed if the dominant allele for blue colour is removed by deletion or changed by mutation. In this study we used the Tradescantia clone 4430, kindly supplied by the Brookhaven National Laboratory (USA) via Dr. A. G. Underbrink (Krakow, Poland). This clone is a diploid hybrid (2n = 12) of T. subacaulis x T. hirsutiflora and is heterozygous for flower colour (blue/pink, blue being dominant) - (Emmerling-Thompson and Nawrocky, 1980). The plants of the Tradescantia clone 4430 were propagated asexually and cultivated in a greenhouse. The response of the Tradescantia system to the application of the chemical mutagen N-methyl-N-nitrosourea (MNU) at following doses: distilled water,S, 50, 500/LM was tested as a positive control. The mutagenic effect of plant tissue culture media BDS (Dunstan and Short, 1977) without sucrose and agar with different combinations of growth regulators was evaluated. The pH of all media was adjusted to 5.5. The combinations of basal medium with growth regulators were following: BDS; BDS + 5 /LM 2,4-dichlorophenoxyacetic acid - 2,4-D; BDS + 5 /LM kinetin (KIN); BDS + 5/LM {3-indoleacetic acid (IAA); BDS + 5/LM 2,4-D + 5 ~ KIN; BDS + 5/LM 2,4-D + 10 /LM KIN + 10 ~ IAA. Criterion for the selection of this media combination was suitability for callus cultivation and plant regeneration in Allium sativum tissue culture. In this system in vitro chromosomal variation was evaluated (Novak, 1974; Novak, 1981). The whole inflorescences of Tradescantia were immersed in the solutions of MNU and/or tissue culture media at 22°C for 24 hours (Gichner et aI., 1980). After treatment the cuttings were washed in distilled water for 1 hour and placed in beakers filled with Hoagland's nutrient solution. The cultivation took place in a controlled chamber at 20°C under 16-hour photoperiod and light intensity of 2,000 Ix. The analysis of stamen hairs and pink mutations was carried out for 13 days in succession after treatment. Each day 10 flowers (i. e. 3,000 stamen hairs) were scored. Pink somatic mutations were scored according to Underbrink et al. (1973). One or more contiguous pink cells were considered to have resulted from one mutant event. The mutation frequencies were calculated as a number of pink mutant events per 100 stamen hairs.
Results
The frequency of spontaneous mutant events was 0.505±0.039 per 100 stamen hairs (28,500 hairs analysed). The maximum frequency of mutant events was reached on the 6th to the 9th day after treatment with MNU (Fig. 1). Thus the interval between the 6th and 9th day was considered as the critical period for analysis. The mean mutation frequencies during this period were statistically different (for P = 0.05) from the control for all three concentrations of MNU (5.0 ILM, 50.0 tLM, 500.0 ILM), the differences between the control and various MNU treatments being 0.162±0.078, 1.078±0.175 and 10.821±0.7oo mutant events per 100 stamen hairs, respectively. Fig. 2 shows the variation in the frequency of mutant events during 13 days of analyses following the treatment with nutrient media BDS with different hormonal composition. No well-defined peaks of the frequency were noted during the interval Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
Culture media effect on somatic mutations
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DAYS AFTER TREATMENT Fig. 1: Pink mutation response between days 4 and 13 after immersion of the inflorescences of Tradescantia clone 4430 cuttings in solutions of different concentrations of MNU at 22°C for 24 hours.
between the 6th and 9th day. The results obtained during this interval are summarized in Table 1. The differences between the control and all treatments were found to be statistically nonsignificant for P = 0.05. We tried to assess the effect of higher concentrations (50.0 JtM) of IAA, KIN and 2,4-D in medium BDS on the frequency of pink mutations in stamen hairs. Probably because of their hormonal nature, the application of KIN increased flower yields during the first days after treatment but after the 6th day the flower yield was Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
J. DOLEZEL and F. J. NOVAK
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Fig. 2: The frequencies of pink mutant events in Tradescantia clone 4430 stamen hairs after immersion of the inflorescences in plant tissue culture medium BDS with different hormonal composition at 22°C for 24 hours (2 a - BDS, 2 b - BDS + 5.0 /LM 2,4-D, 2 c - BDS + 5.0 /LM KIN, 2 d BDS + 5.0 ~ IAA, 2 e - BDS + 5.0 /LM 2,4-D+5.0/LM KIN, 2f - BDS+5.0/LM 2,4-D + 10.0/LM KIN + 10.0 /LM IAA).
Culture media effect on somatic mutations
55
Table 1: Frequencies of pink mutations induced in Tradescantia clone 4430 stamen hairs after immersion of the inflorescences in plant tissue culture medium BDS with different hormonal composition at 22°C for 24 hours. Treatment
No. of hairs Pink events/lOO hairs*) Pink events/lOO hairs scored (minus control) ± S. E. ±S.E.
BDS
12000 12000 12000 12000 12000
BDS+5.0~2,4-D
BDS + 5.0 J.tM KIN BDS + 5.0 ~ IAA BDS + 5.0 J.tM 2,4-D + 5.0 J.tM KIN BDS + 5.0 ~ 2,4-D + 10.0 J.tM KIN + +10.0J.tMIAA 12000 Control (distilled water) 28500
0.475 ±0.057 0.491 ± 0.056 0.483±0.06l 0.508 ± 0.066 0.508±0.056
-0.030±0.069**) -0.014±0.068**) -0.022±0.072**) +0.003 ±0.077**) +0.003±0.068**)
0.500 ± 0.058 0.505 ±0.039
-0.005 ±0.070**)
*) Mutant frequencies are averages of data collected on peak response days 6-9 post treatment with the exception of control which was evaluated during the whole experiment (days 4-13). **) Statistically nonsignificant at P = 0.05.
so low that it made the scoring of appropriate number of stamen hairs impossible, and the application of IAA and 2,4-D caused the inhibition of flowering and drying up of the cuttings. This herbicidal effect was especially intensive after 2,4-D treatment. Discussion
The Tradescantia stamen hair system is extremely sensitive to radiation and chemical mutagens (Ichikawa and Takahashi, 1978; Nauman et al., 1976). A linear relationship between mutation frequency and dosage was found over a wide range of radiation levels with a significant response to a dose as low as 250 mrad with no evidence for a treshold one (Schairer et aI., 1978). Out of all the clones used so far the clone 4430 is the most sensitive to chemical mutagens (Sparrow et aI., 1974). In our work, all test solutions were directly applied to the inflorescences. Although this treatment caused damage to the flowers for the first four days, it was possible to score the flowers at the proper time. We used this method because direct application of test solutions to the inflorescences was shown to increase the rather high sensitivity of Tradescantia stamen hairs (clone 4430) to chemical mutagens (Gichner et aI., 1980) as compared to the immersion of the cutting stems in solutions. Very high sensitivity of the Tradescantia stamen hair system to chemical mutagens was confirmed in our experiment using low concentrations of MNU (Fig. 1). The spontaneous mutation frequency equalled to 0.505 mutant events per 100 hairs. This value is quite high but it does not exceed that of 0.521, which is the highest spontaneous mutation ratio found in Tradescantia clone 4430 (Sparrow and Sparrow, 1976). The differences in the spontaneous pink mutation rates can be explained in Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
56
J. DOLEZEL and F. J. NOVAK
terms of culture conditions, intraclonal variability and seasonal effects (Schairer et al., 1978). In the present study the application of plant tissue culture medium BDS with different hormonal composition (IAA, KIN, 2,4-D) did not induce increased frequencies of pink mutations. The mutagenicity of IAA and KIN has been studied to a limited extent only. It has been shown that higher concentrations (ranging from 5 p.M to 100 p.M) of these substances have an inhibitory effect on mitotic activity but cause no chromosome aberrations (Me Manus, 1959; Sen, 1974; Therman, 1951). On the other hand, it was clearly established that synthetic auxin 2,4-D used at higher (herbicidal) concentrations can induce mitotic and chromosome aberrations (Mohandas and Grant, 1972; Nygren, 1949; Sikka and Sharma, 1976), which suggests its mutagenicity. Low concentrations of 2,4-D, however, used in plant tissue culture media, do not cause such disturbances and probably have no mutagenic effect (Dolezel and Novak, 1983; Styles, 1973). The Tradescantia stamen hair system does not seem to be an ideal model for assessing mutagenicity of the compounds with hormonal activity, either because of their interference with flowering or because of their herbicidal effect. The in vitro culture of Tradescantia stamen hairs (Nagashima-Ishii and Ichikawa, 1982) may help to overcome this problem. Although we have used the same concentrations of growth regulators as they are usually used in plant tissue culture media, total doses (concentration x time) were probably lower than those received by in vitro cultured cells. Unfortunatelly, the experimental system did not allow to prolong the length of treatment nor the use of higher concentrations. With these limitations in mind we can conclude that plant tissue culture medium BDS with different combinations of three most frequently used growth regulators (IAA, KIN, 2,4-D) probably has no mutagenic effect and so has probably no direct effect on induction of mutations in plant tissue cultures. Acknowledgements We gratefully acknowledge the critical reading of the manuscript by Dr. J. Vdemlnsky and Dr. A. Murin and revision of the English manuscript by Miss J. Potomkova.
References BAYLISS, M. W.: Chromosomal variation in plant tissues in culture. In: I. K. VASIL (Ed.): Perspectives in Plant Cell and Tissue Culture, Int. Rev. Cytol. Suppl. IIA, 113-144. Academic Press, New York, 1980. CHALEFF, R. S.: Genetics of Higher Plants. Applications of Cell Culture. Cambridge University Press, Cambridge, 1981. CONSTANTIN, M. J.: Chromosome instability in cell and tissue cultures and regenerated plants. Environ. Exp. Bot. 21, 359-368 (1981). D'AMATO, F.: Cytogenetics of differentiation in tissue and cell cultures. In: J. REINERT and Y. P. s. BAJAJ (Eds.): Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, pp. 343-357. Springer-Verlag, Berlin, 1977.
Z. Pjlanzenphysiol. Ed. 114. S. 51-58. 1984.
Culture media effect on somatic mutations
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DOLEZEL, J. and F. J. NOVAK: Cytogenetic effect of plant tissue culture medium with certain growth substances on Allium sativum L. meristem root tip cells. BioI. Plant. (1983), in press. DUNSTAN, D. 1. and K. C. SHORT: Improved growth of tissue culture of the onion Allium cepa. Physiol. Plant. 41, 70-72 (1977). EMMERLING-THOMPSON, M. and M. M. NAWROCKY: Genetic Basis for using Tradescantia clone 4430 as an environmental monitor of mutagens. J. Hered. 71,261-265 (1980). GHOSH, A. and V. N. GADGIL: Shift in ploidy level of callus tissue: a function of growth substances. Indian J. Exp. BioI. 17, 562-564 (1979). GICHNER, T., J. VELEMINSKY, and A. G. UNDERBRING: Induction of somatic mutations by the promutagen dimethylnitrosoamine in hairs of Tradescantia stamen. Mutat. Res. 78, 381-384 (1980). GRANT, W. F. and K. D. ZURA: Plants as sensitive in situ detectors of atmospheric mutagens. In: H. A. HEDDLE (Ed.): Mutagenicity: New Horizons in Genetic Toxicology, pp. 407-434. Academic Press, New York, 1982. IcHIRAWA, S. and C. S. TAKAHASHI: Somatic mutations in Tradescantia stamen hairs exposed to ethyl methanesulfonate. Environ. Exp. Bot. 18, 19-25 (1978). LARKIN, P. J. and W. R. SCOWCROFT: Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60, 197-214 (1981). Mc MANUS, M. A.: Certain mitotic effects of kinetin, gibberellic acid, maleic hydrazide and indole acetic acid in onion roots. Proc. Iowa Acad. Sci. 56, 74-80 (1959). MOHANDAS, T. and W. F. GRANT: 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 (1972). NAGASHIMA-IsHII, C. and S. IcHIKAWA: Growth and coloration of Tradescantia stamen hairs cultured in vitro. Environ. Exp. Bot. 22, 255-261 (1982). NAUMAN, C. H., A. H. SPARROW, and L. A. SCHAIRER: Comparative effects of ionizing radiation and two gaseous chemical mutagens on somatic mutation induction in one mutable and two non-mutable clones of Tradescantia. Mutat. Res. 38, 53-70 (1976). NOVAK, F. J.: The changes of karyotype in callus cultures of Allium sativum L. Caryologia 27, 45-54 (1974). - Genetic stability of plant tissue cultures (in Czech). Biologicke listy 43, 33-41 (1978). - Chromosomal characteristics of long-term callus cultures of Allium sativum L. Cytologia 46,371-379 (1981). NYGREN, A.: Cytological studies of the effects of 2,4-D, MCPA and 2,4,5-T on Allium cepa. Ann. Roy. Agric. ColI. Sweden 16, 723-728 (1949). SCHAIRER, L. A., J. VAN'T HOF, C. G. HAYES, R. M. BURTON, and F. J. DE SERRES: Exploratory monitoring of air pollutans for mutagenicity activity with the Tradescantia stamen hair system. Environ. Health Perspectives 27, 51-60 (1978). SEN, S.: Differentiated nuclei and elucidation of chromosomal control of differentiation. Nucleus 17, 40-51 (1974). SHAMINA, Z. B.: Cytogenetic study of tissue culture of Haplopappus gracilis. In: Z. LANDA (Ed.): Proc. Symp. The Mutation Process: Mechanism of Mutation and Inducing Factors, pp. 377-380. Academia, Prague, 1966. SIKKA, K. and A. K. SHARMA: The effects of some herbicides on plant chromosomes. Proc. Indian Acad. Sci. 42, 299-307 (1976). SKIRVIN, R. M.: Natural and induced variation in tissue culture. Euphytica 27, 241-266 (1978). SPARROW, A. H., L. A. SCHAIRER, and R. VILLALOBOS-PIETRINI: Comparison of somatic mutation rates induced in Tradescantia by chemical and physical mutagens. Mutat. Res. 26, 265-276 (1974).
Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
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SPARROW, A. H. and R. C. SPARROW: Spontaneous somatic mutation frequencies for flower color in several Tradescantia species and hybrids. Environ. Exp. Bot. 16,23-43 {1976}. STYLES, J. A.: Cytotoxic effects of various pesticides in vivo and in vitro. Mutat. Res. 21, 50-51 {1973}. THERMAN, E.: The effect of indole-3 acetic acid on resting plant nuclei. I - Allium cepa. Ann. Acad. Sci. Fenn. 16, 1-40 {1951}. UNDERBRINK, A. G., L. A. SCHAIRER, and A. H. SPARROW: Tradescantia stamen hairs: a radiobiological test system applicable to chemical mutagenesis. In: A. HOllAENDER {Ed.}: Chemical Mutagens: Principles and Methods for their Detection, pp. 171-207. Plenum Press, New York, 1973.
Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
J. DOLEZEL and F.]. NOVAK Czechoslovak Academy of Sciences, Institute of Experimental Botany, Sokolovska 6, CS-77200 Olomouc, Czechoslovakia Received October 31, 1983 . Accepted December 2, 1983
Summary The mutagenicity of the plant tissue culture medium and its hormonal composition was tested in the Tradescantia stamen hair system. Although this system is highly sensitive to chemical mutagens, no alteration was observed in the frequency of somatic mutations following direct application of the culture medium to Tradescantia inflorescences regardless of its hormonal composition. It is suggested that plant tissue culture media have no direct effect on the induction of mutations in cells cultured in vitro. Key words: Plant tissue culture, genetic variability, somatic mutations, growth regulators, Tradescantia stamen hairs.
Introduction Genetic instability of plant tissue cultures has been reported to be of very general and common occurrence (Bayliss, 1980; Chaleff, 1981; Constantin, 1981; D'Amato, 1977; Novak, 1978; Skirvin, 1978). Due to this fact plant tissue culture may be regarded as a novel source of genetic variability (Larkin and Scowcroft, 1981). For even more general use it would be desirable to know the mechanism and sources of plant tissue culture variability_ No adequate explanation has ever been found, however, for the occurrence of culture variation. Some authors (Ghosh and Gadgil, 1979; Partanen, 1963; Shamina, 1966) suggest that the physical and chemical composition of nutrient media, especially the presence of certain growth substances, may play an important role in the induction of genetic instability. Bayliss (1980) has recently proposed that it is disorganized growth that leads to karyological instability and not any direct effect of nutrient medium. The objective of the experiments presented here was to find out whether one of the plant tissue culture media (BDS; Dunstan and Short, 1977) with a varying content of cytokinin (kinetin, KIN) and auxins (J3-indoleacetic acid, IAA; 2,4-dichlorophenoxyacetic acid, 2,4-D) causes mutagenic activity in Tradescantia tested at the gene level.
Abbreviations: IAA indole-3-acetic acid; KIN kinetin; MNU N-methyl-N-nitrosourea.
Z. Pjlanzenphysiol. Ed. 114. S. 51-58. 1984.
52
J. DOLEZEL and F. J. NovAK
Materials and Methods The Tradescantia stamen hair system was initially developed for a study on mutagenic effects of ionizing radiation but it has recently been used as a detector of mutagenicity of chemical agents (Grant and Zura, 1982; Underbrink et aI., 1973). The system is based on the occurrence of somatic mutations in the stamen hairs of the clones heterozygous for flower colour (Underbrink et aI., 1973). In the heterozygous plants the phenotype is blue but the recessive pink colour can be expressed if the dominant allele for blue colour is removed by deletion or changed by mutation. In this study we used the Tradescantia clone 4430, kindly supplied by the Brookhaven National Laboratory (USA) via Dr. A. G. Underbrink (Krakow, Poland). This clone is a diploid hybrid (2n = 12) of T. subacaulis x T. hirsutiflora and is heterozygous for flower colour (blue/pink, blue being dominant) - (Emmerling-Thompson and Nawrocky, 1980). The plants of the Tradescantia clone 4430 were propagated asexually and cultivated in a greenhouse. The response of the Tradescantia system to the application of the chemical mutagen N-methyl-N-nitrosourea (MNU) at following doses: distilled water,S, 50, 500/LM was tested as a positive control. The mutagenic effect of plant tissue culture media BDS (Dunstan and Short, 1977) without sucrose and agar with different combinations of growth regulators was evaluated. The pH of all media was adjusted to 5.5. The combinations of basal medium with growth regulators were following: BDS; BDS + 5 /LM 2,4-dichlorophenoxyacetic acid - 2,4-D; BDS + 5 /LM kinetin (KIN); BDS + 5/LM {3-indoleacetic acid (IAA); BDS + 5/LM 2,4-D + 5 ~ KIN; BDS + 5/LM 2,4-D + 10 /LM KIN + 10 ~ IAA. Criterion for the selection of this media combination was suitability for callus cultivation and plant regeneration in Allium sativum tissue culture. In this system in vitro chromosomal variation was evaluated (Novak, 1974; Novak, 1981). The whole inflorescences of Tradescantia were immersed in the solutions of MNU and/or tissue culture media at 22°C for 24 hours (Gichner et aI., 1980). After treatment the cuttings were washed in distilled water for 1 hour and placed in beakers filled with Hoagland's nutrient solution. The cultivation took place in a controlled chamber at 20°C under 16-hour photoperiod and light intensity of 2,000 Ix. The analysis of stamen hairs and pink mutations was carried out for 13 days in succession after treatment. Each day 10 flowers (i. e. 3,000 stamen hairs) were scored. Pink somatic mutations were scored according to Underbrink et al. (1973). One or more contiguous pink cells were considered to have resulted from one mutant event. The mutation frequencies were calculated as a number of pink mutant events per 100 stamen hairs.
Results
The frequency of spontaneous mutant events was 0.505±0.039 per 100 stamen hairs (28,500 hairs analysed). The maximum frequency of mutant events was reached on the 6th to the 9th day after treatment with MNU (Fig. 1). Thus the interval between the 6th and 9th day was considered as the critical period for analysis. The mean mutation frequencies during this period were statistically different (for P = 0.05) from the control for all three concentrations of MNU (5.0 ILM, 50.0 tLM, 500.0 ILM), the differences between the control and various MNU treatments being 0.162±0.078, 1.078±0.175 and 10.821±0.7oo mutant events per 100 stamen hairs, respectively. Fig. 2 shows the variation in the frequency of mutant events during 13 days of analyses following the treatment with nutrient media BDS with different hormonal composition. No well-defined peaks of the frequency were noted during the interval Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
Culture media effect on somatic mutations
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DAYS AFTER TREATMENT Fig. 1: Pink mutation response between days 4 and 13 after immersion of the inflorescences of Tradescantia clone 4430 cuttings in solutions of different concentrations of MNU at 22°C for 24 hours.
between the 6th and 9th day. The results obtained during this interval are summarized in Table 1. The differences between the control and all treatments were found to be statistically nonsignificant for P = 0.05. We tried to assess the effect of higher concentrations (50.0 JtM) of IAA, KIN and 2,4-D in medium BDS on the frequency of pink mutations in stamen hairs. Probably because of their hormonal nature, the application of KIN increased flower yields during the first days after treatment but after the 6th day the flower yield was Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
J. DOLEZEL and F. J. NOVAK
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Fig. 2: The frequencies of pink mutant events in Tradescantia clone 4430 stamen hairs after immersion of the inflorescences in plant tissue culture medium BDS with different hormonal composition at 22°C for 24 hours (2 a - BDS, 2 b - BDS + 5.0 /LM 2,4-D, 2 c - BDS + 5.0 /LM KIN, 2 d BDS + 5.0 ~ IAA, 2 e - BDS + 5.0 /LM 2,4-D+5.0/LM KIN, 2f - BDS+5.0/LM 2,4-D + 10.0/LM KIN + 10.0 /LM IAA).
Culture media effect on somatic mutations
55
Table 1: Frequencies of pink mutations induced in Tradescantia clone 4430 stamen hairs after immersion of the inflorescences in plant tissue culture medium BDS with different hormonal composition at 22°C for 24 hours. Treatment
No. of hairs Pink events/lOO hairs*) Pink events/lOO hairs scored (minus control) ± S. E. ±S.E.
BDS
12000 12000 12000 12000 12000
BDS+5.0~2,4-D
BDS + 5.0 J.tM KIN BDS + 5.0 ~ IAA BDS + 5.0 J.tM 2,4-D + 5.0 J.tM KIN BDS + 5.0 ~ 2,4-D + 10.0 J.tM KIN + +10.0J.tMIAA 12000 Control (distilled water) 28500
0.475 ±0.057 0.491 ± 0.056 0.483±0.06l 0.508 ± 0.066 0.508±0.056
-0.030±0.069**) -0.014±0.068**) -0.022±0.072**) +0.003 ±0.077**) +0.003±0.068**)
0.500 ± 0.058 0.505 ±0.039
-0.005 ±0.070**)
*) Mutant frequencies are averages of data collected on peak response days 6-9 post treatment with the exception of control which was evaluated during the whole experiment (days 4-13). **) Statistically nonsignificant at P = 0.05.
so low that it made the scoring of appropriate number of stamen hairs impossible, and the application of IAA and 2,4-D caused the inhibition of flowering and drying up of the cuttings. This herbicidal effect was especially intensive after 2,4-D treatment. Discussion
The Tradescantia stamen hair system is extremely sensitive to radiation and chemical mutagens (Ichikawa and Takahashi, 1978; Nauman et al., 1976). A linear relationship between mutation frequency and dosage was found over a wide range of radiation levels with a significant response to a dose as low as 250 mrad with no evidence for a treshold one (Schairer et aI., 1978). Out of all the clones used so far the clone 4430 is the most sensitive to chemical mutagens (Sparrow et aI., 1974). In our work, all test solutions were directly applied to the inflorescences. Although this treatment caused damage to the flowers for the first four days, it was possible to score the flowers at the proper time. We used this method because direct application of test solutions to the inflorescences was shown to increase the rather high sensitivity of Tradescantia stamen hairs (clone 4430) to chemical mutagens (Gichner et aI., 1980) as compared to the immersion of the cutting stems in solutions. Very high sensitivity of the Tradescantia stamen hair system to chemical mutagens was confirmed in our experiment using low concentrations of MNU (Fig. 1). The spontaneous mutation frequency equalled to 0.505 mutant events per 100 hairs. This value is quite high but it does not exceed that of 0.521, which is the highest spontaneous mutation ratio found in Tradescantia clone 4430 (Sparrow and Sparrow, 1976). The differences in the spontaneous pink mutation rates can be explained in Z. Pjlanzenphysiol. Bd. 114. S. 51-58. 1984.
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terms of culture conditions, intraclonal variability and seasonal effects (Schairer et al., 1978). In the present study the application of plant tissue culture medium BDS with different hormonal composition (IAA, KIN, 2,4-D) did not induce increased frequencies of pink mutations. The mutagenicity of IAA and KIN has been studied to a limited extent only. It has been shown that higher concentrations (ranging from 5 p.M to 100 p.M) of these substances have an inhibitory effect on mitotic activity but cause no chromosome aberrations (Me Manus, 1959; Sen, 1974; Therman, 1951). On the other hand, it was clearly established that synthetic auxin 2,4-D used at higher (herbicidal) concentrations can induce mitotic and chromosome aberrations (Mohandas and Grant, 1972; Nygren, 1949; Sikka and Sharma, 1976), which suggests its mutagenicity. Low concentrations of 2,4-D, however, used in plant tissue culture media, do not cause such disturbances and probably have no mutagenic effect (Dolezel and Novak, 1983; Styles, 1973). The Tradescantia stamen hair system does not seem to be an ideal model for assessing mutagenicity of the compounds with hormonal activity, either because of their interference with flowering or because of their herbicidal effect. The in vitro culture of Tradescantia stamen hairs (Nagashima-Ishii and Ichikawa, 1982) may help to overcome this problem. Although we have used the same concentrations of growth regulators as they are usually used in plant tissue culture media, total doses (concentration x time) were probably lower than those received by in vitro cultured cells. Unfortunatelly, the experimental system did not allow to prolong the length of treatment nor the use of higher concentrations. With these limitations in mind we can conclude that plant tissue culture medium BDS with different combinations of three most frequently used growth regulators (IAA, KIN, 2,4-D) probably has no mutagenic effect and so has probably no direct effect on induction of mutations in plant tissue cultures. Acknowledgements We gratefully acknowledge the critical reading of the manuscript by Dr. J. Vdemlnsky and Dr. A. Murin and revision of the English manuscript by Miss J. Potomkova.
References BAYLISS, M. W.: Chromosomal variation in plant tissues in culture. In: I. K. VASIL (Ed.): Perspectives in Plant Cell and Tissue Culture, Int. Rev. Cytol. Suppl. IIA, 113-144. Academic Press, New York, 1980. CHALEFF, R. S.: Genetics of Higher Plants. Applications of Cell Culture. Cambridge University Press, Cambridge, 1981. CONSTANTIN, M. J.: Chromosome instability in cell and tissue cultures and regenerated plants. Environ. Exp. Bot. 21, 359-368 (1981). D'AMATO, F.: Cytogenetics of differentiation in tissue and cell cultures. In: J. REINERT and Y. P. s. BAJAJ (Eds.): Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, pp. 343-357. Springer-Verlag, Berlin, 1977.
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DOLEZEL, J. and F. J. NOVAK: Cytogenetic effect of plant tissue culture medium with certain growth substances on Allium sativum L. meristem root tip cells. BioI. Plant. (1983), in press. DUNSTAN, D. 1. and K. C. SHORT: Improved growth of tissue culture of the onion Allium cepa. Physiol. Plant. 41, 70-72 (1977). EMMERLING-THOMPSON, M. and M. M. NAWROCKY: Genetic Basis for using Tradescantia clone 4430 as an environmental monitor of mutagens. J. Hered. 71,261-265 (1980). GHOSH, A. and V. N. GADGIL: Shift in ploidy level of callus tissue: a function of growth substances. Indian J. Exp. BioI. 17, 562-564 (1979). GICHNER, T., J. VELEMINSKY, and A. G. UNDERBRING: Induction of somatic mutations by the promutagen dimethylnitrosoamine in hairs of Tradescantia stamen. Mutat. Res. 78, 381-384 (1980). GRANT, W. F. and K. D. ZURA: Plants as sensitive in situ detectors of atmospheric mutagens. In: H. A. HEDDLE (Ed.): Mutagenicity: New Horizons in Genetic Toxicology, pp. 407-434. Academic Press, New York, 1982. IcHIRAWA, S. and C. S. TAKAHASHI: Somatic mutations in Tradescantia stamen hairs exposed to ethyl methanesulfonate. Environ. Exp. Bot. 18, 19-25 (1978). LARKIN, P. J. and W. R. SCOWCROFT: Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60, 197-214 (1981). Mc MANUS, M. A.: Certain mitotic effects of kinetin, gibberellic acid, maleic hydrazide and indole acetic acid in onion roots. Proc. Iowa Acad. Sci. 56, 74-80 (1959). MOHANDAS, T. and W. F. GRANT: 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 (1972). NAGASHIMA-IsHII, C. and S. IcHIKAWA: Growth and coloration of Tradescantia stamen hairs cultured in vitro. Environ. Exp. Bot. 22, 255-261 (1982). NAUMAN, C. H., A. H. SPARROW, and L. A. SCHAIRER: Comparative effects of ionizing radiation and two gaseous chemical mutagens on somatic mutation induction in one mutable and two non-mutable clones of Tradescantia. Mutat. Res. 38, 53-70 (1976). NOVAK, F. J.: The changes of karyotype in callus cultures of Allium sativum L. Caryologia 27, 45-54 (1974). - Genetic stability of plant tissue cultures (in Czech). Biologicke listy 43, 33-41 (1978). - Chromosomal characteristics of long-term callus cultures of Allium sativum L. Cytologia 46,371-379 (1981). NYGREN, A.: Cytological studies of the effects of 2,4-D, MCPA and 2,4,5-T on Allium cepa. Ann. Roy. Agric. ColI. Sweden 16, 723-728 (1949). SCHAIRER, L. A., J. VAN'T HOF, C. G. HAYES, R. M. BURTON, and F. J. DE SERRES: Exploratory monitoring of air pollutans for mutagenicity activity with the Tradescantia stamen hair system. Environ. Health Perspectives 27, 51-60 (1978). SEN, S.: Differentiated nuclei and elucidation of chromosomal control of differentiation. Nucleus 17, 40-51 (1974). SHAMINA, Z. B.: Cytogenetic study of tissue culture of Haplopappus gracilis. In: Z. LANDA (Ed.): Proc. Symp. The Mutation Process: Mechanism of Mutation and Inducing Factors, pp. 377-380. Academia, Prague, 1966. SIKKA, K. and A. K. SHARMA: The effects of some herbicides on plant chromosomes. Proc. Indian Acad. Sci. 42, 299-307 (1976). SKIRVIN, R. M.: Natural and induced variation in tissue culture. Euphytica 27, 241-266 (1978). SPARROW, A. H., L. A. SCHAIRER, and R. VILLALOBOS-PIETRINI: Comparison of somatic mutation rates induced in Tradescantia by chemical and physical mutagens. Mutat. Res. 26, 265-276 (1974).
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SPARROW, A. H. and R. C. SPARROW: Spontaneous somatic mutation frequencies for flower color in several Tradescantia species and hybrids. Environ. Exp. Bot. 16,23-43 {1976}. STYLES, J. A.: Cytotoxic effects of various pesticides in vivo and in vitro. Mutat. Res. 21, 50-51 {1973}. THERMAN, E.: The effect of indole-3 acetic acid on resting plant nuclei. I - Allium cepa. Ann. Acad. Sci. Fenn. 16, 1-40 {1951}. UNDERBRINK, A. G., L. A. SCHAIRER, and A. H. SPARROW: Tradescantia stamen hairs: a radiobiological test system applicable to chemical mutagenesis. In: A. HOllAENDER {Ed.}: Chemical Mutagens: Principles and Methods for their Detection, pp. 171-207. Plenum Press, New York, 1973.
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