II.2. Some observations on the quenching of EMS mutagenic action in barley by the use of sodium thiosulfate solutions*.
T. J. Arnason, Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0.
* Supported by the National Research Council of Canada.
Sodium thiosulfate (ST) has been used to quench ethyl methanesulfonate, (EMS), action in Neurospora (Malling and de Serres, 1968) and in barley caryopses (Narayanan and Konzak, 1969). Some results obtained from additional experiments with barley are reported here.
Results of tests to determine whether 0.1M or 0.2M Na2S203 solutions induce any chlorophyll mutations in Montcalm barley indicated that they did not (Table 1, lots 1 and 2).
Table 1. Mutation frequencies in barley after EMS and sodium thiosulfate treatments.
The concentration of ST required to stop the mutagenic action of fresh EMS solution was determined. Seeds presoaked in water for 2 hours were treated with 0.5% (near .05M) EMS in 0.1M or 0.2M ST solution, pH at 6.7, for 4 hours at 20°C. After rinsing, seeds were immersed in water for 18 hours at 20°C before seeding. One lot of seeds was treated with EMS in P-buffer, pH 6.7, unquenched. The unquenched EMS induced mutations in over 20% of M1 spikes (Table 1, lot 3). Quenching by 0.1M and 0.2M ST resulted in mutation frequencies of <5% and <1% of spikes, respectively (Table 1, lots 4 and 5). Mutagenic action of 0.5% EMS is thus almost completely stopped by a 0.2M concentration of sodium thiosulfate.
Observations were then made on the quenching effects of 0.1M and 0.2M ST solutions, applied to seeds for 18 hours immediately after treatment with EMS. All of the treatments were with 0.5% EMS, pH 6.7 for 4 or 6 hours at 20°C. The pH of the ST solutions was at 7.3. In Lot 6 (Table 1) quenched with a O.1M solution of ST, spike mutations frequency was lower, by 19%, than in unquenched lot 3. Lot 8 quenched with 0.2M ST after 6 hours in EMS also had fewer mutations than unquenched lot 7. With few exceptions, the frequencies of mutated M2 seedlings paralleled rather closely the frequencies of M1 mutated spikes.
In seeds placed in water after EMS treatment the small amounts of the mutagen remaining in the embryos may continue to add mutations during the next 12-24 hours (Arnason and Satpathy, 1971). Such post-treatment additions of mutations may be largely eliminated by quenching EMS action by the application of sodium thiosulfate solutions immediately after the end of the designated treatment.
References:
Malling, H. V. and F. J. DeSerres. 1968. Mutation Research. 6:181-193.
Narayanan, K. R. and C. F. Konzak. 1969. Induced mutations in plants. IAEA pp. 281-304.
Arnason, T. J. and D. Satpathy. 1971. Barley Genetics. II:119-126.
