II. 18. Azide Mutagenesis - varietal response, pregermination conditions and concentration.
A.L. Hodgdon, R.A. Nilan and A. Kleinhofs, Department of Agronomy and Soils and Program in Genetics, Washington State University, Pullman, Washington 99164, U.S.A.
Sodium azide still remains among the most potent mutagens in barley. Its mechanism of action and the nature of mutations it induces are becoming understood. This development has been accelerated by the discovery of a mutagenic metabolite formed by sodium azide in treated barley cells. Much of the biological, especially mutagenic effects, of azide in barley and other organisms have been recently reviewed (Kleinhofs et al., 1979).
To further enhance the mutagenic effectiveness and efficiency of sodium azide and especially the metabolite in barley, more knowledge about the effect of time and temperature of seed soaking and germination prior to treatment and of concentration and varietal differences is required. Three experiments have been recently conducted to provide some information on these parameters.
It has been shown previously that seed germination conditions prior to azide treatment affect the yield of mutations in barley. These conditions include the period of seed absorption of water at 0°C (without metabolism) and the period of germination at 20°C (Nilan et al., 1973; Kleinhofs et al., 1974). Experiment No. 1 and No. 2 analyzes the effect of the duration of cold presoak and of the duration of germination at 20°C, respectively.
1. The effect of cold (0°C) presoak duration on the azide-induced mutation frequency of Himalaya and Steptoe barley.
Seeds of Himalaya (hulless) and Steptoe (hulled) barley were used to determine the effect of 0°C presoak duration on mutation frequencies. The cold presoak periods tested were 2 hr, 8 hr, 16 hr and 24 hr. Each treatment time was replicated four times with lO00 seeds per replication. Seeds were given the cold (0°C) pregermination, followed by a four hr 20°C presoak with aeration. The seeds were then treated with 1 mM NaN3 in 0.1 M phosphate buffer, pH 3, for two hr with aeration. Following the azide treatment, the seeds were washed for one hr in running tapwater and then stored in a cold room at 4°C until planting. The seeds were planted in randomly chosen plots, one plot per replication. Later, one spike was harvested from each mature plant. The spikes were planted in the greenhouse and scored for chlorophyll-deficient and morphological seedling mutations.
Varying the cold presoak time had no statistically significant effect on azide-induced mutation frequency in Steptoe (Table 1). Moreover, in Himalaya, the spike mutation frequency was less with two hr cold presoak than with 16 or 24 hr frequency. This difference may show a faster inhibition or germination with Steptoe than with Himalaya resulting in more azide uptake with shorter cold presoak in Steptoe. Assays of azide levels in embryos of azide treated seeds indicated that this may indeed be the case.
Table 1. Effect of cold seed soak (0°C) duration on azide-induced mutation frequencies.
2. The effect of 20°C presoak duration on the azide-induced mutation yield in Steptoe and Himalaya barley.
In order to complete the study of the effect of seed germination conditions on the azide-induced mutation frequencies, the 20°C germination time was varied.
Both Himalaya and Steptoe barley were used in this experiment and the experimental conditions were the same as in Experiment No. 1, except for the following. The seeds were given 16 hr of cold (0°C) presoak followed by different 20°C germinating periods with aeration prior to azide treatment. These periods were 0, 4, 8, 12, 16 and 20 hr.
Table 2 shows the results of this experiment. The two varieties responded differently to the different germination tines. The response of Steptoe was not great with sterility (seedlings/spike) and spike mutation frequencies peaking at the shorter times. Seedling frequency rate, however, was significantly lower with 0 hr than at 4, 8 or 12 hr. With Himalaya, the maximum responses are at 8 hr with much reduced responses with 0 hr. Work is now underway to correlate this mutation data with azide and azide metabolite level in extracts of embryos from azide treated seeds.
Table 2. Effect of 20°C seed germination duration on azide-induced mutation frequencies.
3. The mutagenic effect of varying sodium azide concentration in Steptoe barley.
With the discovery of the azide metabolite in barley cells, the need for knowledge about the effects of azide concentration per se and metabolite concentration effect has increased. This study provides some basic concentration data for azide in Steptoe barley - a hulled strain.
The effect of concentrations of sodium azide (5 x 10-4 M to 2.5 x 10-3 M) on Himalaya barley was determined by Konzak et al., 1975. A broader range of azide concentration, 10-5 M, 10-4 M, 5 x 10-4 M, 10-3 M, 2 x 10-3 M, 5 x 10-3 M and 10-2 M, was tested in Steptoe barley. Four replications of 1000 seeds/ rep were used with each treatment. The seeds were given a 16 hr cold (0°C) presoak treatment followed by four hr of 20°C presoak under aeration. The seeds were then treated with the appropriate concentration of sodium azide in 0.1 M phosphate buffer pH 3 under aeration for two hr. The rest of the experimental details are as described in Experiment No. 1.
Table 3 presents the data from the eight concentrations. Both the mutagenic effects and sterility (seedlings/spike) induced by sodium azide plateaued at 10-3 M. It confirms the choice of this concentration for standard azide treatments. This data will be used to determine if there is a correlation between azide metabolite level and mutation frequency with a given concentration of azide.
Table 3. Azide concentration experiment with Steptoe barley.
References:
Nilan, R. A., E. G. Sideris, A. Kleinhofs, C. Sander and C. F. Konzak, 1973. Azide--a potent mutagen, Mutation Res., 17:142-144.
Kleinhofs, A., W. M. Owais and R. A. Nilan, 1979. Azide, Mutation Res., (Reviews in Genetic Toxicology). In press.
Kleinhofs, A., C. Sander, R. A. Nilan and C. F. Konzak, 1974. Azide mutagenicity--Mechanism and nature of mutants produced, in: Polyploidy and induced mutations in plant breeding, International Atomic Energy Agency, Vienna, pp. 195-199.
Konzak, C. F., M. Niknejad, I. Wickham and E. Donaldson, 1975. Mutagenic interaction of sodium azide on mutations induced in barley seeds treated wtih diethyl sulfate or N-methyl-N'-nitrosourea, Mutation Res., 30:55-62.
