GrainGenes

II.32. Increasing the mutagenic efficiency of sodium azide in barley.

C. Sander and R. A. Nilan. Department of Agronomy and Soils, Washington State University, Pullman, Washington, 99l63, USA.

The potent mutagenicity of sodium azide in barley was previously reported from this laboratory (Nilan et al. 1973). Furthermore, it is a highly efficient mutagen, as it appears to be unable to produce chromosome breaks. The effectiveness was shown to be dependent on both the pH of treatment and the state of germination of barley seedling. Thus it was shown that the hydrazoic acid form was essential for mutagenicity and the mutagenic effect was enhanced in O2, and particularly if the seeds were germinated for some hours prior to treatment.

We now present additional information on the effect of germination on mutagenesis by azide in barley. Himalaya barley seeds were presoaked for 15 hours at 1-4°C in distilled water, then transferred to aerated distilled water at 20°C for 0, 4, 8, 12, 16, 20, 24, and 28 hours prior to treatment with 10^-3M HN₃. The 0, 4, 8, and 12 hour germinated seeds were treated with azide for two hours while the longer germinated seeds were treated for 1 hour. For the O and 4 hour germination periods, 1,200 seeds were used per treatment. With longer germination the number of seeds was increased. Thus, for the 16 and 24 hour germination treatments, 5,200 seeds were planted per plot. This large number of treated seeds was to allow for the increased mortality observed with treating germinated seeds. The experiments were run in triplicate. For the scoring of M2 mutants an additional replication was done.

During azide treatment (10^-3M pH 3), the solutions were bubbled with O₂. After treatment, the seeds were rinsed 5-6 times in distilled water and planted wet in soaked soil. Three hundred spikes were hand-harvested from each plot, and from these, 150 spikes were planted in the greenhouse and scored for M2 chlorophyll-deficient mutants as described previously.

The results of these experiments are shown in Fig. 1 (next page), where the frequency of chlorophyll-deficient mutant types are shown for the various germination times. Each bar shows the average frequency of mutant types observed in the four replica plantings of M1 spikes. The figure shows that the efficiency of azide as a mutagen is strongly dependent on the stage of germination and thus the cell cycle of the embryonic shoot-tip cells. The maximum level of mutations is observed with seeds germinated for 8 and 16 hours where the frequencies are 85% and 81% respectively. The different times of treatment for the 8 and 12 hour versus the 16 hour germinated material and the increased lethality of azide with longer germination precluded an accurate estimate of the optimal state for azide mutagenesis in barley.

Other studies in this laboratory indicate that the shoot-tip cells of 8-16 hour germinated seeds are in S stage. This suggests that azide is a potent S stage mutagen. The frequency of mutations observed in these experiments is the highest level of mutations reported for any mutagen in the barley system.

Reference:

Nilan, R. A., E. G. Sideris, A. Kleinhofs, C. Sander, and C. F. Konzak. Azide -- A potent mutagen. Mutation Research 17(1):142-144) 1973.

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