BGN 12: Azide metabolite experiments with barley BARLEY GENETICS NEWSLETTER, VOL. 10, II. RESEARCH NOTES
Hodgdon and Nilan, pp. 34-36

II. 15. Azide metabolite experiments with barley.

A. L. Hodgdon and R. A. Nilan, Department of Agronomy and Soils and Program in Genetics, Washington State University, Pullman, Washington 99164, U.S.A.

Preliminary experiments in our laboratory indicated that the azide metabolite produced in Salmonella (Owais et al., 1979) was taken up by Himalaya barley seeds. Therefore, a larger scale experiment was planned to determine if the azide metabolite could induce chlorophyll mutations in barley.

The metabolite was extracted by our standard procedure from eight liters of azide treated Salmonella, giving 250 ml metabolite (Owais et al., 1979). This was diluted to a total of 900 ml and designated 1X strength. Treatments were also done with 1/10X and 1/100X dilutions. Each treatment involved four replications with 500 seeds per rep each in a 250 ml flask with 200 ml treatment volume.

All treatments were given 16 hours of 0°C presoak followed by 8 hr 20°C presoak with aeration. This was followed by a two-hour metabolite treatment in .05 M potassium phosphate buffer (pH 7.2) at 20°C with aeration. After a 1/2 hr wash in running tap water, the seeds were dried overnight in a hood. The treated seeds were then stored in a cold room until planting in a randomized plot design with one rep/plot. M1 spikes were harvested on a one spike per plant basis. The spikes were planted in a greenhouse and scored for chlorophyll mutations. Table 1 shows the results.

Table 1.

The 1X metabolite treatment greatly reduced the M1 seedling viability. However, none of the metabolite treatments significantly increased the chlorophyll mutation over the background rate of the Himalaya control.

Due to these negative results, the uptake of the bacterial metabolite by Himalaya seeds was tested with and without 5% DMSO using the same concentration of the azide metabolite as in the mutation experiment. (Extracts from these treated seeds showed no mutagenic activity on the Ames tester system.) Thus, there has been no evidence that the bacterial azide metabolite is mutagenic in barley or is even taken up by the barley seeds.

Next, an experiment was planned to determine if the barley azide metabolite could be purified by the same steps as the bacterial metabolite (Owais et al., 1979). There have been two major differences noted so far in this experiment. First, the barley metabolite is not retained by the DOWEX column and second, the mutagenic activity is lost in the freeze-drying step in purification. These preliminary results suggest that the barley and bacterial azide metabolites are quite different chemically.

Reference:

Owais, W. M., A. Kleinhofs, and R. A. Nilan, 1979. In vivo conversion of sodium azide to a stable mutagenic metabolite in Salmonella typhimurium. Mutation Research 68:15-22.

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