A Database for Triticeae and Avena
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,
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
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.
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.
BGN 10 toc
BGN Main Index