šPresent address: Tochigi Branch, Tochigi Agricultural
Experiment Station, Tochigi-city, Tochigi, 328 Japan
To meet the various uses of waxy barley for food, it will be better to breed amylose-free cultivar. Since there is no amylose-free cultivar so far, we attempted to induce amylose-free mutation by chemical mutagen and succeeded.
The way we got the amylose-free mutants was as follows; One thousand and a hundred seeds of two-rowed naked non-waxy barley cultivar "Shikoku Hadaka 84" were soaked in water at 0°C for 10 hours, 20°C for 17 hours, then treated with 4x10^-4^ M sodium azide in 0.1M phosphate buffer (pH=3) for two hours at 20°C. They were sown immediately after the treatment in a greenhouse. We selected 140 opaque M/3/ seeds and checked them if they were waxy or non-waxy by iodide reaction. As the result, we got two waxy seeds. Amylose content of purified starch granules of M/6/ seeds of these two strains measured by amperometric titration were 0.0%, which means these strains are amylose-free mutants.
The characteristics of the amylose-free mutants (M/5/) were as follows; There were no significant differences in heading time, maturing time, culm length and ear length between the amylose-free mutants and their original cultivar, and among the amylose-free mutants. These mutants might be originated from single mutation. Seeds of the amylose-free mutants and waxy cultivars were
opaque, while those of non-waxy cultivars were not opaque. Total (1-3),(1-4)-ß-D-glucan (ß-glucan) contents measured by enzymic method (McCleary and Glennie-Holmes, 1985) of waxy cultivars were about 1.2 to 1.4 times higher than non-waxy cultivars. Total ß-glucan contents of the amylose-free mutants (5.3%) were 1.5 times higher than that of the original cultivar (3.5%). F/1/ seeds derived from crosses between the amylose-free mutants and non-waxy cultivars were non-waxy, and F/1/ seeds derived from crosses between the amylose-free mutants and waxy cultivars were waxy.
Proteins bound to starch granules were extracted from endosperms of the amylose-free mutants, waxy cultivars and non-waxy cultivars using the procedure described by Echt and Schwartz (1981) with some modifications. Extracted proteins were separated by SDS-PAGE according to Laemmli (1970) and stained with Coomassie Blue R-250 or silver (Silver Stain Plus Kit; Bio-Rad Laboratories). The protein (probably Wx-protein) with the molecular weight of about 60kD was detected densely in non-waxy cultivars, and faintly in waxy cultivars. But the protein cannot be detected in the amylose-free mutants (Figure 2 and 3).
Seeds of the amylose-free mutants are available upon request.
References:
Fukuba, H. and Kainuma, K. 1977. pp.174-179 In Starch Science Handbook ed. by Nakamura and Suzuki, Asakura Publishing Co. Ltd., Tokyo (in Japanese).
McCleary, B. V. and Glennie-Holmes, M. 1985. Enzymic quantification of (1-3),(1-4)-ß-D-glucan in barley and malt. J. Inst. Brew. 91:285-295.
Echt, C. S. and Schwartz, D. 1981. Evidence for the inclusion of controlling elements within the structural gene at the waxy locus in maize. Genetics 99: 275-284.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.