Artificial induction and characterization of amylose-free mutants of barley
Naoyuki Ishikawaš, Jiro Ishihara, Masamitsu Itoh
Shikoku National Agricultural Experiment Station,
Zentsuji, Kagawa, 765 Japan

šPresent address: Tochigi Branch, Tochigi Agricultural
Experiment Station, Tochigi-city, Tochigi, 328 Japan

Waxy barley is distributed in East Asia. It has been used for Japanese cakes or noodles in Japan, and we are carrying out waxy barley breeding at Shikoku National Agricultural Experiment Station. We collected indigenous waxy cultivars and determined amylose content of endosperm starch by amperometric titration (Fukuba et al., 1977) after purifying starch granules by 0.2% sodium hydroxide and 85% hot methanol. All waxy cultivars contained some amylose in their endosperm starch. Amylose content of starch in waxy cultivars varied much and it ranged from 2.4% to 10% (Figure 1). On the other hand, variation of amylose content in non-waxy cultivars was little, and it was around 27%.

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


Figure 1. Variation of amylose content of endosperm starch in waxy and non-waxy cultivars measured by amperometric titration

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.


Figure 2. Starch granule-bound proteins separated by SDS-PAGE and stained with Coomassie Blue R-250. Lanes : (left) non-waxy cultivar "Shikoku Hadaka 84", (middle) amylose-free mutant "Yon-M-kei 286", (right) waxy cultivar "Sumiremochi". Arrow indicates molecular weight 60kD, i.e. Wx-protein.


Figure 3. Starch granule-bound proteins separated by SDS-PAGE and stained with silver. Lanes : (left) non-waxy cultivar "Shikoku Hadaka 84", (middle) amylose-free mutant "Yon-M-kei 286", (right) waxy cultivar "Sumiremochi". Arrow indicates molecular weight 60kD, i.e. Wx-protein.