Isolation of a rice mutant insensitive to cool temperatures in relation to amylose content

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19. Isolation of a rice mutant insensitive to cool temperatures in relation to amylose

content


Y.	Suzuki1, Y. Sano2 and H.-Y. HIRANO3


1)	National Agriculture Research Center, Kannondai, Tsukuba, Ibaraki, 305-8666 Japan.


2)	Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo. 060-8589 Japan.


3)	Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo,

113-8657 Japan.

In rice (Oryza sativa L.), the amylose content of endosperm starch is an important determinant of eating quality. Higher amylose content lowers rice eating quality, especially the stickiness of cooked rice preferred in Japanese cooking. Amylose content varies from year to year and the site of cultivation of the same variety, causing variation in rice quality. It is thus a major objective in rice breeding to develop rice varieties whose amy- lose content does not change at different temperatures.

Amylose synthesis is controlled by the Waxy (Wx) gene, which encodes ADP glucose starch glycosyl transferase. We found that the amylose content is closely related to Wx gene expression (Sano 1984) and that increased amylose content at cool temperatures is caused by elevated Wx gene expression (Sano eta!. 1985, Hirano and Sano 1998). If we isolate mutants of the Wx gene, insensitive to cool temperatures, these will have suitable amylose content even at cool temperatures. This report describes our procedure for isolating a rice mutant insensitive to cool temperatures and characterization of this mutant.

To find mutants, we used a strain, 76-3/T65, carrying the du gene (Sano 1985). In this du line, Wx gene expression is reduced and amylose content is very low. The amy- lose content in 76-3fF65 seeds matured at a normal temperature (28°C) was about 2% (Table 1). The endosperm appeared chalky. The 76-3/T65 strain responds to cool temperatures the same way as normal nonglutinous rice. The amylose content in seeds maturing at a cool temperature (21°C) was about 12%, and the endosperm appeared slightly dull. This increased amylose synthesis was caused by elevated expression of Wx gene. If mutations occur in the 76-3/T65 strain in the pathway of the cool-temperature response, seeds produced in mutants should show a chalky phenotype even when maturing at cool temperatures. The mutant insensitive to cool temperatures should therefore be easily distinguished from original or temperature-sensitive lines. This strategy enabled us to easily screen mutants from the large population of mutagenized rice plants of 76-3/T65 in field.

Table 1. Amylose content of original 76-3/T65 line and

TM95 mutant under the different temperatures


	Tempera	ture (°C)
Strain	28°C	21°C
76-3/T65	1.9 ± 1.5	12.0 ± 2.6
TM95	2.6± 1.0	3.5 ±2.1

We grew plants derived from 76-3/T65 mutagened seeds in the field. M2 seeds matured at 18-22°C (average temperature) were used for the first screening of rice insensitive to cool temperatures. We screened M2 seeds of M1 plants, and selected four M1 plants, which had chalky M2 seeds as compared to dull seeds of 76-3/t65. Immunoblotting using antiserum against the Wx protein showed that the amount of Wx protein obtained from chalky seeds was lower than those of dull seeds (cool temperature response). After screening M3 and M4 seeds, one line out of four was confirmed as a candidate for the mutant insensitive to cool temperatures, and we named this line TM95.

Since TM95 was screened in the field, we investigated whether TM95 is insensitive to cool temperatures, using temperature-controlled growth chambers. To compare amy- lose content of TM95 and 76-3fF65 when maturing at cool or normal temperatures, the plants grown in the field were divided into two parts at the maximum tillering stage. After 5 days of anthesis, one part of the divided rice plants was grown at 28°C and the other part at 21°C until seed maturation in identical growth chambers differing only in temperature. The amylose content in mature seeds of the original 76-3/T65 line, was about 2% at 28°C and 12% at 21°C (Table 1). That of the TM95 mutant was about 3% at both temperatures. These results indicated that TM95 did not respond to cool temperatures during seed development, resulting in a low-amylose content.

This rice mutant insensitive to cool temperatures should prove to be useful as a breeding material to develop varieties with stable amylose content irrespective of the temperature at seed maturation. This mutant will also be useful for analyzing the gene expressions in response to cool temperatures.

References

Sano, Y., 1984. Differential regulation of waxy gene expression in rice endosperm. Theor. Appl. Genet. 68:

467-473.

Sano, Y., M. Maekawa and H. Kikuchi, 1985. Temperature effects on the Wx protein level and amylose content in the endosperm of rice. J. Hered. 6: 221-222.