The amylose content of endosperm starch is an important characteristic of rice in determining eating and cooking quality. The amylose content is genetically controlled by a major gene, the Waxy (Wx) gene. In addition, amylose content is also affected by several modifying genes and environmental factors such as temperature. The Wx gene encodes the granule-bound starch synthase that is responsible for amylose synthesis in the endosperm. Rice strains having the Wx^b gene contain 15-20% amylose in contrast to rice strains having the Wx^a gene contain 20-25% amylose (Sano 1984). The du loci modify amylose content by controlling expression of the Wx^b gene and mutations at the du loci cause a reduction in amylose content (Okuno et al 1983, Hirano 1993). Although the amylose-extender (ae) mutant shows increasing levels of apparent amylose content, this increase in amylose content is caused by the altered amylopectin structure and the ae mutation is found in the gene for starch-branching enzyme IIb (Nishi et al. 2001). While there are many mutants concerning starch composition, there is no report about the gene, of which mutation gives rise to elevation of amylose content in the endosperm. In this paper, we describe the isolation and characterization of a rice mutant with enhanced amylose content in the endosperm that does not affect the amylopectin structure.

To screen mutants with enhanced amylose content, we used a variety called "Snow pearl". In this variety, the amylose content was very low (about 5%) and the endosperm appeared chalky when seeds are matured at a normal temperature (26C) (Table 1). When seeds were matured at a cool temperature (20C), the amylose content was about 18%, and the endosperm appeared semi-translucent. Because the amylose content in rice seed influences the degree of transparency as previously described (Suzuki et al. 2002), a mutant with enhanced amylose content in (semi-translucent) seeds matured at a normal temperature should be easily distinguished from the original variety with low amylose (chalky) seeds.

We grew mutagenic plants derived from Snow pearl seeds in the field. The first screening used 7,200 M₁ plants that were matured at an average temperature of 24-26C. Although the panicles of almost all of these plants contained exclusively chalky seeds, one of these plants contained a mixture of chalky and semi-translucent seeds. M₂ seeds with chalky endosperm and those with semi-translucent endosperm segregated at a ratio of 3:1 ( chi² = 3.75, Table 2), indicating that this mutation was controlled by a single recessive gene. Next, semi-translucent M₂ seeds were chosen as a candidate (designated as SP14) for a mutant with enhanced amylose content. The M₂ seeds were seeded, and amylose content of SP14 (M₃ seeds) was compared with that of Snow pearl and Koshihikari using a temperature-controlled growth chamber. The amylose content in mature seeds of SP14 was about 12% at 26C and 25% at 20C (Table 1), indicating amylose content of SP14 is responding to maturation temperatures and 6-7% higher than that of Snow pearl. Furthermore, to clarify whether amylose content in

SP14 was increased by the ae locus, the amylopectin chain length distribution in rice endosperm was analyzed by high performance anion exchange chromatography with a pulsed amperometric detector. Because the chain length distribution of amylopectin were almost the same between the two strains maturated at both temperatures (data not shown), the mutation in SP14 would not be the ae mutation reported previously (Nishi et al. 2001), but a new mutation that enhances amylose content in rice endosperm. Though it should be also noted whether SP14 is derived from "Snow pearl" or not, our preliminary data on the nucleotide sequences in Wx gene indicates SP14 is derived from Snow pearl. Thus, this mutant should be useful in breeding programs designed to produce rice of an abundant quality and in understanding genetic and molecular mechanisms for amylose synthesis.

References

Hirano, H. -Y. 1993. Genetic variation and gene regulation at the wx locus in rice. Gamma-Field Symp. 24: 63-79.

Nishi, A., Nakamura, Y., Tanaka, N., and Satoh, H. 2001. Biochemical and genetic analysis of the effects of amyloseextender mutation in rice endosperm. Plant Physiol. 127: 459-472.

Okuno, K., Fuwa, H., and Yano, M. 1983. A new mutant gene lowering amylose content in endosperm starch of rice, Oryza sativa L. Jpn. J. Breed. 33: 387-394.

Sano, Y. 1984. Differential regulation of waxy gene expression in rice endosperm. Theor. Appl. Genet. 68: 467-473.

Suzuki, Y., Sano, Y., and Hirano, H. -Y. 2002. Isolation and characterization of a rice mutant insensitive to cool temperatures on amylose synthesis. Euphytica 123: 95-100.