Various combinations of temperature and time for soaking seed were tested to establish a system for transient assay of transcriptional factors controlling anthocyanin biosynthesis to prove that the isolated gene is an inducible gene for anthocyanin expression at the aleurone layer of mature rice seed. The aleurone of mature seed of RIB-Pl with purple pericarp was sufficiently exposed under five combinations. Both Cl and B-Peru, transcriptional factors controlling anthocyanin biosynthesis of maize, were introduced by particle bombardment into the aleurone exposed under these five conditions. The highest expression of anthocyanin was obtained at the aleurone of the seeds soaked at 28°C for 4 hours. The effect of concentration of plasmids with Cl and B-Peru for introduction into the aleurone of A-58 mature seed with white pericarp soaked at 28°C for 4 hours was examined. Both Cl and B-Peru were found to give high expression of anthocyanin at the same concentration. Especially, 5 μg of Cl with 5 μg of B-Peru and 5 lig of Cl with 10 μg of B-Peru showed the highest expression. In conclusion, soaking of mature rice seed at 28°C for 4 hours and introduction of 5 lig of Cl with 51μg of B-Peru into the exposed aleurone was found to be a highly efficient system for transient assay of transcriptional factors controlling anthocyanin biosynthesis.

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For the purpose of monitoring gene regulation in the seminal root of rice (Oryza sativa L.) under a low pH condition, we carried out gene expression profiling by cDNA microarray constructed from 1, 265 rice cDNA clones. Total RNA was isolated from the rice seminal root with or without low pH treatment (pH 3.5), and fluorescence labeled by reverse transcription before hybridization. In order to identify up-regulated genes under a low pH condition, principal component analysis (PCA) was applied to analyze the gene expression profiles. The first two components appear to represent (1) the variance of signal intensity specific for each gene and (2) the variance resulted from a low pH treatment. Genes corresponding to upper 2.5 % based on the score of the second principal component were assumed to be acid response genes. Several genes, suggested to be responsible for metabolic regulation of intracellular pH in plants, were apparently up-regulated after acid treatment. On the basis of the above results, we concluded that PCA was a suitable method for gene expression analysis by cDNA microarray.

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DNA polymorphism among 15 japonica varieties, which includes 10 local varieties and 5 improved varieties, and 2 indica varieties were investigated by restriction fragment length polymorphism (RFLP), random amplified polymorphic DNAs (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) markers. Small numbers of polymorphisms with all types of markers were found among 15 japonica varieties compared with that between indica and japonica varieties. The percent polymorphism was 7.5 to 17.6% for RFLP, 3.3 to 7.8% for RAPD, 1.2 to 3.9 % for AFLP, and 11.4 to 34.3 % for SSR markers among 15 japonica varieties. Multiple alleles (5 or 6 alleles) were detected by some SSR markers. Use of both RFLP and SSR markers would facilitate the genetic analysis using crosses between temperate japonica varieties.

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