To evaluate quantitatively the protective effects of the Pb1 gene which is derived from an Indica rice variety “Modan” and confers field resistance to rice panicle blast, tests for blast resistance were conducted in paddy fields over a period of two years at two testing sites using three pairs of near-isogenic lines (NILs) for the Pb1 locus selected from about 2,000 recombinant inbred lines (RILs) which were developed from the cross between the Japanese rice cultivars “Koganebare” (susceptible to rice blast without Pb1 gene) and “Tsukinohikari” (resistant to the disease with Pb1). A NIL pair of cultivar “Koshihikari” for Pb1 and Stvb-i, a gene conferring resistance to rice stripe virus, was also used for the evaluation, in which the protective effect was given as follows:

Protective effect
=(rate of infected part without Pb1−rate of infected part with Pb1)/(rate of infected part without Pb1)×100

The protective effect of the Pb1 gene against rice blast increased with the progression of the growth stages of the rice plants; leaf (vegetative growth) stage < flag leaf stage < panicle (reproductive growth) stage. The average protective value of the Pb1 gene for the percentage of diseased grains was very high (93) for each year, which showed that Pb1 could reduce the percentage of diseased grains to less than 1/10, compared with sib-lines without Pb1 gene. The head brown rice yield ratios of NILs with Pb1 to the sib-lines without Pb1 were 2.40:1-16.2:1 under conducive conditions for blast disease, revealing a very high protective effect on yield loss associated with panicle blast infection, while 0.78:1-1.29:1 ((1.07± 0.18):1) under conditions less conducive to the disease. The values of the head/gross brown rice rate and thousand kernel weight in brown rice of the NILs with Pb1 under disease-conducive conditions, were significantly higher, while the protein content was significantly lower than the values of the sib-lines without Pb1, respectively. Thus, the Pb1 gene also showed a secondary protective effect on rice quality under the blast fungus-conducive environment.

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In the present study, 4 sets of multiplex PCR primers were developed to identify 130 varieties of rice-paddy cultivated in Japan (12 of which being suitable for sake brewery,) covering approximately 99% of the domestic rice planted area. Gene arrangements to distinguish each of these samples were defined, and 15 sets of STS (Sequence-Tagged Sites) primers were designed. Each of these sets was used for PCR analysis of 130 varieties to detect discriminating bands that are unique to each variety. These 15 sets of primers were integrated into 4 multiplex PCR primer sets by examining their base sequence, combination of primers and PCR conditions, so that they would not interfere with each other. Multiplex PCR was then performed to confirm that they could identify each of the 130 varieties chosen. The rice varieties that are cultivated in several prefectures in Japan, namely Koshihikari, Hitomebore, Hinohikari, Akitakomachi, Kinuhikari, Nihonbare, Sasanishiki, Hanaechizen, Matsuribare and Asahinoyume were analyzed by PCR using the 4 multiplex PCR primer sets. The results showed that there were no differences in the discrimination of the bands among the samples of a variety collected from different prefectures. The present study suggests that the multiplex PCR primer sets developed were effective in distinguishing the rice varieties cultivated in Japan.

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To test the applicability of the temperature gradient chamber (TGC) method to the evaluation of the high temperature ripening ability of rice, five varieties of rice were cultivated in TGC environments. The temperature gradient occurred in the TGC as expected, and experiments under different conditions of gradual temperatures became possible. As an index of the high temperature ripening ability, the relationship between the milky white kernel ratio, basal white kernel ratio, white back kernel ratio and the average temperature during the ripening stage was investigated. As a result, it became evident that the frequency of the types of white immature kernels differed from variety to variety, such as milky white kernel of “Hitomebore” and basal white kernel and white back kernel of “Niigatawase”. These results indicated that the TGC could be applied to evaluate the high temperature ripening ability of rice. The implications of the subsidiary findings indicating that the occurrences of white immature kernels depended on the rice varieties, and also the ripening stage of the exposure to high temperature were examined.

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We examined the effect of the anther density on callus induction and plant regeneration in floating anther culture and the effect of the removal of the liquid medium adhering to the induced calli before transfer to agar medium on plant regeneration in floating anther culture of rice. Anthers with mid-to late uninucleate pollen grains were collected from a restricted position on the ears on the main culms of the rice plants densely planted in pots. The anthers were cultured by a three-step method consisting of callus induction (liquid medium), callus proliferation (agar medium) and plant regeneration (agar medium) stages. The effect of the anther density was examined using a Japonica rice cultivar, Kita-ake. The rate of callus induction decreased with the increase in the density of anthers in liquid medium from 0.9 to 6 anthers/ml, while further increase in the density up to 12 anthers/ml did not effect any significance. The effect of the removal of the liquid medium adhering to the induced calli before the transfer to agar medium on plant regeneration was examined using two Japonica rice cultivars, Kita-ake and Kirara 397. The wet calli from the liquid medium were blotted with a filter paper for a few seconds before being transplanted onto agar medium. By this procedure, the rate of green-plant regeneration from the calli increased by 13-18%. Thus, we recommend that the density of anthers in the liquid culture be reduced to 3 anthers/ml or less, and that the liquid medium adhering to the calli be blotted with a filter paper for a few seconds before the anthers are transferred to agar medium for plant regeneration.

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