In order to develop Enethods for the transfer of organelle genetic information in alfalfa (Medicago sativa L.) from one species to another, we investigated asymmetric somatic hybridization and succeeded in developing a stable method for cybrid callus formation. Protoplasts of a cultivar (SpredorII) were inactivated with iodoacetamide (IOA) and protoplasts of the cytoplasmic male sterile (CMS) Iine L-2905 were treated with X-rays. Both types of protoplasts were fused electrically. Hetero-fusion frequency could be increased by the increase of the ratio of CMS protoplasts to cultivar protoplasts in a fusion chamber. Inactivation of cultivar protoplasts could be almost achieved by treatment with 6mM IOA when the agarose block method was used. A higher X-ray dose was needed to inactivate CMS protoplasts compared with other plants. Analysis of restriction fragment length polymorphism (RFLP) of mitochondrial DNAs (mtDNAs) of the parents using the Southern blot hybridization method revealed that the digestion of total DNA with Xho I and hybridization of atpA enabled to differentiate mtDNAs between the cultivar and CMS. Analysis of calli from asymmetric fusion product by using a combination of Xho I and atpA confirmed that hybrid type calli showing the bands corresponding to those of both parents could be obtained. Based on the investigations on the conditions of IOA concentration and X-ray dose, it was observed that treatnent with 6 mM IOA was suitable for decreasing cultivar type escape callus formation. Isozyme analysis revealed that the malate dehydrogenase isozyme pattern of some of the hybrid type calli was different frum that of CMS control, suggesting the presence of cybrid clones. Although mtDNA of regenerated plants was analyzed, RFLP pattern of all of them was the same as that of cultivarcontrol.

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Diallel cross analysis for eight varieties of barley (Hordeum vulgare L.) differing in their salt tolerance at germination and at the seedling stage was performed. Salt tolerance at germination was evaluated from average shoot length after ten days of incubation in 250 mM NaCl solution. Salt tolerance at germination was mainly controlled by recessive genes. Non-allelic gene interaction was not observed. Non additive genetic variance was larger than additive genetic variance, and the mean degree of dominance was 1.47. The broad and narrow sense heritabilities were as high as 0.99 and 0.75, respectively. On the other side, a leaf injury index was used to assess tolerance at the seedling stage after exposure to 500 mM NaCl solution for four weeks. Salt tolerance at the seedling stage was predominantly controlled by additive genes, and the mean degree of dominance was 0.52. Non-allelic gene interaction was absent, and dominance was directed toward tolerance. Heritabilities were 0.85 and 0.75 in a broad and a narrow sense, respectively. No correlation was observed between salt tolerance at germination and the seedling stage in the 8×8 diallel tables. Thus the mode of inheritance for salt tolerance was different between germination and the seedling stage.

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Hybrid lethality system is one of the most serious barriers in wide hybridization. Hybrid plants between N. suaveolens and N. tabacum exhibit lethality and die at the cotyledonary stage. Intact seedling culture on solidified media containing cytokinins is a simple method for over-coming the lethality in this cross-combination. In order to improve the method, we cultured F_l seeds in liquid media containing cytokinins. Two types of cytokinins, purine-type (kinetin, 6-benzylaminopurine (BAP) and t-zeatin) and urea-type (N-phenyl-N'-(1, 2, 3-thidiazol-5-yl) urea (TDZ), N-(2-chlor0-4-pyrizyl)-N'-phenylurea (CPPU)), were used at various concentrations (0-10 mg/l ). In the liquid media containing 0.01-10 mg/l cytokinin, shoot-like structures were formed at the hypocotyl and the primary root of the dying seedlings. The shoot-like structures did not exhibit lethality and grew up to vigorous shoots without developing any lethal symptoms within a month. Vigorous multiple shoots developed from the shoot-like structures in the liquid media containing more than 0.2 mg/l cytokinins. The regenerated shoots were transferred to the medium containing 2 mg/l indole-3-acetic acid (IAA) to accelerate rooting. The hybrid seedlings displayed a vigorous growth without developing any lethal symptoms up to maturity in a greenhouse. Forty chromosomes were counted in root tip cells of the seedlings and morphological characters of the seedlings were intermediate between those of parental species. In this experiment, we developed a simplified method using liquid media with urea-type cytokinins and obtained more hybrid seedlings in a shorter period of time.

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Leaf segments of Rosa hybrida cv. Carl Red and R. canina were cultured on Murashige and Skoog medium (1962) (MS) containing various kinds and concentrations of auxins. In Carl Red, pale yellow calli with embryogenic potential were predominantly induced on media containing a-naphthaleneacetic acid (NAA) at a wide range of concentration (0.25-5 mg/l), whereas low concentration (0.05 mg/l ) of thidiazuron (TDZ) inhibited embryogenic calli (EC) formation. Abnormal somatic embryos were produced from EC on MS without plant growth regulator (PGR) or with 100 ml /l coconut water (CW) and shoot formation was initiated from abnormal embryos on media with 6-benzylaminopurine (BA) or TDZ in Carl Red. The regenerated shoots of Carl Red were rooted on PGR-free MS medium and successfully transplanted on the soil. In R. canina. abnormal somatic embryos were initially induced at low frequencies on media containing high concentration of NAA (5 mg/l) or 2, 4-dichlorophenoxyacetic acid (2, 4-D) (10 mg/l ). EC with abnormal embryos was further induced after the explants were transferred onto PGR-free MS medium. Green friable calli were also initiated from EC on MS with CW and differentiated to nodular structure which further initiated leaf primordia on MS with 2 mg/l BA or 0.1 mg/l TDZ. Shoot regeneration was induced from leaf primordia and abnormal embryos on MS with 2 mg/l BA or 0.1 mg/l TDZ at 6 g/l gellan gum. Normal somatic embryos with germinability were also initiated secondarily from abnormal embryos on MS with 0.1 mg/l TDZ at 6 g/l gellan gum.

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Intergeneric reciprocal crosses between Raphanus sativus and three Brassica species, such as B. fruticulosa (ssp. cossoneana strain 201 and ssp. mauritanica strain 401), B. maurorum and B. oxyrrhina, were carried out in order to analyze their crossability and to produce their F_l hybrids. Crossability was analyzed based on the pollen germination index (P. G. I.) and the development of hybrid embryos which was evaluated in vivo by conventional crossing and in vitro by ovary culture. When R. sativus was used as the pistillate parent, pollen grains of the Brassica. species germinated well on the stigmas while only a small number of pollen tubes could elongate near the ovules in a few R. sativus pistils, resulting in a low development of hybrid embryos. Using the Brassica species as the pistillate parent in the intergeneric crosses, the crossability was relatively higher than that of the reciprocal cross. When R. sativus was used as the pistillate parent, one F₁ hybrid was produced by embryo culture in the cross of R. sativus × B. fruticulosa-401. When the Brassica species were used as the pistillate parent, 200 F₁ hybrids were obtained through ovary culture followed by embryo culture from all cross combinations and 12 F₁ hybrids were obtained in the cross of B. oxyrrhina × R. sativus by conventional crossing without embryo rescue. F_l hybrids were intermediate between their parents in several characteristics except for flower color and root thickening. In the cross of the Brassica species × R. sativus, 12 amphihaploid F_l hybrids formed trivalents and a number of bivalents at metaphase I(MI)and their pollen fertility ranged from O% to 70.2%. Induced 6 amphidiploid F_l hybrids showed relatively stable chromosome associations at MI and a higher pollen fertility than that of the corresponding amphihaploid. It is suggested that these new F_l hybrids could become valuable materials in the breeding of the tribe Brassiceae.

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Electrofusion was performed to produce somatic hybrids between Solanum tuberosum (2n=4x=48) and dihaploid S. acaule (2n=2x=24). Among the 18 regenerated plants obtained, 14 were found to be hexaploid hybrids based on RAPD analysis and cytological observation. In order to identify their cytoplasmic genome constitution, RFLP analysis of chloroplast and mitochondrial DNA was carried out. Six and eight hybrids were found to have S. tuberosum and S. acaule chloroplast genomes, respectively, suggesting that the chloroplast genomes of the somatic hybrids segregated randomly. In addition, two regenerated plants derived from the same callus had different chloroplast genomes, indicating that the segregation of the chloroplast genome occurred before shoot formation from callus. Concerning the mitochondrial genome constitution, 13 hybrids harbored the genome of S. tuberosum type and the others the S. acaule type genome. This mode of segregaion is obviously in favor of S. tuberosum mitochondrial genome. Regarding cytoplasmic genome segregation in somatic hybrids, the mitochondrial genome was not associated with the chloroplast genome. For the cytoplasmic genomes of both species, no hybrid was observed with recombinant or heterogeneous genome from both parents. In most of the somatic hybrids, some characters such as plant habit, shape of foliage and shape of tuber, were generally similar to those of S. tuberosum. The high level of resistance to potato virus X (PVX) detected in dihaploid S. acaule and somatic hybrids, suggests that the resistance to PVX is incorporated by fusion with S. acaule.

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Isozyme types were determined for six cultivar groups carrying different resistance to rice bacterial blight in Asia. The temporarily proposed Isozyme genotypes would be associated with the differentiation of the reaction of resistance. The predominant type of the Java 14 group was Isozyme type VI-1, and type II-4 was involved as the second predominant type. The majority of the DZ 192 group belonged to type II-1 and II-2. A newly assigned TN 1 group consisted of Isozyme type I and VI / . The predomi-nant occurrence of I-1 and I-2 was common among the TKM 6, Cas 209, Mond Ba and TN I groups. However, frequent involvement of IM-1 and IM-2 were specific to the TKM 6 group. The frequent presence of I-4 and I-5 was the characteristic of the Cas 209 and Mond Ba groups. Based on these observations the ecotypic diversity of each group was discussed.

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Rice (Oryza sativa L.) root proteins were separated by two-dimensional electrophoresis and 292 protein spots were detected by Coomassie Blue staining. The N-terminal and internal amino acid sequences of 76 proteins of 292 were determined so as to construct a detailed data-file of rice protein sequences. Sequences for a total of 126 proteins including 50 proteins reported previously have been determined so far. In the Rice Genome Research Project, more than 20, 000 cDNA clones randomly selected from rice cDNA Iibraries have been sequenced to construct a cDNA catalog. The possibility of identifying cDNAS encoding particular proteins from the catalog by homology search based on the protein sequence data-file was investigated. Attempt was made to screen cDNAs encoding 126 proteins from the catalog by homology search. Complementary DNAs encoding 42 proteins were identified in the catalog, indicating that about 30% of the cDNA species could be identified. It was deduced that 20, 000-40, 000 genes are present in the rice genome. Although more than 20, 000 cDNAs were sequenced in the Rice Genome Research Project, only half should have unique sequences when gene redundancy is considered. This corresponds to 25-50 % of genes present in the entire rice genome. This appears consistent with the fact that about 30% of the cDNAs encoding proteins sequenced could be identified in the catalog. If the size of the cDNA catalog is increased enough, all cDNAs encoding proteins separated by two-dimensional electrophoresis can be easily picked up from the catalog by computer search using the sequences determined.

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Soybean plant shape evaluation is an important part of the soybean plant breeding process in Japan. This selection process is currently performed by visual inspection by the soybean plant breeder. This paper describes a method to evaluate soybean plant shape quality automatically. The method developed was an expert system using fuzzy logic rule sets to evaluate soybean shape quality. The evaluator operated on 4 shape indicators extracted from digitized images of each soybean plant. The evaluator placed the shape of each soybean plant into one of three categories: good (3), fair (2), and poor (1). Only those rated as good were selected by the soybean breeders. The goal was to develop an evaluator that would give the same ratings as those given by the soybean plant breeders. The shape quality evaluation results based on the fuzzy logic rule sets developed in this study were slightly better than those obtained using statistical discriminant analysis. The efficiency of the correct evaluation was about 76% for both the plants to be selected (good) and the plants to be removed (fair or poor). Fuzzy logic evaluation has two advantages in contrast to statistical discriminant analysis. One is that it does not require any assumption on statistical distribution of the shape features and the other is that its structure is easy to understand.

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In order to obtain basic information for the design of a more detailed linkage map of sugi (Cryptomeria japonica D. Don), 15 isozyme loci with 13 enzyme systems were analyzed for their linkage relationships using crossed families including selfing. Moreover, Iethal genes which led to a deviation of the genotypic segregation ratio, and eventually resultes in abnormal recombination values were analysed. A Iinkage relationship was detected in Mnr : Lap (r= 0.365), and a weak linkage was found in the following 4 pairs of isozyme loci; Shd-2 : 6Pg-1 (r=0.382), 6Pg-2 : Lap (r=0.477), G6p : Pgm-2 (r=0.409), Dia-3 : Est-1 (r =0.293). In the cross of a family of Boka sugi×Kumotooshi, a linkage relationship was observed between the isozyme locus (Shd-1) and dwarf locus(dw-1) with a recombination value of 0.318. The analysis of the abnormal segregation ratio in isozyme loci, revealed the presence of 5 Iethal genes (including embryonic lethal genes). In particular, the lethal genes were strongly linked to G6p, Dia-3 and Lap.

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One of the important factors in the use of rice for sake brewery is represented by the characters of the white-core in kernels. Rice breeders and sake brewers require information about the size and position of the white-core in grains of rice cultivars for sake brewery. However, it had hitherto been difficult to obtain the information, because the size and position of the white-core in grains vary with the cultivars. We reported that the evaluation of the size and position of the white-core is possible by using image analysis, in which the standard image for a cultivar was obtained by superimposing the image of 85 grains (1996). In this study, we aimed at detecting the difference in the white-core characters in grains among rice cultivars for sake brewery by using the standard images. It was reported that the size of the white-core correlated wath the thickness of the rice kernel. Then we classified the grains into 5 groups according to their thickness and made the standard image of each group. The results showed that the size of white-core correlated with kernel thickness, i. e. the thicker the grain, the greater the size of the white-core. On the other hand, the positions of the white-core in grains did not differ among the 5 groups (Fig. 2, 3). Therefore, the standard images of 34 cultivars were obtained, using grains more than 2.0 mm thick from rice plants cultivated in Nagaoka city, Niigata prefecture in 1995. Based on the comparison of the 34 standard images, we classified the cultivars into 5 categories (Fig. 4). Then we reviewed the genealogical tree of the rice cultivars for sake brewery based on the literature, and investigated the relationship between the lineage and the characters of the white-cores. It was found that the cultivars which were closely related gave a similar standard image (Fig. 5). These findings support the assumption that characters of the white-core are genetically controlled. The results obtained suggest that the method of evaluation applied is not valid for the selection of raw materials for sake brewery, but also could be used as an index of breeding.

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Snow damage in winter wheat (Triticum aestivum L.), which is mainly caused by snow mold diseases, is influenced by the physiological activity of the plants under the snow. This study was conducted to examine the shoot weight (SW), percentage of crown weight against the shoot weight (crown %), percentage of dry matter (DM %) and freezing tolerance (FT) by measuring median lethal ternperature (LT₅₀) before wintering in the 1992/93 winter season, FT twice in December and in the following March, and snow mold damage in May in the 1993/94 winter season, using 45 winter wheat varieties. Moreover, we attempted to evaluate the changes in SW, DM. FT and etiolated growth (EG) in the 1992/93 winter season, using 10 representative varieties (Table 1). The correlation coefficients before wintering and 70 days after snow cover were significant for SW and DM % but not significant for FT and EG. Moreover, there was no significant correlation between the respective combinations of DM %, FT and EG. However, the relation became significant 70 days after snow cover (Table 2), mainly due to the slower decrease in physiological indicators such as FT and EG in the snow-tolerant varieties (P.I.173438, Niederndorferberg. Haunsberg, Norin 62) that were sensitive to low temperature and exhibited a higher dry matter percentage than the others (Fig.1). Correlations of FT and EG with snow mold damage were not significant before wintering, but became significant 70 days after snow cover (Fig.4, 5). On the other hand, in all the 10 varieties, DM % decreased under snow cover. As a result, a highest correlation between DM % and snow mold damage was recognized both before wintering (r=-0.618) and 70 days after snow cover (r = -0.879 ^{* * *} ). Snow-tolerant varieties were characterized by a high DM %, comparatively low FT and upright type with a crown % of around 35%, while freezing-tolerant varieties showed a high DM % and FT and a rosette type with a crown % of nearly 50% (Fig.2, 3).

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To obtain basic information for the micropropagation and future breeding of endangered Cypripedium species, plant regeneration from immature seed-derived callus of Cypripedium macranthas var. taiwanianum was studied. Calli, induced from immature seeds on T medium (Tsutsui and Tomita 1990) supplernented with 200 mg/l yeast extract, 10 g/l sucrose and 2 g/l gellan gum, were used for the plant regeneration test. The regeneration was observed in three lines of callus on media containing 10^-5M NAA with or without the addition of 10^-6M BAP. After vernalization, regenerated plants were established.

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Cumulative after-effects of successive vernalization treatments for 20 to 30 generations lvere observed using three wheat varieties; Danchi-komugi, Norin 61 and Lutescens 62. The results are summarized as follolws: 1. Heading date was advanced by the successive treatments by a maximum of 1.7 days in the 4th generation in the case of Danchi-komugi and 6.5 days in the 17th generation in Norin 61. Thereafter the advancement gradually decreased. In this experiment, it appears that the cumulative after-effects did not convertge to a particular level, and that they lvere not significantly larger than those in the Ist generation. In contrast to these varieties, the heading date was not significantl\* affected bv the vernalization treatment in Lutescens 62. 2. In Danchi-komugi, the culm length decreased until the 4th generation and tended to increase up to the 8th generatlon, and then decreased again. Remarkable cumulative after-effect on culm length was observed in Norin 61. Culm became shortest in the 12th generation and the length recovered by the 16th generation, and decreased gradually thereafter. Culm length of Lutescens 62 rather increased until the 4th generation and then decreased. 3. Ear length tended to increase for all the varieties, though the changes were not conspicuous after the 12th generation in Danchi-komugi, the 8th generation in Norin 61 and the 16th generationin Lutescens 62, respectively. 4. Plant height at the internode elongation stage in Danchi-komugi had shown a periodicity covering eight genelations, with reversed cumulative and non-cumulative effects every four years in both the successive treatment lines (D₁-D₃₀) and the non-treatment lines after successive treatment (D_5-0-D_24-0). This periodicity suggested that Danchi-komugi developed a dynamic homeostasis over generations for the sensibility to low temperature.

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In 5 sugarcane clones, cell suspension cultures were initiated and maintained in modified N6 Iiquid medium containing 2 mg/l 2, 4-D, 500 mg/l casein hydrolysate and 3% sucrose. These suspension cultures were able to regenerate plants for more than three months. Protoplasts isolated from these suspensions were embedded in 1.2% agarose (modified KM8P medium) and cultured in modified KM8P medium with the addition of nurse culture cells from the suspension culture. After several weeks of culttl[re, calluses were obtained in all the 5 clones. Calluses derived from protoplasts were cultured for 2 weeks on PR4 medium (pre-culture medium for regeneration), then transferred to R9 regeneration medium. In the subsequent 30-day period of culture, protoplast-derived calluses of US 76-9 and NiF 8 regenerated green shoots and albino shoots, respectively, while the other 3 clones did not form any organs. In the 6 experiments using suspension cultures of US 76-9 differing in age (8 to 31 weeks after initiation), regeneration of fertile green shoots from protoplast-derived calluses was observed. After the transfer to R1 medium for rooting, a few shoots developed to plants, while most of the shoots died without rooting.

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