Field performance of the progeny of protoplast-derived barley plants (cvs. Igri and Dissa) was investigated. The least significant differences for five agronomic characteristics (culm length, heading date, fertility, spike length and spikelet density) were calculated to compare the field performance of the protoplast-derived plants with that of control plants. Our results indicated that most of the progeny plants showed no abnormal traits and set seeds, but the field performance of those plants changed in some agronomic characteristics. These facts suggest that we should make an effort to establish in vitro culture conditions which are less mutagenic to cultured cells of barley.

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Genetic diversity of 19 cherimoya cultivars classified into five types based on their skin texture was taxonomically identified by Amplified Fragment Length Polymorphism (AFLP). AFLP analysis revealed 33 reproducible amplified bands with an average number ranging from 10 to 47 bands per primer combination. The size of the amplified bands ranged from 50 to 450 bp in all the primer combinations. More than 30% (88/264) of the bands were polymorphic among the cultivars. The most efficient combination (E-AGT/M-8) yielded 21 polymorphic bands. A total of 264 AFLP bands enabled to detect the genetic variation among cherimoya cultivars. The consensus of eight trees with the highest degree of parsimony reflected known relationships of some cultivars. Cladogram constructed by AFLP revealed that all the 19 cultivars could be clearly differentiated. The similarity indexes were estimated for cherimoya, and the values ranged from 0.33 to 0.95. The minimum value was observed between‘Fino De Jete' and‘Ott' or ‘Sallmon' and the maximum was detected between‘Bays' and‘Bay Ott'. Although these results were not consistent with morphological classifications, AFLP provided a better resolution in revealing the phylogeny of the cherimoya cultivars. It is concluded tbat AFLP enables to analyse the phylogenetic relationships of cherimoya cultivars.

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Three somatic hybrid plants (no. 1, no. 2 and no. 3) were obtained from fused protoplasts between barley (Hordeum vulgare L.) and carrot (Daucus carota L.) (Kisaka et al. 1997). They closely resembled carrot in terms of morphology. In the present study, cold and salt tolerance of the somatic hybrid calli which were induced from these somatic hybrid plants were investigated to know whether both tolerances were transferred. No. 2 was more tolerant to cold than carrot and other somatic hybrids. Furthermore, NaCl and KCI tolerance of no. 2 was as high as that of barley, while those of no. 3 were intermediate between those of barley and carrot. How-ever, no. 1 was not tolerant to NaCl and KCI, as was carrot. Levels of betaine in no. 2 and no. 3 were higher than that in carrot, and the levels of beta.ine were increased by treatment with NaCl, as in barley. The calli induced from hybrids and their parents grew very well in medium supplemented with betaine, but the betaine was not effective in reducing the inhibition of cell growth by NaCl. These results indicate that the cold- and salt-tolerant traits might have been transferred from barley by protoplast fusion in the somatic hybrid (no. 2).

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Using the Nuclear Magnetic Resonance (NMR) method, the detection of the activity of rutin-degrading enzyme in buckwheat was examined. Mixture of dimethylsulfoxide (DMSO) and acetate buffer (i.e., water) solvent was selected and double homogate (DHMG) decoupling technique was used in NMR measurement in order to suppress large solvent signals and to detect the signals of rutin and quercetin. NMR signals of aromatic H of the flavonoid in rutin were detected at different positions from those in quercetin. Then, the activity of the enzyme in buckwheat was examined by the NMR method. The crude extract solution of the enzyme (acetate buffer solution pH 5.0) was added to rutin in a DMSO solution, and the activity was measured by the NMR method. The enzyme activity could be detected by the appearance of the NMR signals of quercetin, which were observed in the shifted position, i.e., in a higher field than that of rutin. When the extracts from common buckwheat were added, NMR signals did not change. However, when the extracts from tartary buckwheat were added, NMR signals which shifted from that of rutin in the higher frequency region were observed. The NMR method was useful to detect the enzyme activity. Furthermore, it was confirmed that common buckwheat did not show the rutin-degrading enzyme activity, unlike tartary buck-wheat.

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Variation of virulence in Rhynchosporium secalis(Oud.) Davis distributed in the Hokuriku district was investigated by seedling infection test using 14 differential barley cultivars. A total of 38 cultures were isolated from the infected leaves of Minorimugi, a leading variety in the Hokuriku area, in 1992, 1993 and 1995 from various locations. The 38 isolates showed a complex variation in the virulence pattern and could be classified into 36 different pathotypes according to the virulence spectra to the differentials. Based on the cluster analysis of the reactions of 14 differentials, a geographical cline for virulence was suggested; the isolates from the southern part of the district (Fukui, Ishikawa and Toyama) were more virulent to the differentials than those from the northern part (Yamagata and Niigata). This fact suggests that the virulence of the fungus is conditioned by not only race-specific gene(s) but also by some genes with interactions such as additive or complementary effects. Among the 14 differentials, Brier, Turk and Osiris were highly resistant to the 38 isolates. It appeared that the resistance of these 3 cultivars was controlled by different gene(s) from Rh, Rh⁴. Rh5, rh6 or RhlO, since other differentials with the same genotype were attacked by many of the isolates. The 17 cultivars, which had been already selected for the resistance to the Niigata isolate, were tested by 32 of the 38 isolates above mentioned, and 3 cultivars, Turkey 22, Trukey 208 and Carre 26, were confirmed to be highly resistant to all the isolates. It was concluded that these cultivars including Brier, Turk and Osiris are highly suitable materials for breeding for resistance to scald in the Hokuriku district.

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Production of intergeneric somatic hybrids between two sexually incompatible leguminous species, alfalfa (Medicago sativa L.) and birdsfoot trefoil(Lotus corniculatus L.) was carried out. Donor protoplasts of alfalfa were given lethal doses of X-irradiation, and recipient protoplasts of birdsfoot trefoil were inactivated with iodoacetamide. Donor and recipient protoplasts were fused with PEG. Fusion products initiated cell division and colony formation in the culture medium and resulted in callus formation. By using isozyme analysis, it was confirmed that 17 out of 155 calli were somatic cell hybrids. Sorne of the hybrid calli regenerated shoots. By using isozyme analysis it was clarified that the chromosomes or chromosome segments were randomly eliminated from both parents at an early stage of hybrid callus culture. However, after a long period of subculture the isozyme pattern of the recipient type was seen in all of the regenerated shoots which were analyzed. This indicates that the genomes in the calli rapidly converged on the recipient type as subcultures progressed. Southern blot analyses of the chloroplast genomes indicated that either genome of both parents sorted out in all calli investigated.

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Photoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm to common wheat (Triticum aestivum) under long-day conditions (≥15 h light period) has been reported as a new means of producing hybrid wheat, that is a “two-line system”. The PCMS Iine is Inaintained by self-pollination under short-day conditions (≤14.5 h light period), and F₁ seeds can beproduced by outcrossing of the PCMS Iine with a pollinator under long-day conditions. A cross pollination study for producing F₁ seeds was carried out using three PCMS Iines and three restorer lines under a long-day condition at Tanno, Hokkaido, Japan. Seed setting rate was 14-33%, and 19-55 g/m² F₁ seeds with 47-88% hybrid purity were obtained. Variation in seed setting rate attributable to male parents was highly significant. High seed setting rate was achieved by using a pollinator taller than the PCMS Iine. Shriveled and preharvest sprouting kernels were found in the out-crossed F₁ seeds, resulting in lower volume weight and germination rate than the selfed seeds of original cultivars of the PCMS Iines. A field test of evaluating four F₁ hybrids with high hybrid purity (>65 %) was conducted under a short-day condition at Kasai, Hyogo, Japan. All F₁ hybrids exhibited higher grain yield than their respective better-parent because of more spikelet number/ear and higher 1000 grain weight. The top F₁ hybrid yielded 40% more than the mid-parent and 37% more than the better-parent, suggesting a possibility of the practical use of this system for hybrid wheat breeding. F₁ hybrids tended to show higher volume weight than their parental cultivars. This indicates that grain quality of the F₁ hybrids with the Ae. crassa cytoplasm is not inferior to those of parental cultivars with the wheat cytoplasm.

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The genetic control of regeneration from rice seed callus in a highly regenerating rice cultivar Joshu was studied. The reciprocal F₁s, F₂, B₁F₁ and B₁F₂ populations from the crosses of Joshu with the low regenerating cultivars (Moritawase and Norin 1) were tested for their rege-eration ability, which was expressed by shoot regeneration rate. The results revealed that the high regeneration ability in Joshu was controlled by a dom-nant and a recessive gene independently and expression of any one of the two genes or both of them would result in high regeneration ability. The allelism test indicated that the dominant gene in Joshu was the same allele as in Aikoku which we reported previously. Moreover, there was no reciprocal cross difference on regeneration ability in this study.

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In the screening of rice mutants having altered seed protein compositions, two mutant lines lacking α-globulin were selected from the M₂ seeds of a white panicle mutant of cv. Koshihikari irradiated with gamma-rays. The mutant character, Iack of α-globulin, was found to be controlled by a single recessive gene. Northern blot analysis of endosperm RNA using the probe of α-globulin cDNA revealed that the α-globulin gene is not expressed in the α-globulin-less mutants. The mutated gene was considered to be α-globulin gene, and named glb-1(t). RFLP analysis of F₂ plants between the mutant and indica cultivar‘Kasalath' using land marker probes suggested that glb-1(t) is on chromosome 5 and that the distances of glb-1(t) from the nrarkers, C246 and X'Npb366, were 15.4cM and 30.8cM, respectively. The mutant line lacking α-globulin was expected to have a higher content of lysine in rice grains, but the lysine content of the mutant line was found not to be higher than the original cultiver. The possible use of the mutant as a breeding material is discussed.

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The indica rice cultivar Kasalath (KA) has been extensively used as a model cultivar in the molecular genetics of rice in Japan. To advance our knowledge of the nrolecular genetics of heading thrre in rice, we performed a genetic analysis of KA with special reference to two major photoperiod sensitivity loci, E₁ and Se-1. We also discussed the relationships of these two loci with the five QTLs (Hd-1∼Hd-5) that Yano et al.(1995) identified using the progenies from the cross‘KA × the Japanese japonica cultivar Nipponbare'.The F₁ and F₂ generations from the crosses ‘11 heading time tester lines × KA' were subjected to genetic analyses. The Rc (brown pericarp and seed coat) and Pgi-2 (phosphoglucose isomerase-2) Ioci were used as linkage markers of the E₁ and Se-1 Ioci, respectively. Experimental results showed that KA carries a new photoperiod sensitivity allele E₁ k(t) and a photoperiod insensitivity allele Se-1^e. The E₁^k(t) proved to stimulate the photoperiod sensitivity of rice more than a strong photoperiod sensitivity allele E₁. Based on the findings of the present study and previous works, we concluded that the E₁ and Se-1 Ioci were identical with the Hd-4 and Hd-1 Ioci, respectively.

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Common buckwheat, Fagopyrum esculentum, is a heteromorphic sporophytic self-incompatibility species while an annual wild species, F. homotropicum, is homomorphic self-cornpatible. An F₂ population was generated from an interspecific hybrid between them and used to determine the genetic basis of self-compatibility due to homomorphic phenotype. Homostyly appears to be controlled by a single dominant gene (designated “Ho”) as F₂ progenies segregated in a 3 homomorpic : 1 heteromorphic flower types. We used RAPD and bulked segregant analysis to identify three markers, OPB14₁₂₅₀, OPP8₁₀₀₀ and OPQ7₈₀₀, Iinked to the Ho gene. All three markers were mapped to the same side of the Ho gene with OPB14₁₂₅₀ being the closest at 0.6 cM. The mapping of this gene represents a first step toward better understanding of sexual reproduction system in buckwheat. These markers should be useful for marker-assisted selection for introgression of self-compatibility into common buckwheat.

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We demonstrated developmental transgene silencing and resetting in torenia (Torenia fournieri Lind.). The experiments used transformants carrying the neonrycin phosphotransferase II gene, the β-glucuronidase (GUS) gene, and tbe hygrounycin phosphotransferase gene. GUS activity was measured at various developmental stages in GUS-homozygous and GUS-hemizygous plants derived from a primary transformant, R₀-1. Rapid silencing of the GUS gene occurred 12 to 19 days after germination in the homozygous plants, while it occurred at a later developmental stage, 54 days after germination, in the hemizygous plants. The silenced state seemed to be stable through vegetative propagation in both the homozygotes and the hemizygotes. Northern blot analysis showed that mRNA reduction correlated with a reduction in GUS activity. A single GUS gene locus was sufficient to trigger the silencing, and initial levels of GUS activity correlated with the timing of silencing. These results suggested that the GUS gene silencing observed in torenia is not dependent on allelic interaction of the GUS genes, but is correlated with a dose effect. This type of silencing can be explained by the RNA threshold model.

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Successful hybridization between Lilium×elegance and L. Ionglflorum has not been reported, although its reciprocal cross has been developed. Ovary slice culture technique, after cut-style pollination, was used to produce interspecific hybrids between these species. Crosses between diploid cultivars (2n=2x=24) of Lilium X elegance and L. Ionglflorum were conducted. On the days 5, 10, 15, 20, 25, 30, 35, 40, and 45th after pollination (DAP) ovaries were sliced and cultured on a modified MS (Murashige and Skoog 1962) medium without growth regulators and ammonium nitrate (NH₄N0₃), supplemented with 6% sucrose, 50 mg/l yeast extract and 0.25% gelrite at pH 6.3. Embryo germination was found only at 35 DAP. After transfer to a MS (half-strength) medium supplemented with 1.5% sucrose, 0.25% gelrite and 0.2% active charcoal at pH 5.8, seven hybrid plants were developed. All regenerated plants were identified as hybrids on the basis of karyotype, isozyme and PCR analyses.

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[in Japanese]

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