Callus induction and morphogenesis from different black-gram explants were tested on MS basal medium supplemented with B5 vitamins, IAA, NAA, IBA, KIN and BAP individually and in combinations. The explants were hypocotyl, epicotyl, axillary bud, cotyledonary node and immature leaf. The optimal levels of the frequency of callus induction was 22.8μM of IAA or 16.1μM NAA and in combination with 2.2μM of BAP. Among the seedling explants, hypocotyl was found to be more efficient in producing callus. Shoots were induced from callus cultures of hypocotyls, epicotyls, axillary bud, cotyledonary node and immature leaf with varying frequencies in the medium containing KlN (2.3-9.3μM) or BAP (2.2-8.8μM) and in combination with IAA (2.8μM) or NAA (2.6μM). Multiple shoots were obtained using cotyledonary node segments. The regenerated shoots rooted best on MS basal medium containing 9.8μM IBA. Seventy three per cent of the shoots produced roots, and 80-85% of the plantlets survived under greenhouse condition.

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High density molecular map of semidwarfing gene, sd-1, was constructed using 178 F₂ plants derived from the cross between the semidwarf nearisogenic line and its recurrent parent, japonica cultivar “Shiokari”. The total size of the map including nine molecular markers (five RAPD and four RFLP markers) and sd-1 was 6.4 cM. The most closely linked RAPD markers in both sides of sd-1 were OPVI0₅₀₀ and OPAE14₁₀₀₀, and the distance between them was 1.7 cM. Since sd-1 is a recessive gene, these closely linked RAPD markers will be very powerful to monitor the gene for large scale selection in breeding program.

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Aegilops crassa cytoplasm causes photoperiod-sensitive Introductron cytoplasmic male sterility (PCMS) to some Japanese common wheat (Triticum aestivum ) cultivars: the PCMS line with the Ae. crassa cytoplasm is almost completely male sterile under long-day conditions (≥l5 h light period), but highly male fertile under short-day conditions (≤14.5 h). Using the PCMS, a “two-line systerm” is applicable for hybrid wheat production. To examine the effects of Ae. crassa cytoplasm on the agronomic characters in the PCMS Iines and F₁ hybrids, five PCMS Iines and 23 F₁ hybrids with the Ae. crassa cytoplasm were compared their agronomic characters to the corresponding lines with the wheat cytoplasm under a short-day condition (natural condition at Kasai, Hyogo, Japan). In the PCMS Iines, the Ae. crassa cytoplasm delayed heading date and reduced spikelet number/ear. Shriveled kernels were found in the PCMS seeds, resulting in decreased volume weight and germination rate. Although the Ae. crassa cytoplasm reduced selfed and open-pollinated seed fertilities in the PCMS Iines, the averaged 69 % open-pollinated seed fertility indicates that the PCMS Iines can be maintained by self-pollination under the natural condition at Kasai. In the F₁ hybrids, selfed and open-pollinated seed fertilities were also reduced by the effect of Ae. crassa cytoplasm. The reduced seed fertility resulted in decreased grain numberlear and grain weight/plant, but increased 1, 000-grain weight. No significant effect of the cytoplasm was detected on volume weight of the seeds produced by the F₁ hybrids, suggesting no seriously deleterious effects of Ae. crassa cytoplasm on the grain quality. In spite of reduced seed fertility, the F1 hybrids with the Ae. crassa cytoplasm showed on the average 14 % heterosis for grain weight/plant over the mid-parent value of the female and male parents with the wheat cytoplasm. Correlation analysis revealed that grain weight/plant was more strongly related with the ear number/plant and grain numberlear than the 1, 000-grain weight in both F₁ hybrids with the Ae. crassa and wheat cytoplasm.

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Many somatic hybrid plants between L. esculentum × L. peruvianum and S. Iycopersicoides were obtained by symmetric electrofusion. Twenty putative somatic hybrid plants were randomly selected and analyzed. The hybrid nature of these plants was evaluated according to their morphological characteristics and then confirmed by RAPD analysis. The nuclear genomes in the somatic hybrid plants contained three parental specific RAPD markers, using three random primers. Furthermore, the chloroplast genotype was analyzed using tobacco chloroplast DNA probes and species-specific RFLPs. Out of 15 somatic hybrid plants, 10 plants inherited tomato chloroplast DNA and 5 plants S. Iycopersicoides one. The number of chromosomes was varied, and more than half of the somatic hybrids were aneuploid in tetraploid level and none of them developed flowers. However, euploid somatic hybrids predominantly consisted of highly fertile hexaploids and tetraploids which gave rise to many progenies.

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PCR-RFLP of 14 accessions of six Annona species namely A. cherimola Mill., A. squamosa L., A. reticulata L., A. glabra L., A. muricata L. and A. moutana Macfad. and A. cherimola× A. squamosa were studied. One set of Sequence Tagged Site (STS) primers was used in order to amplify the position of cpDNA fragment:‘rbc L-ORFI06'. The amplified fragment was digested with six kinds of restriction enzymes (four base cutter). Enzymes average generated two to eight bands in each sample. Seven different restriction patterns were observed when digested with Rsa I, but other enzymes showed four to six. The pair-wise distance values ranged from 0.017 to 0.607 with the minimum between A. squamosa and A. cher-imola × A. squamosa and the maximum between A. glabra and A. cherimola × A. squamosa. Eight most parsimonious trees were obtained using Swofford parsimony method, and 50 % majority-rule consensus tree was obtained from them. These results indicated that the cultivated and wild species grouped separately and the A. cherimola × A. squamosa was placed between its parents in the cultivated species. A. glabra clustered singly in the dendrogram demonstrating that this species is different from the others. We believe that these results would give basic information in the phylogenetic studies of Annona species using a large number of species.

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Varietal differences in embryogenesis and plant regenerative ability from isolated microspores of Chinese cabbage, Brassica rapa L. ssp. pekinensis, were investigated. There were significant differences (< I%) among the varieties in total embryoid yield and regenerative embryoid yield per 1 × l0⁵ microspores. Tropical varieties had high embryogenesis and regenerative ability.‘Hsifu Early 30 Days' showed the highest embryoid yield and ‘Homei' showed a higher efficiency of plant regeneration from microspores. However, domestic Japanese varieties showed very low embryogenesis and plant regeneration ability in microspore culture. Though ‘Hsifu Early 30 Days' showed a higher embryoid yield than ‘Homei', the latter showed more efficient plant regeneration from embryoids than the former. There was no correlation between the total embryoid yield and the percentage of embryoids with plant regeneration. These results suggest that plant regeneration from embryoids is controlled by some genetic factors different from those controlling embryogenesis. In order to introduce high regenerative ability to domestic Japanese varieties, microspores isolated from the F₁ hybrid of ‘Homei'בNozaki No. 2' (domestic Japanese variety) were cultured. Doubled haploid lines, which had both high regenerative ability and characteristics comparatively similar to domestic Japanese varieties, could be selected among these regenerated plants. If high regenerative lines with domestic Japanese characteristics are developed, it is expected that the application of microspore culture to practical plant breeding will become easier for the breeding of Chinese cabbage in Japan.

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Breeding of alfalfa (Medicago sativa L.) for resistance to Sclerotinia crown and stem rot (SCR; causal agent Sclerotinia trifoliorum Eriks.), which is a serious disease in Japan, was started in 1983. Increase of resistance to this disease has been examined following repeated selection of surviving plants from artificially inoculated field plots to the 9th generation. The strains selected for SCR resistance showed a higher resistance than Natsuwakaba and Tachiwakaba which were used as breeding materials and control cultivars. The effectiveness of selection based on the survival rate of progenies increased gradually as generations in which the recurrent selection was performed advanced. Realized heritability was low (h²=0.078) in the 1st and the 2nd generations in which selection was performed but was high (h²=0.364) in the 3rd to 9th generations. It is considered that the selection effect on SCR resistance became higher in the later generations, probably due to the accumulation of genes which have minor or polygenic effects on resistance.

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Heritability of salt tolerance in germinating seeds of barley (Hordeum vulgare L.) was estimated by parent-offspring correlation and selection response. Germination tests in 250 mM NaCl solution were conducted using the seeds of F₁, F₂, and F₃ plants (F₂, F₃, and F₄ generations, respectively) derived from the crosses between salt tolerant (OUJ417 and OUK666) and sensitive (OUE211) varieties. Salt tolerance of the two crosses showed polygenic segregations in F₂, F₃, and F₄ generations. Correlation coefficients between the parents and offsprings in two crosses were about 0.5 between F₂-F₃ generations and 0.6∼0.7 between F₃-F₄ generations. Realized heritabilities estimated from the ratio of (selection response)/(selection differential) were 0.2∼0.4 in the F₂-F₃ and 0.8∼0.9 in the F₃-F₄. Although dominant genetic variance of salt tolerance at germination is larger than additive genetic variance in the F₂ generation, heritability of this trait is relatively high even between F3-F4 generations and higher heritability may be obtained in lat-er generations. We have already selected highly salt tolerant barley varieties from over 6, 700 accessions, Using such genetic resources of salt tolerance as the parents, selection for increasing salt tolerance at germination may be carried out.

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In garlic, molecular markers associated with the trait of pollen fertility were searched, using random amplified polymorphic DNA (RAPD). Twelve pollen fertile and sterile garlic clones were screened, using 60 RAPD primers to find the DNA fragments related to pollen fertility. With the 60 arbitrary 10-mer primers, 625 discrete fragments of DNA were amplified, and 397 (64%) of them showed polymorphisms among the 12 clones. Two RAPD markers, OPJ12₁₃₀₀ and OPJ12₁₇₀₀ were amplified only in the pollen fertile clones with the primer OPJ12 (5'-GTCCCGTGGT-3'). The selected primers were tested on 60 pollen fertile and sterile garlic clones. The two RAPD markers were present commonly in all of the 31 pollen fertile clones, and they were not amplified in the 29 sterile clones with the exception of one, No. 395-2 which produces sterile pollen. The two RAPD markers were related to the pollen fertility of male organ, but the female fertility was not confirmed in this clone yet. The first step towdrds marker-assisted selection of fertile clones of garlic was made in this study.

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A unique protein which was induced in husk of rice strongly under high temperature (in greenhouse) and weakly under low temperature (out-of-doors) was found in a survey with two-dimensional electrophoresis. The N-terminal 16-amino-acid sequence of this rice husk protein (RHP) showed a significant homology with chitinases in other plant species and a complete identity with a peptide deduced from a partial sequence of registered cDNA clone SS2594 in the library of the Rice Genome Research Program, Japan. Thus, the deduced amino-acid sequence for SS2594 was identified as a kind of chitinases. The amino-acid sequence for SS2594 has a unique structure among other chitinases reported for rice. It is concluded that RHP is translated from the gene for SS2594 and a product of a new locus, Chi-4 (S12594) in rice. On the basis of RFLP linkage analysis, the gene Chi-4 was located on chromosome 4.

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Environment-sensitive genic male sterility (EGMS) Iine is useful in hybrid rice seed production. A parental line of rice, Norin-PL12, has a thermosensitive genic male sterility (TGMS) gene, tms-2. We estimated the locus of tms-2 using RFLP markers in F₂ population derived from a cross between Norin-PL12 and Aus variety, Dular. As a result of quantitative trait locus (QTL) analysis using RFLP markers, it was revealed that the tms-2 is located between R643A and R1440 on chromosome 7.

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Asparagus officinalis is important horticultural species. Genus Asparagus includes some ornamental species and some medicinally important species, and distributed on dry land in the Old World. To clarify the phylogenetic relationships within this genus, we compared the restriction sites of chloroplast DNAs(ctDNAs)among ten species: A. officinalis. A. cochinchinensis. A. schoberioides, A. asparagoides, A. falcatus, A. macowanii, A. plumosus, A. scandens, A. virgatus and A. sprengeri. Phylogenetic relationships within this genus, derived from the ctDNA restriction sites, yielded values for inter-specific divergence (100 × p) that ranged from 0.4 to 2.4. Compared to other groups, such as Zea (0.0 to 0.3) and Aegilops /Triticum (0.24 to 1.0), the species of Asparagus exhibited higher levels of variation in their ctDNA. A phylogenetic tree showed that the Asparagus species used in this study could be divided into two clusters. The phylogenetic relationships derived from variations in the ctDNA were slightly different from those proposed by Bailey(1944) and Clifford and Conran(1987). The ctDNAS of A. falcatus (tetraploid, 2n=40) and A. sprengeri(hexaploid, 2n=60)gave the same restriction pattern, indicating that these species are very closely related. Moreover, it is of considerable interest that the polyploid species of Asparagus were grouped in one cluster. The three dioecious species, A. ofncinalis, A. schoberioides and A. cochinchinensis, were also grouped into one cluster, which suggests the possibility that dioecy in Asparagus might have had a monophyletic origin.

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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. Therefore, rice breeders and sake brewers require information about the size and position of the white-core in grains of rice cultivars. In order to gain the inforrnation relating to the white-core in kernels, however, it is necessary to cut kernels transversally or longitudinally. Generally, breeders investigated the characters of white-core by using transverse sections of kernels because of the difficulty of cutting kernels longitudinally with the sharp blade of a knife. One of the important factors in the use of rice for sake brewery is represented by the characters of the white-core in kernels. Therefore, rice breeders and sake brewers require information about the size and position of the white-core in grains of rice cultivars. In order to gain the inforrnation relating to the white-core in kernels, however, it is necessary to cut kernels transversally or longitudinally. Generally, breeders investigated the characters of white-core by using transverse sections of kernels because of the difficulty of cutting kernels longitudinally with the sharp blade of a knife.

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Recent complete sequence data of the cyanobacteria genome and the plastid genome of algae and land plants clearly show that plastids originated from the endosymbiotic integration of a photosynthetic prokaryote into eukaryotic host cells. One indication of this event is that the organization of ribosomal protein genes has been conserved from bacteria to plastids of land plants. We are interested in the rpl16 and rpl14 genes, which are interrupted by the rpl29 and rps17 genes in both the cyanobacteria genome and the plastid genome of red alga, but are adjacent in liverwort to higher plants. This suggests that a linker between rpl16 and rpl14 was made by deletion of the rpl29 and rps17 genes at the appearance of land plants, and therefore the rpl16-rpl14 Iinkers of land plants have diverged from a common ancestral sequence. The rpl16 and rpl14 genes are highly conserved (more than 84% homology) among rice, spinach and tobacco, whereas their linkers show low homology (28 % ). The plastid rpl16-rpl14Iinker sequences of higher plants, such as barnyard grass, gentian and oak tree could be amplified and sequenced using a pair of common primers, even if their sequences are unknown. We propose short DNA sequence in the 3' noncoding region from the stop codon of the rpl16 gene as plastid subtype ID sequence(PS-ID). PS-IDs of ca. 50 bp are thought to be long enough to address plastid subtypes of higher plants and the establishment of a PS-ID database could contribute to plant phylogeny.

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Oryza glaberrima Steud, the indigenous domesticated Oryza species in West Africa, is a potential source of useful genes for a range of economically important characters. Progenies from interspecific hybridization of O. sativa and O. glaberrima show high sterility in the F₁ hybrids and early generations. However, in some populations fertility was obtained(5-95%)and retained after two to four back-crossings. Although true intermediates between O. glaberrima and O. sativa were generally rare, their occurence in some populations was as high as 30%. Desirable morphological traits introgressed from O. glaberrima into segregating BC₂ F₃ populations included early maturity(75-100 days), rapid seedling growth and high tillering.

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[in Japanese]

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