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Plant regeneration was achieved through anther culture of three wild species of Hordeum (H. murinum, H. rnarinum and H. bulbosum). Calli or embryoids were formed from microspores in anthers cultured on a medium containing 6-benzylaminopurine (BAP) and ficoll. These calli or embryoids regenerated green or albino shoots and roots after transfer to regeneration media. Green plantlets which developed on regeneration media were transferred to soil where they showed further growth. [ABSTRACT FROM AUTHOR]

In yellow soybeans, inhibition of seed coat pigmentation by RNA silencing of CHS genes is suppressed by low temperature and a viral suppressor, resulting in 'cold-induced seed coat discoloration' and 'seed mottling', respectively. Differences exist in the degree of cold-induced seed coat discoloration among Japanese yellow soybean cultivars; for example, Toyomusume is sensitive, Toyohomare has some tolerance, and Toyoharuka is highly tolerant. In this study, we compared the degree of seed mottling severity due to soybean mosaic virus (SMV) among these three soybean cultivars. Obvious differences were found, with the order of severity as follows: Toyohomare > Toyomusume > Toyoharuka. RNA gel blot analysis indicated that CHS transcript abundance in the seed coat, which was increased by SMV infection, was responsible for the severity of seed mottling. Quantitative reverse transcription PCR analysis revealed why mottling was most severe in SMV-infected Toyohomare: the SMV titer in its seed coat was higher than in the other two infected cultivars. We further suggest that a major gene ( Ic ) for tolerance to cold-induced seed coat discoloration can relieve the severity of seed mottling in SMV-infected Toyoharuka., (Copyright © 2020 by JAPANESE SOCIETY OF BREEDING.)

Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world's leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement.

Lodging tolerance (LT) is an important trait for high yield and combine-harvesting efficiency in soybean [Glycine max (L.) Merr.]. Many previous studies have investigated quantitative trait loci (QTLs) for lodging score (LS) in soybean. Most of the investigated QTLs were located in the proximal region of maturity or growth habit loci. The aim of this study was to identify genetic factors for LT not associated with maturity or growth habit. QTL analysis was performed using a recombinant inbred line (RIL) population derived from a cross between 'Toyoharuka' (TH), a lodging-tolerant cultivar, and 'Toyomusume' (TM). The genotypes of TH and TM were estimated as both e1e2E3E4 and dt1. The average LS over 4 years was used for QTL analysis, identifying a major and stable QTL, qLS19-1, on chromosome 19. The LS of the near-isogenic line (NIL) with the TH allele at Sat_099, the nearest marker to qLS19-1, was significantly lower than the NIL with the TM allele at that position. The TH allele at Sat_099 rarely had a negative influence on seed yield or other agronomic traits in both NILs and the TM-backcrossed lines. Our results suggest that marker-assisted selection for qLS19-1 is effective for improving LT in breeding programs.

The length of the reproductive period affects the grain yield of soybean (Glycine max [L.] Merr), and genetic control of the period might contribute to yield improvement. To detect genetic factor(s) controlling the reproductive period, a population of recombinant inbred lines (RILs) was developed from a cross between Japanese landrace 'Ippon-Sangoh' and, Japanese cultivar 'Fukuyutaka' which differ in their duration from flowering to maturation (DFM) relative to the difference in the duration from sowing to flowering (DSF). In the RIL population, the DFM correlated poorly (r = -0.16 to 0.34) with the DSF in all field trials over 3 years. Two stable QTLs for the DFM on chromosomes (Chr-) 10 and 11 as well as two stable QTLs for the DSF on Chr-10 and -16 were identified. The QTL on Chr-11 for the reproductive period (designated as qDfm1; quantitative trait locus for duration from flowering to maturation 1) affected all three trials, and the difference in the DFM between the Fukuyutaka and Ippon-Sangoh was mainly accounted for qDfm1, in which the Fukuyutaka allele promoted a longer period. qDfm1 affected predominantly the reproductive period, and thus it might be possible to alter the period with little influence on the vegetative period.

While the cultivated soybean, Glycine max (L.) Merr., is more recalcitrant to pod dehiscence (shattering-resistant) than wild soybean, Glycine soja Sieb. & Zucc., there is also significant genetic variation in shattering resistance among cultivated soybean cultivars. To reveal the genetic basis and develop DNA markers for pod dehiscence, several research groups have conducted quantitative trait locus (QTL) analysis using segregated populations derived from crosses between G. max accessions or between a G. max and G. soja accession. In the populations of G. max, a major QTL was repeatedly identified near SSR marker Sat_366 on linkage group J (chromosome 16). Minor QTLs were also detected in several studies, although less commonality was found for the magnitudes of effect and location. In G. max × G. soja populations, only QTLs with a relatively small effect were detected. The major QTL found in G. max was further fine-mapped, leading to the development of specific markers for the shattering resistance allele at this locus. The markers were used in a breeding program, resulting in the production of near-isogenic lines with shattering resistance and genetic backgrounds of Japanese elite cultivars. The markers and lines developed will hopefully contribute to the rapid production of a variety of shattering-resistant soybean cultivars.

In yellow soybean, seed coat pigmentation is inhibited by post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. A CHS cluster named GmIRCHS (Glycine max inverted-repeat CHS pseudogene) is suggested to cause PTGS in yellow-hilum cultivars. Cold-induced seed coat discoloration (CD), a commercially serious deterioration of seed appearance, is caused by an inhibition of this PTGS upon exposure to low temperatures. In the highly CD-tolerant cultivar Toyoharuka, the GmIRCHS structure differs from that of other cultivars. The aim of this study was to determine whether the variation of GmIRCHS structure among cultivars is related to variations in CD tolerance. Using two sets of recombinant inbred lines between Toyoharuka and CD-susceptible cultivars, we compared the GmIRCHS genotype and CD tolerance phenotype during low temperature treatment. The GmIRCHS genotype was related to the phenotype of CD tolerance. A QTL analysis around GmIRCHS showed that GmIRCHS itself or a region located very close to it was responsible for CD tolerance. The variation in GmIRCHS can serve as a useful DNA marker for marker-assisted selection for breeding CD tolerance. In addition, QTL analysis of the whole genome revealed a minor QTL that also affected CD tolerance.

Seed coat pigmentation is inhibited in yellow soybean. The I gene inhibits pigmentation over the entire seed coat. In yellow soybean, seed coat discoloration occurs when plants are exposed to low temperatures after the onset of flowering, a phenomenon named 'cold-induced discoloration (CD)'. Inhibition of seed coat pigmentation results from post-transcriptional gene silencing (PTGS) of the chalcone synthase (CHS) genes. PTGS is a sequence-specific RNA degradation mechanism in plants and occurs via short interfering RNAs (siRNAs). Similar post-transcriptional suppression is called RNAi (RNA interference) in animals. Recently, we identified a candidate of the I gene designated GmIRCHS. In this study, to elucidate the molecular mechanism of CD, CHS mRNA and siRNA levels in the seed coat were compared between CD-sensitive and CD-tolerant cultivars (Toyomusume and Toyoharuka, respectively). In Toyomusume, the CHS siRNA level was reduced markedly by low temperature treatment, and subsequently the CHS mRNA level increased rapidly after treatment. In contrast, low temperature treatment did not result in severe reduction of the CHS siRNA level in Toyoharuka, and the CHS mRNA level did not increase after the treatment. These results suggest that the rapid increase in CHS mRNA level after low temperature treatment may lead to enhanced pigmentation in some of the seed coat cells and finally in seed coat discoloration. Interestingly, we found a Toyoharuka-specific difference in the GmIRCHS region, which may be involved in CD tolerance.

Low temperature is among the critical environmental factors that limit soybean production. To elucidate the genetic basis for chilling tolerance and identify useful markers, we conducted quantitative trait loci (QTL) analysis of seed-yielding ability at low temperature in soybean (Glycine max), using artificial climatic environments at usual and low temperatures and recombinant inbred lines derived from a cross between two contrasting cultivars in terms of chilling tolerance. We identified a QTL of a large effect (LOD > 15, r (2) > 0.3) associated with seed-yielding ability only at low temperature. The QTL was mapped near marker Sat_162 on linkage group A2, where no QTL for chilling tolerance has previously been identified. The tolerant genotype did not increase the pod number but maintained the seed number per pod and single seed weight, namely, the efficiency of seed development at low temperature. The effect of the QTL was confirmed in a segregating population of heterogeneous inbred families, which provided near-isogenic lines. The genomic region containing the QTL also influenced the node and pod numbers regardless of temperature condition, although this effect was not primarily associated with chilling tolerance. These results suggest the presence of a new major genetic factor that controls seed development specifically at low temperature. The findings will be useful for marker-assisted selection as well as for understanding of the mechanism underlying chilling tolerance in reproductive organs.

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39,936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5' and 3' ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22,674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5'- and 3'-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.

cDNA of buckwheat (Fagopyrum esculentum Moench) was isolated from immature seeds harvested 14 days after pollination. Two genes, designated FA02 and FA18, were found to encode legumin-like proteins and were expressed during seed development. The deduced amino acid sequence of FA02 was identical to the N-terminal amino acid domain of BW24KD, which was believed to be a major buckwheat allergen (Urisu, A.; Kondo, Y.; Morita, Y.; Yagi, E.; Tsuruta, M.; Yasaki, T.; Yamada, K.; Kuzuya, H.; Suzuki, M.; Titani, K.; Kurosawa, K. Isolation and characterization of a major allergen in buckwheat seeds. In Current Advances in Buckwheat Research; Shinshu University Press: Matsumoto, Japan, 1995; pp 965--974). It was predicted that FA02 would be cleaved to generate two separate components, a 41.3 kDa alpha-subunit and a 21 kDa beta-subunit. Antiserum was raised against the deduced FA02 beta-subunit, and immunoblotting of total protein from buckwheat seeds (F. esculentum M. and Fagopyrum tartaricum Gaertn.) revealed that several groups of proteins reacted with the antiserum. Polypeptides in the 23--25 kDa range displayed the greatest reactivity.

Rice (Oryza sativa L.) seedlings, when kept at 42 degrees C for 24 h before being kept at 5 degrees C for 7 d, did not develop chilling injury. Chilling resistance was enhanced in parallel with the period of heat-treatment. The level of APX activity was higher in heated seedlings whereas CAT activity was decreased by heat stress. There was no significant difference in SOD activity between heated and unheated seedlings. The elevated activity of APX was sustained after 7 d of chilling. The cytosolic APX gene expression in response to high and low temperature was analysed with an APXa gene probe. APXa mRNA levels increased within 1 h after seedlings were exposed to 42 degrees C. Elevated APXa mRNA levels could also be detected after 24 h of heating. The APXa mRNA level in preheated seedlings was still higher than unheated seedlings under cold stress. The promoter of the APXa gene was cloned from rice genomic DNA by TAIL-PCR, and characterized by DNA sequencing. The promoter had a minimal heat shock factor-binding motif, 5'-nGAAnnTTCn-3', located in the 81 bp upstream of the TATA box. Heat shock induction of the APXa gene could be a possible cause of reduced chilling injury in rice seedlings.

We have isolated five cDNA clones (osk1-5) for protein kinases from rice which are related to SNF1 protein kinase of Saccharomyces cerevisiae. Based on the sequence homology, these cDNAs can be classified into two groups, group 1 (osk1) and group 2 (osk2-5). The products of these genes were demonstrated to be functional SNF1-related protein kinases by in vitro and in vivo experiments. Recombinant proteins expressed from both groups of genes were fully active as protein kinases and could phosphorylate SAMS peptide, a substrate specific for the SNF1/AMPK family, as well as themselves (autophosphorylation). Moreover, expression of osk3 cDNA in yeast snf1 mutants restored SNF1 function. Northern blot analyses showed differential expression of these two gene groups; group 1 is expressed uniformly in growing tissues (young roots, young shoots, flowers, and immature seeds), whereas group 2 is strongly expressed in immature seeds. SNF1-related protein kinases have been reported from different plant species, such as rye, barley, Arabidopsis, tobacco, and potato, while the type of gene strongly expressed in immature seeds is known only in cereals such as rye, barley, and, from our findings, in rice. Expression levels of the group 2 genes were further analyzed in seeds during seed maturation. Expression is transiently increased in the early stages of seed maturation and then decreases. The expression peak precedes those of the sbe1 and waxy genes, which are involved in starch synthesis in rice. Taken together, these findings suggest that group 2 OSK genes play important roles in the early stages of endosperm development in rice seeds.

With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.