The interaction between Agrobacterium and soybean has been studied at the transcriptome level but not at the metabolic level. However, it is necessary to investigate the difference in metabolites between susceptible and non-susceptible cultivars for high efficiency transformation. We investigated the difference in metabolites from sonicated soybean cotyledons of Korean cultivars and Bert cultivar. To identify difference in metabolites, sonicated extracts were analysed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). The soybean cultivars were classified by susceptibility using green fluorescent protein expression. We found a difference in metabolites between the high susceptible and low susceptible cultivars. The FT-ICR/MS experimental m/z data of different metabolites were compared with theoretical m/z in KNApSAcK database. The candidate list was made using KNApSAcK and focused on phenolic compounds. These candidate metabolites are speculated to influence factors in the interaction. This list of candidates may be useful to investigate the interaction between Agrobacterium and plants to increase transformation efficiency.

Wheat is the third largest crop in the world behind corn and rice. Wheat is grown over a wide range of environments, and an essential source of carbohydrates. However, the genomics of wheat, a non-model species, is still challenging despite of corn and rice was done. The recent advent of RNA-Sequencing, a massively parallel sequencing method for genome and transcriptome analysis, provides opportunity to identify gene discovery and molecular mechanisms of cellular processes. We performed a RNA-Seq experiment to find differentially expressed genes under high temperature condition. More than 344 million shot reads were generated using Illumina HiSeq technology. A comprehensive and integrated 285,324 transcripts were assembled via Trinity by combining tentative consensus sequences. Transcripts annotated by BLAST2Go and differently expressed transcripts were analyzed. A total of 208 up-regulated and 182 down-regulated transcripts were found that involve in plastid, starch and sucrose metabolism, glycerolipid metabolism and glycerolipid and dicarboxylate metabolism. Our results demonstrate that RNA-Seq can be successfully used for gene identification, transcript profiling in wheat. Furthermore these sequences will provide valuable resources for wheat researchers.

The objective of this study is to develop the gene based sequence tagged site (STS) markers in wheat. The euchromatin enriched genomic library was constructed and the STS primer sets were designed using gene based DNA sequence. The euchromatin enriched genomic (EEG) DNA library in wheat was constructed using the Mcr A and Mcr BC system in DH5α cell. The 2,166 EEG colonies have been constructed by methylated DNA exclusion. Among the colonies, 606 colonies with the size between 400 and 1200 bp of PCR products were selected for sequencing. In order to develop the gene based STS primers, blast analysis comparing between wheat genetic information and rice genome sequence was employed. The 227 STS primers mainly matched on Triticum aestivum (hexaploid), Triticum turgidum (tetraploid), Aegilops (diploid), and other plants. The polymorphisms were detected in PCR products after digestion with restriction enzymes. The eight STS markers that showed 32 polymorphisms in twelve wheat genotypes were developed using 227 STS primers. The STS primers analysis will be useful for generation of informative molecular markers in wheat. Development of gene based STS marker is to identify the genetic function through cloning of target gene and find the new allele of target trait.

The immature embryos during seed development were examined to predict the suitable embryos for an efficient regeneration system. Five spring wheat genotypes and five winter wheat genotypes were tested using immature embryos as explants. Spring wheat genotypes showed much higher levels of plant regeneration than those of winter wheat genotypes. The highest frequencies of embryogenesis and regeneration were obtained when embryos at 13-14 days after anthesis (DAA) were used as explant and decreased using embryos at 21-22 DAA during seed development. Significant differences were also found for callus induction and regeneration as affected by immature embryo size. The regeneration efficiency was drastically decreased in spring and winter wheat genotypes when embryos larger than 2.0 mm of length were used. The optimum developmental stage and embryo length for regeneration efficiency were at 13-14 DAA and 1.0-1.5 mm, respectively. The selection of suitable embryos for the high frequencies of embryogenesis and regeneration leads us to efficient genetic improvement of wheat.

For sensitive and accurate gene expression analysis, normalization of gene expression data against housekeeping genes is required. There are conventional housekeeping gene (e.g. ACT) that primarily function as an internal control of transcription. In this study, we performed an in silico analysis of 278 rice gene expression samples (GSM) in order to identify the gene that is most consistently expressed. Based on this analysis, we identified novel candidate housekeeping genes that displayed improved stability among the cross experimental conditions. Furthermore four of the most conventional housekeeping genes were included in our 30 other housekeeping genes among the most stable genes. Therefore, these 30 genes can he used to normalize transcription results in gene expression studies on rice at a broad range of experimental conditions.

MYB proteins are a superfamily of transcription factors (TF) that play regulatory roles in developmental processes and resistance mechanism in plants. We identified 130 and 109 genes in the MYB superfamily from an analysis of the complete Arabidopsis and rice genome sequence. Although microarray based transcriptome analysis approach allows the investigation of the biological networks of MYB TF in DNA level, the underling mechanisms related to their functional role is not fully understood. In this work, we performed meta-analysis of public microarray data that analyzed with Arabidopsis and rice using co-expression analysis. A phylogenetic comparison of the members of this superfamily were performed with Sorghum bicolour to suggested that MYB super family underwent a rapid expansion their evolutionary times. We identified conserved expression pairs which play important role in transcription. Our comprehensive analysis of this huge transcription factor of Arabidopsis and rice may shed further light on the possible biological roles of the MYB TF in various plants.

Rapid extension of genomic database leads to the remarkable advance of functional genomics. This study proposes a novel methodology of functional analysis using 5-methyltrytophan (5 MT) mutant together with their 2-DE analysis and public microarray database. A total of 24 proteins was changed in 5 MT mutant and four remarkably different expressed proteins were identified. Among them, three spots were converted to Affymetrix probe. A total of 155 microarray samples from Gene Expression Omnibus (GEO) in NCBI was retrieved and followed by constructing gene co-expression networks over a broad range of biological issues through Self-Organising Tree Algorithm. Three co-expressing gene clusters were retrieved and each functional categorization with differential expression pattern was exhibited from 5 MT resistance mutant rice. It was indicated new co-expression networks in the mutant. This study suggests that on investigating possibility which correspond 2-DE to microarray database with their full potential.

Mature dry seeds of Korean cultivars, Daepungkong, Muhankong, Myeongjunamulkong, Somyeongkong, Sowonkong, Jinpumkong, and Pungsannamulkong were used. The influence of antibiotics on elimination of Agrobacterium growth and shoot regeneration was estimated with cotyledonary node. Cefotaxime and timentin at the concentration of 250 and 500 mg/l suppressed Agrobacterium, especially cefotaxime was an efficient antibiotic to suppress Agrobacterium in all cultivars. While carbenicillin and timentin at the concentration of 50 and 100 mg/l were not sufficient to control the development of Agrobacterium, respectively. Cefotaxime and timentin represented high shoot formation rates compared with carbenicillin. Carbenicillin at low concentrations did not effectively suppress Agrobacterium and also had no effect on shoot development. Cefotaxime at the concentration of 250 mg/l showed maximum frequency of shoot regeneration in cvs. Somyeongkong and Sowonkong. Furthermore, on medium containing cefotaxime, shoot was more quickly formed than the other antibiotics. The use of cefotaxime was very useful for elimination of Agrobacterium growth with cotyledonary node of Korean soybean cultivars.

The purpose of this study is to develop the EEG (Euchromatin Enriched Genomic) DNA library of wheat, barley, rye and oat. Mcr A and Mcr BC system in DH5 alpha bacteria cell line and Kuemkangmil, Olbori, Olhomil and Olgwiri were used for materials in our experiments. EEG colonies have been constructed by using junk DNA exclusion. We analyzed the genetic information of the colonies using blast searches of NCBI and GRAMENE web sites. One hundred eighty-four, 65, 79 and 119 STS primer pairs were developed using sequencing data of selected colonies in Kuemkangmil, Olbori, Olhomil and Olgwiri respectively. Twenty-eight and forty-two percent of designed primer pair showed polymorphism using six endoucleases in Kuemkangmil, Olbori, Olhomil and Olgwiri germplasm respectively. These primers could be useful for specific allele tagging in mapping populations and germplasm and for the study of functional genomics of wheat, barley, rye and oat.

The puroindoline genes (Pina and Pinb) losely linked on the short arm of the 5D chromosome (Hardness locus (Ha)) of hexaploid wheat (Triticum aestivum L.) are of great importance to wheat and product quality. In this study, the barley cultivar Harrington was transformed with wheat gene pina and pinb by particle bombardment and performed in order to develop softer barley lines. The result, an efficiency of 3.9% of the bombarded calli regenerated transgenic plants. Each transgenic event was obtained from the callus of a different immature embryo. They showed the multiple shoots compared with control (no bombardment). Two plants from T0 lines were PCR positive for only the 'soft type' pinb transgene, 10 plants were positive for the 'soft type' pina transgene only, whereas they did not show both 'soft type' pina and pinb transgene. These results supply more evidence to support the hypothesis that puroindolines are potential genes for barley grain hardness and used to be suitable target for the production of transgenic barley plants to their availability biolistic transformation system.

The C3HC4 zinc RING finger proteins seem to be a family of protein-protein interactions. Little is information regarding the role of the C3HC4 zinc RING finger proteins in rice plant. We have attempted to assess their genome localization, phylogenetic relationship and expression patterns of members via in silico analysis as well as semi-quantitative RT-PCR. A total of 132 genes encoding C3HC4 zinc RING finger proteins appear to be distributed over 12 rice chromosomes, reflecting evolutionary dynamics of the rice genome, e.g. whole genome duplication and tandem duplications. A genome-wide dataset including 155 gene expression omnibus sample (GSM) plates evidenced a high degree of functional specialization of the rice C3HC4 zinc RING finger proteins, especially during developmental stages and against abiotic stresses. We have retrieved co-expression genes with each of the rice C3HC4 zinc RING finger proteins, probably providing some clues on specialized functions of individual genes. Expression patterns of 13 co-expression genes with one gene encoding C3HC4 zinc RING finger protein (Os04g51400) against salt and dehydration stresses were evaluated in crown tissues and leaf tissues, evidencing highly similar patterns among members. These findings might provide clues to shed further light on comprehensive functions of C3HC4 zinc RING finger proteins.

Previously, the wheat non-specific lipid transfer proteins (TaLTP), members of a small multigene family, appear to show a complex pattern of expression regulation. For further assessment of expression diversity of the TaLTP genes, we have attempted to evaluate their expression profiles of responses to abiotic stresses via the semi-quantitative RT-PCR method. The expression profiles revealed that the TaLTP genes in group A evidenced highly similar (but not identical) responses against abiotic stresses, whereas much differential expression pattern among genes in each group. The four promoters of TaLTP1, TaLTP7, and TaLTP10 of group A and TaLTP3 of group B were fused to a GUS reporter gene and the recombinant genes were introduced into Arabidopsis. The promoters of TaLTP1, TaLTP7 and TaLTP10 of group A, drove strong but various GUS expression in cotyledons, hypocotyls, epidemic and sub-epidemic cells of young shoots and leaves, floral organs as well as siliques. By contrast, the promoter of TaLTP3 just directed trace expression in cotyledons, young emerged leaves and epidemic cells of flower ovaries. The promoter of TaLTP1 directed the expression in root system whereas the promoters of TaLTP1 and TaLTP10 showed some degree of expression during seed development. The expression diversity of TaLTP genes suggests their multiple physiological functions, evidencing subfunctionalization over evolutionary time.

Most gene functions of biochemical pathways were still unexplored, especially interactions of constituent genes. We attempted to uncover interaction network of biochemical pathways via a survey of co-expression clusters, which we have constructed from the NCBI GEO database, and then to define key genes of networks with expression correlations between members. Top 20 pathways with high numbers of individual genes were retrieved from 178 pathways. One pathway, ‘removal of superoxide radicals’ was excluded for further study, evidencing somewhat low degree (16%, 13 out of 79 genes) of mapped probes. We employed expression correlations of random pairs of 1,000 randomly selected genes for determining a cut off r-value for gene networks. Numbers of interactions with a significant expression correlation values between members might evidence that “hub genes” play key roles among a given pathway genes. For example most interactive pathway, ‘tRNA charging pathway’, that is composed of 60 probes corresponding to genes showed 264 positive significant interactions between members of 47 genes while 5 negative interactions between members of 7 genes., evidencing ‘Os10g26050’ (methionyl-tRNA synthetase) gene with highest interactions is suggestive of a hub gene. These findings might provide some clues on evolutionary fate of co-expression genes including each of biochemical pathways, e.g. convergent evolution

Spikelet proteins expressed at the young microspore stage in rice were separated and analysed by two-dimensional polyacrylamide gel electrophoresis (2DE). The separated proteins were electro blotted onto a polyvinylidene difluoride (PVDF) membrane, and 50 proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino acid sequences of 20 out of 50 proteins were determined. N-terminal regions of the remaining proteins could not be sequenced because of blocking. The internal amino acid sequences of proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method. Results revealed the presence of the photosynthetic apparatus at rice young microspore stage. Major proteins identified in this study could be used as a marker for various studies on physiological stresses.

Mature embryos were aseptically excised with a scalpel and sliced in fragments measuring 0.5 mm in diameter (sliced mature embryo fragment; 4 ~~ 5 fragments/one embryo). Sliced mature embryo fragments of six wheat cultivars were cultured to develop an efficient method of callus induction and plant regeneration. Callus derived from sliced mature embryo fragments showed a good capacity to embryogenesis and regeneration. Furthermore sliced mature embryo fragments decreased contamination from fungi and bacteria. The high efficiency of callus induction were obtained Keumkangmil and Bob­white. For plant regeneration, selected embryogenic calli were transferred to two types regeneration media. An average number of green spots per callus was 4 to 5 in regeneration media after about one week. Percentage of plant regeneration showed high in regeneration medium containing 0.1 mg/l 2,4-D and 5 mg/l zeatin. Especially, Keumkangmil (27.5~% ) and Bobwhite (33.3~% ) showed high regeneration efficiency. This regeneration system from sliced mature embryo fragments may provide an effective and convenient explant for plant transformation studies

Thirty-two germplasms of Korean adlay landraces were examined to analyse the genetic relationship through the amplified fragment length polymorphism (AFLP) approach. Total number of AFLP products generated by 12 selective primer combinations was 882. The number of polymorphic fragments by each primer combination greatly varied from 4 to 51 with a mean of 20.3, bands visible on the polyacrylamide gel. A genetic similarity coefficient was used for cluster analysis following UPGMA (unweighted pair grouping method of averages) method. The resulting clusters were represented in the form of a dendrogram. The clustering was not tight in the dendrogram. There was generally no clear grouping of the adlay according to the geographic regions in which germplasms were collected. The present AFLP analysis imply that although Korean adlay displayed a larger amount of AFLP variation within germplasms, the variation was shown independently without reflecting a clinal variation. This study demonstrated that AFLP method can be used to examine the genetic relationships among different germplasms of adlay.

The effects of sonication and vacuum infiltration on transformation efficiency was investigated by using immature embryos of Korean wheat as explants. Two Agrobacterium tumefaciens strains, KYRT1 and EHA105, carrying pCAMBIA 1305.1 were used. Transformation efficiency was demonstrated by the detection of β-glucu-ronidase (GUS) activity. GUS expression showed clear difference among Korean wheat cultivars. Geurumil showed higher GUS expression efficiency 79.1~% compared with other cultivars. The effects of the duration of vacuum infiltration and sonication treatment showed a tendency high GUS expression efficiency by their combination. In comparison with other Agrobacterium strains, KYRT1 showed high efficiency in most Korean cultivars.

To observe and analysis ultra-microscopically barley aleurone cell surface, atomic force microscope (AFM) was used. Seed coat of early maturing germplasm, eam9, was dehulled and scanned by non-contact mode. We have obtained the high resolution topographic 3-dimensional image of barley aleurone layer with high resolution. These images showed the membrane proteins in barley aleurone cell. One channel protein and numerous peripheral or integral proteins were detected in a area of 100 ~mu~textrmm2 . Furthermore, we found that their widths were ranged from 50 to 750nm and lengths from 0 to 66 ~mu~textrmm . The thickness of aleurone layer was measured by scanning electron microscope. The thickness at early developmental stage was about 16 and then the aleurone cell enlarged upto 57 ~mu~textrmm ~mu~textrmm at least until 42 days after anthesis. In this study, we firstly reported on the ultrastructural AFM analysis of living aleurone cell as a biological specimen. It was clearly suggested that AFM will become an powerful tool for probing both the structural properties of biological samples

Immature and mature embryos of 18 Korean wheat genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration, and to compare the responses of both embryo cultures. Immature and mature embryos were placed on a solid agar medium containing the MS salts and vitamins, 30g/l maltose, 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and amino acids. The developed calli were maintained on regeneration medium containing MS salts and B5 vitamins, 20 g/l sucrose, and the combination of two plant growth regulators, 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). Immature embryos in most genotypes showed high efficiency of callus induction except three genotypes; Eunpamil, Chunggemil, and Namhaemil, and significant differences among the genotypes. Plant regeneration of calli induced from immature embryos showed high efficiency in Geurumil (56.5%), Tapdongmil (50.5%), Gobunmil (45.5%), and Urimil(42.2%). The analysis of variance showed significant differences for regeneration frequency among the genotypes. Mature embryos showed low callus induction frequency compared with that in immature embryos, and significant differences among the genotypes. Plant regeneration of calli induced from mature embryos showed high efficiency in Keumkangmil (33.33%), Tapdongmil(28.13%), and Geurumil (27.78%). The analysis of variance showed significant differences for plant regeneration frequency among the genotypes.

Mature embryos of five oat genotypes were cultured to develop an efficient method of callus induction and plant regeneration. Murashige and Skoog(MS) and N6 media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin were used for callus induction. Percentage of callus induction showed significant among the combinations of plant growth regulators. Callus induction showed high efficiency in medium containing 3 mg/~ell of 2,4-D. The high frequency of callus induction was obtained in Gwiri37. For plant regeneration, calli induced from mature embryos were transferred onto MS and N6 media supplemented with combinations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) for 5 weeks. Percentage of plant regeneration showed high in MS medium containing 0.2 mg/~ell of NAA and 1 mg/~ell of BA. The callus initiation medium affected the subsequent plant regeneration. Treatment with 3 mg/~ell of 2,4-D, and 3 mg/~ell of 2,4-D and 3 mg/~ell of kinetin in callus induction media showed high frequency for plant regeneration. Plant regeneration frequency among the genotypes showed significant. Especially, Gwiri37 showed high regeneration frequency. Regenerated shoots were treated with 200, 350 and 500 mg/~ell of indole-3-butyric acid (IBA) transferred onto half-strength MS medium without plant growth regulators. Treatment of shoots with IBA induced root formation rapidly