Recently, in Korea various kinds of genetically modified (GM) crops have been imported and used as a raw material to manufacture foods and livestock feeds, but the different social concerns about the benefits and the potential risks of GM crops are being shown with a different reaction from the public. Thus a persistent management is required for the safe utilization of genetically modified organism (GMO). PCR analysis of transgene into crop is generally performed for the efficient post management of GMOs. The most important prerequisite for the application of nucleic acid detections is to decide the effective DNA-extraction methods. Particularly, in the case of processed feeds, the nucleic acids of which may be damaged by heating, high pressure, pH treatments, fermentation, etc. in processing, DNA must be extracted with high sensitivity from the samples to perform the PCR successfully. In this study, seven of DNA-extraction methods used commercially and non-commercially were compared with respect to the yields and quality of DNA extracted from livestock feeds and those crop materials. Amounts of genomic DNA obtained from the extraction methods varies according to feed configurations and crop materials. The DNA yield and uniformity of samples extracted with PG, CTAB, and QF method is greater than that obtained from other extraction methods. In the DNA integrity of the selected extraction methods, PCR analysis showed distinct amplifications and similar patterns in detecting crop endogenous genes and GMO genes. These results would be applicable for the selection of an adequate DNA-extraction method in extracting processed feeds and/or crop materials.

AtSAGT1 encodes a salicylic acid (SA) glucosyltransferase enzyme that catalyzes the formation of SA glucoside and SA glucose ester. Here, the AtSAGT1 gene expression patterns were determined in AtSAGT1 promoter::GUS transgenic Arabidopsis plants. As a result, the factors regulating the induction of AtSAGT1 were identified as pathogen defense response, wound response, exogenous application of SA, and jasmonic acid treatment.

Natural and artificially induced mutants have provided valuable resources for plant genetic studies and crop improvement. Some variations induced in the process of plant transformation have often been observed in regenerated plants. In this study, we investigated the insertion number of transgene and the flanking sequences of T-DNA in tall-induced line BP23, which was unexpectedly gained in the process of transformation of insect-resistant rice with cryBP1 gene, and also analyzed the whole-genome sequencing by using the NGS technologies to gain a better understanding of the sequence and structural changes between tall line or natural cultivar and rice reference. than others, was confirmed with two copies of foreign gene insertion, which was inserted in one genomic site facing each other between the position 2,430,152~2,430,151 of rice chromosome 12 without any deletion of genomic sequences. Sequencing analysis also revealed that 18bp-unknown sequences were added in the 5′ insertion site of T-DNA. This position in rice genome was confirmed with none of expressed gene sites. By the NGS analysis, we detected 86560 SNPs and 1091/1472 large insertion/deletion (indel) sites (100bp) between BP23 and rice reference, and 84743 SNPs and 1094/1451 large indels between natural cultivar Nagdong and rice reference. The possible mechanisms for the gene mutation, the developmental and tissue expression of the taller height in BP23 line may need to be scrutinized a few more.

가뭄저항성벼의 복수세대에 대한 후대안정성을 서던 분석과 PCR로 분석한 결과, 가뭄저항성벼의 Agb0103 T4～T6 세대에서는 도입된 모든 유전자들이 안정적으로 도입되어 있으며, T-DNA 구성요소 이외의 backbone DNA는 가뭄저항성벼에 삽입되지 않았음을 확인하였다. 목적 유전자인 CaMsrB2와 제초제 저항성 선발 마커인 bar 유전자가 가뭄저항성벼 Agb0103의 T4～T6 세대에서 안정적으로 발현됨을 검증하였다. 제초제 저항성 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 Agb0103 T4～T6의 3세대에서 생육시기별, 부위별로 안정적으로 발현됨을 입증하였다. 도입유전자의 삽입 위치를 확인한 결과, 가뭄저항성벼 Agb0103의 도입유전자가 벼 8번 염색체 내에서 intergenic한 상태로 안정적으로 유지되고 있음을 확인하였다. 이상의 분석 기법을 통해 복수세대에서 가뭄저항성벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

Four transgenic rice lines harboring insect-resistant gene cry3A showed ideal field performances characterized by high considerable resistance to rice water weevil (Lissorhoptrus oryzophilus Kuschel). In this study, we estimated the insert number of foreign genes, and analyzed the flanking sequences of T-DNA in rice genome. As a result, The T-DNA of Btt12R 3-1-1-1 line was inserted in exon region of rice chromosome 10 and Btt12R 6-1-1-1 line was inserted in two copies of foreign gene. Btt12R 9-1-1-1 line was analyzed at only left border flanking sequence. The T-DNA of Btt12R 13-1-1-1 line was inserted one copy of foreign gene between position 24,516,607~24,516,636 of rice chromosome 5 and 30bp known genomic sequences were deleted. The Btt12R 13-1-1-1 line confirmed to be inserted in intergenic region having not any expressed gene and no any deletion/addition of T-DNA sequence. From these results, we demonstrated that the molecular data of rice water weevil resistant Bt rice could be acceptable to conduct the biosafety and environment risk assessment for GM crop commercialization

Genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by various agricultural biotechnology companies. Commercialization of GM crops will be required the assessment of risk associated with the release of GM crops. The purpose of this research is a molecular characterization of introduced T-DNA in transgenic rice T4 ∼ T6 generation lines harboring a pepper MsrB2 gene under the control of stress inducible Rab21 promoter, as a part of biosafety evaluation for drought-tolerant transgenic rice (Agb0103). We identified the structure and sequence of transformation vector of T-DNA and analyzed insertion sites, flanking sequences, and generational stability of inserted T-DNA in transgenic rice lines. The transformation vector was consisted of right border, a drought-tolerant CaMsrB2 gene unit (Rab21 promoter::CaMsrB2::PinII terminator), a selectable marker herbicide resistance unit (CaMV 35S promoter::bar::Nos terminator), and left border in sequential order. Based on the adaptor-ligation PCR and whole genome sequence database, we confirmed that T-DNA was introduced 2 copies (head to head type) at the position of 2,471,957 ∼ 2,472,049 bp of chromosome No. 8. From the generational stability study, T-DNAs were stably inherited through the T4 to T6 generations, and also stable expression of bar gene from T-DNA was confirmed. It was also confirmed that the backbone DNA of transformation vector containing antibacterial gene (aadA) was not present in Agb0103 rice genome. These results will be filed to biosafety assessment document of Agb0103

Two carotenoid biosynthetic genes, phytoene synthase (Psy) and carotene desaturase (CrtI) linked via synthetic 2A sequence under control of CaMV 35S promoter (two T0 plants 5 and 6) or β- conglycinin promoter (three T0 plants 7, 13 and 16) were transformed into soybean variety Kwangan. After agronomic and phenotypic selection at early generations, T5 progeny of PAC soybean were analyzed by Southern blot to confirm T-DNA copy numbers. A total of 27 homologous lines derived from one of three T0 plants (line 7 under the control of β- conglycinin promoter) with one copy T-DNA insertion, were separated and planted into greenhouse. Flanking sequence analysis was carried out on one of homologous line 6-2-3 and results indicated the T-DNA was intergenic inserted into chromosome 14 from 10,873,131 to 10,872,998 base of soybean chromosome. T-DNA insertion structure, flanking sequence and inserted gene expressions need to be analyzed in the further study.

Resveratrol rice Iksan526 was developed by overexpession of T-DNA (RB::P-Ubi::RS::T-NOS::P-35S::PAT::T-35S::LB) in rice variety Dongjin. To confirm one locus insertion of T-DNAs, Mendelian genetic analysis was carried out on selection marker bar gene and objective RS gene separately by using a F2 population derived from a cross of Dongjin/Iksan526 (T6). A total of 450 four-leaf-old plants from F2 population were treated by 0.3% basta, and a phenotypic separation ratio of 3:1 (321 survival: 129 dead, p>0.90) complied with Mendelian inheritance indicating one locus insertion of bar gene. Genotypic separation was analyzed by using PCR with specific primers for 300 plants, which were selected from 321 survival plants after phenotypic separation. Results revealed a ratio 1:2 of homologous to heterozygous (92:208, p>0.90), which further confirmed one locus insertion of RS gene. In addition, comparison on agronomic traits and resveratrol contents between transgenic rice and the donor variety were launched to evaluate the phenotypic performance over multi-generations (years).

가뭄저항성벼의 복수세대에 대한 후대안정성을 서던 블롯과 PCR로 분석한 결과, 가뭄저항성벼의 CaMsrB2-8 T4 ~ T6 세대에서는 도입된 모든 유전자들이 안정적으로 도입되어 있으며, T-DNA 구성요소 이외의 backbone DNA는 가뭄저항성벼에 삽입되지 않았음을 확인하였다. 목적 유전자인 CaMsrB2와 제초제 저항성 선발 마커인 bar가 가뭄저항성벼의 CaMsrB2-8 T4 ~ T6 세대에서 안정적으로 발현됨을 검증하였다. 제초제 저항성 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 CaMsrB2-8 T4 ~ T6의 3세대에서 생육시기별, 부위별로 안정적으로 발현됨을 입증하였다. 도입유전자의 삽입 위치를 확인한 결과, 가뭄저항성벼 CaMsrB2-8의 도입유전자가 벼 1번 염색체 내에서 intergenic한 상태로 안정적으로 유지되고있음을 확인하였다. 이상의 분석 기법을 통해 복수세대에서 가뭄저항성벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

Pollen development in flowering plants is regulated by a comprehensive pattern of genes. One way to produce hybrid rice based on nuclear male sterility is to find out firstly the potential promoters that function specifically in anthers since it is a specific site for transcription initiation and play key roles for the spatial and temporal expression of the genes. To implement this objective, we were selected promoter region of 16 genes based on the expression pattern of microarray and then those were introduced into the promoterless final destination vector which containing the GFP and GUS reporters genes. The resulting twelve vectors were transformed into monocotyledonous rice (Oryza sativa L) and a dicotyledonous Arabidopsis as heterologous system. Minimum 20 plants for each vector were analyzed by histochemical GUS assay at the flowering stage in Arabidopsis. 9 vectors out of 12 vectors constructed were expressed exclusively at the anther, especially in pollen, however one vector exhibited expression in stigma. For rice, T-DNA insertion were confirmed with specific primers in each promoter and GFP region. All T0 transgenic plants contained T-DNA insertion in their genome. This study would provide valuable information for biotechnological application for the induction of male sterility in plants.

Organ size control is a fundamental developmental processes for higher plants as well as a promising target trait for molecular breeding in crop plants. Genetic mechanisms how plant organs grow to a certain size remains still unclear. Here we present the identification and characterization of a genetic mutant, big flower1-1 (bif1-1) in Arabidopsis that exhibits bigger organ size primarily due to increased cell size. Genetic analysis indicated that it is a single, semi-dominant mutation. Phenotypic analysis showed that bif1-1 exerts pleiotropic effects: it caused bigger seed size, bigger seedling, bigger leaf, thicker stem, increased trichome branching, smaller fruit, and bigger pollen. Microscopic analysis suggested that the bigger organ size in bif1-1 mutant is primarily attributed to increased cell size. Gene expression analysis indicated that most of growth-control genes tested were not altered in bif1-1 mutant. Instead, expression of ARGOS and auxin-inducibility of ANT were reduced in bif1-1 mutant. Our ongoing positional on the corresponding gene would not only shed light on the molecular mechanisms how plants adopt final organ size but also provide a promising genetic resource for genetic engineering of flower- and seed-size in crop plants.

Toward molecular understanding of flower senescence/abscission, we have identified a mutant, designated as dea1-1D (dealyed abscission1-1D), with delayed flower senescence/abscission syndrome from activation-tagged pools. Phenotypic analysis revealed pleiotropic effects of dea1-1D mutation including delayed flowering as well as smaller serrated leaves. Genetic analysis showed that it is a dominant mutation. Molecular analysis on the flower senescence syndrome indicated that dea1-1D might define novel regulatory branch of flower abscission, controlling expression of ethylene-responsive AP2 transcription factor. On the contrary, triple responses was not affected by dea1-1D mutant. Though the penetrance was not complete, the mutant phenoytpes was shown to be tightly linked with the T-DNA selection marker, BASTA-resistance. We identified the T-DNA insertion site through molecular cloning of the T-DNA flanking genomic DNA and found that a neighboring gene was overexpressed in the dea1-1D mutant. Together with gene expression analysis, we will discuss possible function of DEA1 during flower senescence and abscission.

The transport of nascent messenger RNA from the nucleus to the cytoplasm is mediated by the THO/TREX complex and is evolutionary conserved from yeast, metazoa and humans. However, in plants, it is still yet unclear if the similar mechanism of transport exists. Here we identified and characterized a mutant gene, AtTHO2, a putative Arabidopsis thaliana THO2 component protein, homologous to yeast THO2 of the THO/TREX pathway required for mRNA transport. The mutation from this gene resulted to various developmental defects that include semi-dwarfism and abnormal floral development which further leads to sterility. Gene expression analysis revealed that AtTHO2 is expressed in all organs and pollen developmental stages. In addition, the homozygote progeny of null mutants did not persist until mature stage. These results suggest an indispensable role of AtTHO2 in the development of Arabidopsis. Differential gen expression and silencing were also observed between the null mutants and wild type depending on T-DNA insertion. Furthermore, alternative splicing which was tightly linked with the THO/TREX pathways was also defective on AtTHO2 and null mutants. A similar pattern of defect in SR34a was observed in the AtTHO2 and null mutants. In terms of microRNA biosynthesis, no significant differences were seen on the wild-type and mutant plants; however this data should be validated. Thus this work provides some evidences that a similar THO/TREX complex exist in plants and gave a foundation for further studies on the mechanism of nuclear export in plants.

A diverse number of genes are involved in the floral transition and development to ensure the proper timing on the switch from vegetative to reproductive development in Arabiodopsis. MADS-box genes play a major role in floral development especially in the case of vernalization process, In this study we mapped a mutation in MAF5 encoding a MADS-domain protein which was reported to be up-regulated during vernalization and regulates flowering time. The mutant in MAF5 showed several pleiotropic phenotypes that includes semi-dwarfism, delayed senescence and abnormal pollen phenotype, High percentages of vacuolated and aborted pollen phenotype were observed in the mutant plant. Transmission efficiency showed that mutation from this gene was defective in both male and female gametes. Furthermore, gene expression analysis revealed that this gene was predominantly expressed in reproductive organs and gave a strong expression in the mature pollen which coincides with the defect in pollen phenotype. The results from this study provide some evidences on the additional role of MAF5 in pollen development however more specific approaches should be done to determine the specific stages of pollen development altered in this mutant.

Pollen development in flowering plants is regulated by a comprehensive pattern of genes. One way to produce hybrid rice based on nuclear male sterility is to find out firstly the potential promoters that function specifically in anthers since it is a specific site for transcription initiation and play key roles for the spatial and temporal expression of the genes. To implement this objective, we were selected promoter region of 16 genes based on the expression pattern of microarray and then those were introduced into the promoterless final destination vector which containing the GFP and GUS reporters genes. The resulting twelve vectors were transformed into monocotyledonous rice (Oryza sativa L) and a dicotyledonous Arabidopsis as heterologous system. Minimum 20 plants for each vector were analyzed by histochemical GUS assay at the flowering stage in Arabidopsis. 9 vectors out of 12 vectors constructed were expressed exclusively at the anther, especially in pollen, however one vector exhibited expression in stigma. For rice, T-DNA insertion were confirmed with specific primers in each promoter and GFP region. All T0 transgenic plants contained T-DNA insertion in their genome. This study would provide valuable information for biotechnological application for the induction of male sterility in plants.

A total of 162 accessions of Perilla germplasm collected from diverse habitats in South Korea. The oil content of the seeds varied from 19.8 to 49.6%, the average being 33.3%. Oil content showed wide ranges of variation. The fatty acid composition of the seed oil showed the following overall ranges: palmitic 4.5 to 8.1%, stearic 1.6 to 7.4%, oleic 13.4 to 23.6%, linoleic 9.5 to 19.5%, and linolenic acid 48.6 to 66.4%. No correlation was found between oil content and fatty acid composition. The results obtained in this study provide useful background information for developing new cultivars with a high oil content and different fatty acid. Several accessions could be used as parental lines in breeding programs aiming to increase perilla oil quantity and quality.