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Beta-carotene-enhanced transgenic soybeans, harboring genes encoding phytoene synthase and carotene desaturase under the control of a seed-specific promoter, were developed to alleviate vitamin A deficiency in populations, the diet of which was deficient in this vitamin. However, metabolic engineering of carotenoid biosynthetic pathways often has unintended effects, leading to major metabolic changes in plants that harbor endogenous beta-carotene biosynthesis pathways. In the present study, we performed transcriptome profiling analysis using RNA-seq to investigate the changes in the transcriptome and some unintended pleiotropic effects on the leaves, stems, roots, and seeds of beta-carotene-enhanced transgenic soybean lines, and compared them to those of their non-transgenic counterpart donor variety Kwangan. We observed that transgenic soybeans showed significant changes in secondary metabolic biosynthesis in leaves and down-regulated galactose metabolism in roots. Differentially expressed genes in the transgenic group, which were significantly up-regulated, included those encoding glycine-aspartic acid-serine-leucine-motif esterase/lipase, known as cutin synthase and cutinase. These results suggested enhanced beta-carotene biosynthesis may affect related enzymes to carbohydrate metabolism and fatty acid metabolism. Hence, we speculated that upregulation of cutin polymerization resulted in thickened seed coat and delayed seed germination of transgenic soybeans. Furthermore, downregulation of raffinose family oligosaccharide biosynthesis may cause redundancy of myo-inositol, a substrate of phytin formation. This could lead to phytic globoids accumulation in transgenic soybean seeds. The present imformation would be important for transgenic plant development via carotenoid metabolic engineering, with focus on beta-carotene over-production. Gene expression changes related to secondary metabolite biosynthesis, cutin metabolism, and raffinose-family oligosaccharide biosynthesis in leaf, stem, and root, and phytin globoid accumulation in seeds, occurred in beta-carotene-enhanced transgenic soybeans.

Bacterial communities in rhizosphere and root nodules have significant contributions to the growth and productivity of the soybean (Glycine max (L.) Merr.). In this report, we analyzed the physiological properties and dynamics of bacterial community structure in rhizosphere and root nodules at different growth stages using BioLog EcoPlate and high-throughput sequencing technology, respectively. The BioLog assay found that the metabolic capability of rhizosphere is in increasing trend in the growth of soybeans as compared to the bulk soil. As a result of the Illumina sequencing analysis, the microbial community structure of rhizosphere and root nodules was found to be influenced by the variety and growth stage of the soybean. At the phylum level, Actinobacteria were the most abundant in rhizosphere at all growth stages, followed by Alphaproteobacteria and Acidobacteria, and the phylum Bacteroidetes showed the greatest change. But, in the root nodules Alphaproteobacteria were dominant. The results of the OTU analysis exhibited the dominance of Bradyrhizobium during the entire stage of growth, but the ratio of non-rhizobial bacteria showed an increasing trend as the soybean growth progressed. These findings revealed that bacterial community in the rhizosphere and root nodules changed according to both the variety and growth stages of soybean in the field.

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

Nutritional composition is important for assessing the safety of genetically modified (GM) crops for human consumption. Three beta-carotene-enhanced soybean lines were developed by introducing the β-conglycinin promoter::Phytoene synthase-2A-Carotene desaturase/t35S gene cassette into the genome of the commercial Kwangan (Glycine max L.) soybean variety. Transgenic soybeans were successfully detected on beta-carotene productions ranged from 170.47 to 213.58 µg/g. Comparative assessments of nutrition were conducted with 3 transgenic soybeans, their non-GM counterpart, and several commercial soybean varieties. Results indicated that most levels of proximate, fatty acids, amino acids, and vitamins showed non-significant differences between transgenic soybeans and their counterpart, and fit within the reference ranges established for other commercial soybeans and Organization for Economic Cooperation and Development Guidelines. However, significant differences on levels of crude fat, carbohydrate, δ-tocopherol, and oleic acid of transgenic soybeans comparing to those of non-transgenic counterpart Kwangan cannot eliminate the influences of transgene insertion. Alternations on compositions should be definite by further studies, such as transcriptome and metabolome profiling.

Public concern about the safety of genetically modified (GM) crops and potential pleiotropic effects of selectable marker gene insertion and expression can be avoided by developing a selectable marker-free transformation system. Various techniques have been developed to produce marker-free transgenic plants; however, an approach that does not require the use of selectable marker genes would not require post-transformation processes. We report such a method for generating of GM rice plants. To develop transgenic rice resistant to lepidopteran insects, a marker-free binary vector harboring the Bacillus thuringiensis mcyr1Ac gene was constructed and used for Agrobacterium tumefaciens-mediated transformation of rice scutellar calli. Initial screening of putative T0 transgenic rice plants was performed using polymerase chain reaction (PCR) on pools of DNA from 15 to 30 regenerated shoots, followed by genomic DNA PCR and Cry1Ac ImmunoStrip assays of individual plant extracts. Although the overall rice transformation efficiency of the non-selection approach is lower than that of traditional rice transformation by using marker genes, we derived six independent homozygous T3 events from Cry+ T0 shoots. Transgenes were detected and inheritance confirmed using DNA PCR, RT-PCR, and Southern blot analysis, and insertion sites were characterized by the sequencing of DNA flanking the transgenes. The transgene expression of cry1Ac was also stable and effective against rice leaf folder at levels comparable to those of a transgenic plant that had been derived by selection. This method could be applied to other plant species with similar transformation efficiencies to create selectable-marker-free transgenic plants for research and commercial uses.

Most Bt transgenic rice events have an underlying grain cost depending on the insect pressure in the field. This study was conducted (1) to determine if the cry1Ac/bar genetically modified (GM) rice (designated Agb0101) based on a Korean japonica rice cultivar has any underlying grain cost and (2) to develop cry1Ac/bar GM rice lines with yield performance similar to those of Korean elite cultivars via crossbreeding. A three-year field experiment showed that Agb0101 had a high underlying grain cost equivalent to 18% grain yield loss, and this cost was mainly due to the grain number per panicle. Moreover, it was found that the cry1Ac/bar transgene was inherited as a single dominant gene. BC1F4―F5 crossbred progeny derived from an anther culture of Agb0101―showed phenotypic resemblance to non-transgenic Korean elite cultivars, resulting in a decrease in the grain yield cost of Agb0101 from 27% to 10%. These results suggest that Bt-GM rice-oriented cross-fertilization with a non-GM elite cultivar followed by two- or threefold selection is a feasible method to minimize or remove the underlying grain cost of the Bt transgenic insect-resistant rice line.

본 연구는 국내에서 개발된 토코페롤 합성 TC 유전자가 형질전환 된 GM 벼와 그 모본인 흑남벼 및 일반벼 품종인 일미벼, 동진벼 현미의 일반성분, 무기질 및 아미노산함량을 분석하여 조성 차이가 있는지를 비교하였다. 모본벼인 흑남벼와 비교하여 GM 벼 현미의 일반성분 조성중 수분 함량과 조회분 및 단백질이 다소 높았지만 기존에 보고된 일반품종 벼 현미의 성분함량 범위 안에 포함되었다. 또한 GM 벼 현미에 포함된 아미노산 함량과 무기질 함량은 전반적으로 모본벼과 유의적 차이가 없었다. 따라서 본 실험에 사용된 GM 벼는 유전자 형질전환에 의한 비의도적 영양성분 변화는 없는 것으로 판단된다.

Four transgenic rice lines harboring insect-resistant gene cry3A showed ideal field performances characterized by high considerable resistance to rice water weevil ( Lis-sorhoptrus oryzophilus Kuschel). In this study, we estimated the insertion number of foreign genes, and analyzed the flanking sequences of T-DNAs in rice genome. As a result, T-DNA of BT12R1 line was inserted in exon region of rice chromosome 10. Two copies of T-DNAs were inserted in line BT12R2. BT12R3 line was analyzed at only left border flanking sequence. BT12R4 line was confirmed one copy of foreign gene insertion at the position 24,516,607 ~ 24,516,636 of rice chromosome 5, accompanied by a deletion of 30 bp known genomic sequences. This intergenic position was confirmed none of expressed gene and any deletion/addition of T-DNA sequence. In conclusion, these molecular data of rice water weevil resistant Bt rice would be used to conduct the biosafety and environment risk assessment for GM crop commercialization.

Confirmation of T-DNA inheritance and stability requires testing of several parameters such as phenotypic performance of transgenic lines, transgene integrity structure and expression stability for at least three consecutive generations, and Mendelian inheritance analysis, which are very important to transgenic breeding and commercialization of genetically modified crops (GMOs). In the present study, we measured nine agronomic and yield-related traits of Iksan526 and confirmed stable phenotypic performance comparing to the donor variety Dongjin over three years. Transgene integrity structure, flanking sequence, functional element copy numbers, absence of backbone sequence and transgene expression were all verified in Iksan526 over three consecutive generations, indicating a stable inheritance of the transgene. In addition, phenotypic and genotypic segregation were analyzed on F2 and F3 populations derived from a cross of Dongjin/Iksan526, and results complied with Mendel’s law of segregation based on single transgene locus. This study demonstrated detailed analyses and confirmation of transgene event Iksan526 which is crucial for commercialization of GMO.

Tocopherols (α-, β-, γ-, and δ-tocopherol) represent a group of lipophilic antioxidants that are synthesized only by photosynthetic organisms. It is widely believed that the main functions of tocopherols are protection of pigments and proteins of photosystem and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species. In the present study, we report on the cloning and characterization of NtTC, which is a tocopherol cyclase (TC) ortholog isolated from tobacco. To enhance tocopherol contents, we generated independent transgenic rice events in expressing NtTC or NtTC along with Perilla γ-tocopherol methyltransferase genes. The transgenic TC line significantly increased α-, total tocopherol, total glutathione, and total antioxidant status activity levels compared with the wild type. Furthermore, TC rice plants showed higher tolerance to drought than wild-type rice plants. On the basis of these studies, we concluded that overexpression of NtTC could increase the tolerance to drought stress and that the increase in tocopherol affects cellular signaling and antioxidant defense of plants in response to drought.

Photoautotrophic culture has advantages over conventional micropropagation methods, and its potential benefits appear to be promising. Nevertheless, little is known regarding the effects of such culture conditions on plant physiology during ex vitro acclimation. In this study, Doritaenopsis plantlets were grown under three different microenvironmental conditions: (1) in vitro under photoautotrophic conditions (high photosynthetic photon flux density [PPFD], high CO2 concentrations and an increased number of air exchanges), (2) photomixotrophic conditions (photoautotrophic conditions, with 30 g L−1 sucrose added to the culture medium) and (3) heterotrophic conditions (conventional culture conditions; low PPFD, low CO2 concentration and no air exchange). The plantlets were subsequently transferred into ex vitro conditions, and the physiological and biochemical changes were investigated during the acclimation process. Photoautotrophic and photomixotrophic in vitro cultivation markedly stimulated the growth of Doritaenopsis plantlets during ex vitro acclimatization. The development of photosynthetic function (PEP-carboxylase activities and carbohydrate metabolism) appeared to be fully achieved in ex vitro plantlets that had been cultured in photoautotrophic and aerated photomixotrophic conditions in vitro. The strong enhancement of growth by CO2 enrichment, along with the exposure of photoautotrophic plantlets to high PPFD, may explain why the plants adjust more effectively (exhibiting higher rates of photosynthesis under high-light conditions) during ex vitro acclimatization. The results of this study suggest that the growth and survivability of Doritaenopsis during the acclimatization process can be enhanced by CO2 supplementation and increased light levels during in vitro culture.

Resveratrol, a plant phenolic compound, has potential therapeutic benefits due to its antioxidant properties. This is substantiated by previous studies that show that resveratrol derived from rice grains is an effective treatment agent for metabolic syndrome. Here, we characterized the T-DNA sequence, inserted T-DNA structure, copy number, integrity of the transgene locus, resveratrol synthase gene expression and resveratrol contents in the grains of two resveratrol transgenic rice lines, Iksan515 and Iksan526. The T-DNA transformation vector contained two expression cassettes of the resveratrol synthase gene under the control of the ubiquitin promoter and the bar selection marker gene under the control of the CaMV35S promoter. Flanking sequence analysis indicated that the T-DNAs were inserted into intergenic regions of chromosome 4 for Iksan515 and chromosome 12 for Iksan526. Two T-DNAs connected in an inverted repeat structure at a single locus of the rice genome were identified by whole genome sequencing and Southern blot hybridization in both Iksan515 and Iksan526. No novel open reading frames (ORFs) around insertion sites, sequences encoding allergenic or toxic protein, or other unintended effects by T-DNA insertion were found in either case. In addition, resveratrol synthase gene expression in leaves and resveratrol detection in brown rice grains suggested the successful expression of the inserted foreign resveratrol synthase gene in two transgenic rice lines.

Stacked genetically modified (GM) crops are becoming popular for their enhanced production efficiency and improved functional properties. In this study, we developed an event-specific PCR method for simple qualitative detection of stacked events combining more than 2 transgenic traits. Ten primer sets were designed, including 9 that were event-specific and 1 that was specific for a maize endogenous gene. Five event-specific multiplex-PCR systems were built, based on the main type of stacked GM events approved in Korea. Multiplex PCR was performed with mixtures of template DNA extracted from certified reference materials. PCR amplicons (3 or 4 by type) of expected sizes and mutually similar intensities were detected. The limit of detection was approximately 0.1%(v/v) for stacked GM maize in all event-specific PCRs. This method may be useful for the specific detection and monitoring of stacked GM maize lines and individual parent GM maize lines, by effectively distinguishing genestacked events.

The rice water weevil (RWW), Lissorhoptrus oryzophilus Kuschel, is the most widely distributed and destructive early season insect pest of rice, Oryza sativa L. worldwide. The rice plants were transformed with cryIIIA insecticidal gene as well as with the bar gene coding phosphinothricin acetyltransferase. CryIIIA gene under the control of a modified RCg2 promoter drives the insect-toxic gene expression in roots and/or leaves. The cryIIIA gene was transferred into O. sativa L. cv. Nakdong by Agrobacterium-mediated transformation. Stable integration of the transgene was confirmed in putative transformed rice by Southern blot analysis. The expression of the cryIIIA toxin gene in the roots of transgenic rice plants was verified by RT-PCR and immunoblot analysis. Transgenic rice plants were also evaluated for resistance to natural infestations of the RWW under field conditions between 2007 and 2011. The transgenic Btt8R and Btt12R lines reduced the growth rate of RWW larvae and pupae populations compared with non-transgenic control plants by approximately 52 and 58 %, respectively. To further examine the efficacy of the RWW bioassay, we used pots and performed experiments in trays and under field conditions in 2012. The Btt12R line reduced the total populations of RWW larvae and pupae in trays and under field conditions by 56 and 45 %, respectively. The bioassay experiments conducted over 6 years, showed a significant reduction rate of RWW larvae and pupae populations demonstrating that the cryIIIA gene in transgenic rice confers resistance to RWW.

A less simple approach developed for generation of marker-free transgenic plants is to select transformants without the use of selective marker genes. Some results about development of marker-free transgenic plants were obtained using a non-selective approach in several crops such as rice, potato and tobacco. However, the study did not provide evidence on detailed characterization of introduced gene on genome, a critical step for confirming the stable integration and transmission of a foreign gene. In this study, we evaluated structure and integration sites of transgene (m Cry1Ac ) in the transgenic Bt rice plants which were made via conventional Agrobacterium -mediated transformation by non-selective method. Structure and integration sites of transgene in these transgenic plants had similar fashion as those recovered under selection. Keywords Non-selective, Marker-free transgenic rice, Agrobacterium -mediated transformation, Transgene, integration site 서 론 선발마커유전자(Selective marker gene)는 목표유전자와 연결하여, 도입유전자가 형질전환 된 식물체를 선택적으로 선발하는데 사용된다. 통상적으로

살충성 유전자 mcrylAcl을 포함하고 있는 해충저항성 유 전자변형 （GM) 벼 Agb0101이 국내에서 개발되었다． 향후 Agb0101 벼의 환경방출에 따른 모니터링과 이력추적을 위해서는 신뢰성 있는 검출방법의 개발이 필요하다． 따라서， 본 연구에서 해충저항성 GM벼의 사후 안전관리를 위한 정성적 및 정량적 PCR 검정 방법을 개발하였다． 벼 녹말분지효소 유전자 RBE4를 PCR 분석의 내재유전자로 사용하였고， 이의 primer쌍 RBEgh- 1/ - 2는 101bp의 PCR 증폭산물을 형성하였다． 정성 PCR 분석을 위해서 삽입된 T- DNA를 바탕으로 특이 Primer를 제작하였고， 이벤트 특이적 검출 primer의 경우 Agb0101의 도입유전자 및 벼 염색체 DNA 사이의 5``또는3``인접염기부위를 정확하게 특이적으로 PCR 증폭하였다． 반면， 대조구인 각종 작물， 국 내 벼 품종 및 Agb0101과 동일 형질전환 벡터를 갖는 해 충 저 항성 벼 에 서 는 어 떠 한 PCR 증폭산물도 형성하지 않았다. 표준물질로써 내재유전자 및 이벤트 특이적 단편으로 제조된 pRBECrR을 이용한 real- time PCR 분석에 의해 서 정량한계 （LOQ） 가 10 copies 농도의 범위인 것으로 확인되었고, 이의 유효성을 검증하기 위하여 상이한 농도 의 Agb0101시료 （1O, 5, 3 및 I%를 real-time PCR 분석하여 정량검정에 대한 표준편차 및 상대표준편차가 각각 0. 06~0.40 및 3.80, 7.01％ 의 낮은 범위에 포함되는 것을 확 인할 수 있었다． 이들 결과로 본 연구에서 개발된 정성 및 정량 PCR 검정 방법이 해충저항성 GM벼 Agb0101의 모니터링 및 이력추적에 효과적으로 이용될 수 있을 것으로 본다.

The cultivation of genetically modified (GM) crops has increased due to their economic and agronomic advantages. Before commercialization of GM crops, however, we must assess the potential risks of GM crops on human health and environment. The aim of this study was to investigate the possible impact of Bt rice on the soil microbial community. Microbial communities were isolated from the rhizosphere soil cultivated with Bt rice and Nakdong, parental cultivar and were subjected to be analyzed using both culture-dependent and molecular methods. The total counts of bacteria, fungi, and actinomycetes in the rhizosphere of transgenic and conventional rice were not significantly different. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that the bacterial community structures during cultural periods were very similar each other. Analysis of dominant isolates in the rhizosphere cultivated with Bt and Nakdong rice showed that the dominant isolates from the soil of Bt rice and Nakdong belonged to the Proteobacteria, Cloroflexi, Actinobacteria, Firmicutes, and Acidobacteria. These results indicate that the Bt rice has no significant impact on the soil microbial communities during cultivation period. Further study remains to be investigated whether the residue of Bt rice effect on the soil environment.

A novel genetically modified organism (GMO) detection method was developed using a simple and rapid procedure to identify a regulatory gene introduced into genetically modified (GM) maize. After DNA extraction from GM maize flours, fragments of the conserved sequence of the cauliflower masaic virus (CaMV) 35S promoter gene along with a competitive internal control gene were amplified using duplex polymerase chain reaction (PCR). Amplification products were then detected using a disposable detection device. The quantitative detection limit for GM maize 59122 was found to be 1.0% (w/w). Because the combination-method involving PCR technology coupled with a disposable detection device does not require expensive instrumentation or expertise, it will serve as a valuable alternative to immunoassays and traditional PCR-based tests in the detection of GMOs.

Composition analysis of genetically modified crops is an important consideration in the assessment of food safety. Agb0101 (Oryza sativa L. cv. Nakdongbyeo) developed in Korea is a form of insect-resistant rice that contains a synthetic truncated cry1Ac gene isolated from Bacillus thuringiensis (Bt), and demonstrates high resistance to rice leaf folder under field conditions. Nutrients, anti-nutritive components, and secondary metabolites of Agb0101 were analyzed and compared with those of its non-transgenic counterpart. The amounts of proximates, amino acids, fatty acids, minerals, vitamins, trypsin inhibitors, phytic acid, and ferulic acid in brown rice from Agb0101 were comparable to those of its non-transgenic counterpart. Statistical comparisons to test for equivalence showed that all the analyzed components in the insect-resistant rice plants were substantially equivalent to those of its non-transgenic counterpart. Furthermore, most of the measured values from Agb0101 were within the range of values reported for other commercial rice varieties.

We developed 14 transgenic lines of Chinese cabbage (Brassica rapa) harboring the T-DNA border sequences and CryIAc1 transgene of the binary vector 416 using Agrobacterium tumefaciens-mediated DNA transfer. Six lines had single copy cryIAc1 gene and four of them contained no vector backbone DNA. Of the left border (LB) flanking sequences six nucleotides were deleted in transgenic lines 416-2 and 416-3, eleven nucleotides in line 416-9, and 65 nucleotides including the whole LB sequences in line 416-17, respectively. And we defined 499 bp of genomic DNA (gDNA) of transformed Chinese cabbage, and blast results showed 96% homology with Brassica oleracea sequences. PCR with specific primer for the right border (RB) franking sequence revealed 834 bp of PCR product sequence, and it was consisted of 3` end of cryIAc1, nosterminal region and 52 bp of Chinese cabbage genomic DNA near RB. RB sequences were not found and the 58 nucleotides including 21 bp of nos-terminator 3` end were deleted. Also, there were deletion of 10 bp of the known genomic sequences and insertion of 65 bp undefined genomic sequences of Chinese cabbage in the integration site. These results demonstrate that the integration of T-DNA can be accompanied by unusual deletions and insertions both in transgenic and genomic sequences.

Lactoferrin is an 80-kDa iron-binding glycoprotein that is found in high concentrations in human milk. Human lactoferrin (hLF) has several beneficial biological activities including immune system modulation and antimicrobial activity. In the present study, we devolved a method of hLF expression through introducing the hLF gene construct into Oriza sativa cv. Nakdong using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the rice glutelin promoter was detected in the seeds of transgenic rice plants. Transformed rice plants were selected on media containing herbicide(DL-phosphinothricin) and the integration of hLF cDNA was confirmed by Southern blot analysis. The expression of the full length hLF protein from the grains of transgenic rice plants was verified by Western blot analysis. The lactoferrin expression levels in the transformed rice grains determined by enzyme-linked immunosorbant assay accounted for approximately 1.5% of total soluble protein. Taken together, these data indicate that rice grains expressing hLF can be directly incorporated into infant formula and baby food.

Selectable marker gene systems are vital for the development of transgenic plants, but the presence of selectable marker genes encoding antibiotic or herbicide resistance in genetically modified plants poses a number of problems. A lot of research results and various techniques have been developed to produce marker-free GM plants. The aim of this review is to describe the principal methods used for eliminating selectable marker genes to generate marker-free GM plants, concentrating on the three significant methods(co-transformation, site-specific recombinase-mediated ex-cision, non-selected transformation) in several marker-free techniques. 서 론 1996년 처음 상업화된 유전자변형 (Genetically Modified, GM) 작물의 재배면적은 매년 급속한 성장을 기록하여 2009년에는 25개국 1억 3천 4백만 헥타르에 이르고 있다(James 2009). 이와 같은 GM작물의 성장세는 개발도상국과 선진국의 재배농민 모두에게 GM작물이 경제적으로 도움이 되며 식물형질전환기술의 급속한 발전으로 주요작물에서 높은 효율로 형질전환 (transformation)이 가능해졌기 때문이다. GM작물 개발을 위한 식물형질전환기술의 급속한 발전은 새로운 배지나 형질전환법의 개발이 빠르게 이루어졌기 때문이기도 하지만, 항생제 내성 유전자와 같은 선발표지 시스템을 식물형질전환에 적용했기 때문이다 (Miki and McHung 2004). 일반적으로 식물체 내에 목적유전자를 형질전환하기 위해서는 운반체 (vector)라는 DNA 구성물을 사용하여 목적유전자와 함께 항생제 내성 유전자와 같은 선발표지 유전자를 토양미생물인 아그로박테리움 (

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is one of the most commonly used in the development of genetically modified (GM) crops. In this research, we analyzed Bt rice showing lepidopteran pest-resistance. The Bt gene is a synthetic Cry1Ac composed of optimal codons for plants, and the Bt protein is targeted to the chloroplast by a transit peptide. Three Cry1Ac rice events (C103-3, C127-1, and C7-1) were analyzed for molecular characterization. C103-3 contains two copies of T-DNA where the left border (LB) region is truncated. Both C7-1 and C127-1 have a single copy of T-DNA, but a part of the vector backbone DNA is inserted into the genome of C127-1; thus, only C7-1 had intact T-DNA. Progenies of C7-1 crossed with the original cultivar, Nakdong, and double-haploid lines from anther culture of lines crossed with the elite cultivar, Dongjin, were analyzed for T-DNA flanking genomic DNA and genotyping. Results showed that an intact T-DNA region without the vector backbone was inserted into the genome and was stably inherited through generations. The C7-1 homozygous event could be used as breeding material to develop GM rice with pest resistance.

Genetic components introduced into approved GM crops are a key subject for safety assessment and provide a basis for the development of detection methods for GM crops. In order to understand the genetic components in approved GM crops comprehensively, we screened the genetic vector maps of GM crops that had been approved for commercialization around the world. A total of 64 varieties from 5 major GM crop species (maize, canola, cotton, soybean, and tomato) were subjected to analysis. The genetic components included genes, promoters, terminators, and selection marker. This survey may be useful for researchers who develop GM crops and methods for detecting GM crops.

In recent years, several genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by various agricultural biotechnological companies. Commercialization of GM crops will be required the assesment of risks associated with the release of GM crops. In advance of the commercial release of GM crops, developer should submit the several information on GM crops for approval. In this study, we carried out to provide the molecular data for the risk assessment of GM rice containing insect-resistant gene, modified Cry1Ac (CryIAc1). Through the molecular analysis with CryIAc1 induced GM rice, we confirmed the steady integration and expression of transgene, the transgene copy number, the adjacent region sequences of inserted gene into rice genome, and the transgene stability in progenies. For the qualitative PCR detection methods, specific primer pairs were designed on the basis of integration sequences, and construct- and event-specific detection markers were developed for leaf folder-resistant rice, Cr7-1 line. From these results, we demonstrated that the molecular data and the PCR detection methods of leaf folder- resistant GM rice could be acceptable to conduct the biosafety and environment risk assessment.

For the development of qualitative PCR detection method of genetically modified (CM) rice, rice species-specific gene, OsCc-1 (rice cytochrome c gene), was selected as suitable far use as an endogenous gene in rice. The primer pair OsCytC-5`/3`with 111 bp amplicon was used for PCR amplification of the rice endogenous gene, OsCc-1 and no amplified product was observed from 8 different crops as templates. Qualitative PCR method was carried out with stack traits of L528CryIAc1 GM rice developed in Korea. For the qualitative PCRs, some primer pairs were designed with a construct-specific and event-specific type based on T-DNA and junction sequences of T-DNA in GM rice. Actck-5`/3` amplifying between actin promoter and OsCK1 gene introduced in LS28 gave rise to an amplicon 306 bp; also, CrLB-5`/3` from CryIAcl and CKRB-5`/3`amplifying the junction region of T-DNA and genome sequence from LS28 as event-specific primers gave rise to an amplicon 142 bp and 91 bp, respectively. These primer pairs for the detection of event-specific targets not produced PCR amplicons on non-CM rice and various crops in contrast to event lines. Therefore, in this study we verified that event-specific primers were effective to specifically detect stack trait lines and demonstrated that this method presented could be provided with the detection-method data for risk assessment analysis of GM rice to be commercialized.

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13-41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.

Tocopherols are essential lipophilic antioxidant in human cells, while little is known about its function in plant tissues. To study the impact of composition and content of tocopherols on stress tolerance, tobacco (Nicotiana tabacum) was transformed with a construct containing a cDNA insert encoding -tocopherol methyltransferase (-TMT/VTE4) from perilla under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic tobacco was confirmed by PCR and RT-PCR. The total content and composition of tocopherols in the transgenic lines were similar with wild type controls. However, chlorophyll-a and carotenoid content in the transgenic lines were increased by up to 45% (P-TMT increased the salt stress tolerance in tobacco plants. These results demonstrate that over-expression of -TMT gene in tocopherol bio-synthetic pathway can increase salt stress tolerance and contents of chlorophyll-a and carotenoid in transgenic tobacco plants.

The Agrobacterium-mediated transformation has been successfully used method to introduce foreign genes into some monocotyledonous as well as a large number of dicotyledonous plants genome, We developed transgenic Chinese cabbage plants with insect-resistance gene, modified CryIAc, by Agrobacterium-transformation and confirmed transgene copy number by Southern blot analysis. We confirmed that twenty-nine out of 46 transgenic Chinese cabbage plants have single copy of CryIAc. To obtain the sequences information on the transferred DNA (T-DNA) integration into plant genome, we analyzed left border (LB) flanking sequences by genome walking (GW) PCR method. Out of 46 transgenic Chinese cabbage plants examined, 37 carried the vector backbone sequences. This result indicates that the transfer of the vector backbone from the binary vectors resulted mainly from inefficient termination of LB site. Analysis of T-DNA LB flanking region of 9 transgenic Chinese cabbage plants without vector backbone revealed that all LB ends were not conserved and nucleotides up to 36bp from the LB cleavage site were deleted.

The influence of light quality on growth and development of in vitro grown Doritaenopsis hort. (Orchidaceae) plants was investigated. Growth parameters like leaf and root fresh/dry mass and leaf area were highest with plants grown under red plus blue light emitting diodes (LEDs). Leaf length was greater with the plants grown under red LED. Carbohydrate (starch, sucrose, glucose and fructose) and leaf pigment (chlorophylls and carotenoids) biosynthesis of the plants was significantly increased in plants grown under red plus blue LEDs compared to red or blue LED and fluorescent light treatments. This study suggests that the production of quality Doritaenopsis plants is possible by culturing the plants in vitro under a mixture of blue plus red light sources.

Commercialization of genetically modified (GM) plants will be required the assessment of risks associated with the release of GM plants that should include a detailed risk assessment of their impacts in human health and the environment. Prior to GM plant release, applicants should provide the information on GM crops for approval. We carried out this study to provide the molecular data for risk assessment of the GM Chinese cabbage plants with insect-resistance gene, modified CryIAc, which we obtained by Agrobacterium-transformation. From the molecular analysis with GM Chinese cabbage, we confirmed the transgene copy number and stability, the expression of the transgene, and integration region sequences between the transgene and the Chinese cabbage genome. Based on the unique integration DNA sequences, we designed specific primer set to detect GM Chinese cabbage and set up the GM cabbage detection method by qualitative PCR analysis. Qualitative analysis with GM Chinese cabbage progenies analysis was revealed the same as the result of herbicide treatment. Our results provided the molecular data for risk assessment analysis of GM Chinese cabbage and demonstrated that the primer set proposed could be useful to detect GM Chinese cabbage.

Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, α-, γ-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice.

The effects of endogenous levels of ethylene and phenolic compounds on somatic embryogenesis, medium-browning, and peroxidase activity were evaluated in thin section cultures ofDoritaenopsis. Cultures were maintained for 8 weeks with four different treatments: i) thick leaf segment culture, ii) thin leaf section culture, iii) thin leaf section culture with ventilation, or iv) thin leaf section culture after expiants were first washed. Expiants cultured in closed vessels produced a larger number of somatic embryos than those reared in the ventilated vessels. This enhanced formation confirmed the greater involvement of accumulated ethylene under non-ventilated conditions, because wound-induced tissues from thin leaf sections normally release high level of ethylene. When expiants were washed in the liquid medium and inoculated on the same solid medium, somatic embryo production was 1.7 and 18.5 times higher than in the thin section cultures and thick segment cultures, respectively. Reducing the level of phenolics in expiants at the initial stage of culturing apparently stimulated this embryo regeneration.

We investigated the effects of various qualities of light-emitting diode (LED) light sources on the growth and carbohydrate accumulation of grape rootstock ‘Teleki 5BB’ culturedin vitro. Shoot fresh and dry weights and net photosynthetic rates were increased when plants were exposed to fluorescent lighting (control), red light, or a mixture of blue plus red, but were unaffected by blueonly radiation. Shoot elongation was significantly stimulated by red light whereas the combination of blue and red light was associated with the shortest shoots. However, the number of nodes did not differ among these treatments. Under monochromic blue or red light, sugar content and starch accumulation increased under the mixed-radiation treatment.

Post-transcriptional RNA processing and translational regulations are important steps for gene expression. To analyze the 5′UTRof psbA that enhances translation of the sweet protein monellin in chloroplasts, we cloned the monellin gene, with and without thepsbA 5′UTR, into the chloroplast expression vector for chloroplast transformation. Transgenic plants were identified as being transplastomic via PCR and Southern blot analyses. We also observed non-specific recombination during tobacco chloroplast transformation. Analyses of the transcription patterns showed that intercistronic cleavage of the psbA mRNA 5′ untranslated (UTR) region was functional at the mature stage, with the monocistronic mRNA ofmonellin increasing while its dicistronic mRNA decreased. Moreover, monellin accumulation accounted for 2.3% of the total soluble protein at the mature stage, but only 1.3% at the young stage in transplastomic lines that contained the 5′UTRof psbA. These results suggest that activation of the endonucleolytic cleavage of thepsbA 5′UTR element depends on chloroplast developmental conditions, and that it enhances the accumulation of sweet protein monellin in those chloroplasts.

An efficient protocol has been developed for rapid mass propagation of sweetpotato from shoot-tips derived embryogenic callus. Optimal embryogenic callus was induced from shoot apical meristem explants on Murashige and Skoog (MS) medium supplemented with 1mg/L 2,4-D. The addition of casein hydrolysate in the media increased the embryogenesis efficiency of sweetpotato. Somatic embryos were easily induced from the embryogenic callus on MS basal medium containing 300-500mg/L casein hydrolysate without phytohormon. Treatment of casein hydrolysate (100∼300mg/L) with 1mg/L 2,4-D also improved the secondary embryonic efficiency from somatic embryos below 2mm in length. Plant regeneration was achieved via somatic embryogenesis and direct organogenesis. Regenerated planlets with well developed shoots and roots on MS basal medium were successfully transferred to soil.

Chloroplast molecular markers can provide useful information for high-resolution analysis of inter- and intra-specific variation in Brassicaceae and for differentiation between its species. Combining data generated from nuclear and chloroplast markers enables the study of seed and pollen movement, and assists in the assessment of gene-flow from genetically modified (GM) plants through hybridization studies. To develop chloroplast DNA markers for monitoring of transgene introgression in Brassica napus L., we searched for sequence variations in the chloroplast (cp) genome, and developed a simple cpDNA marker that is reliable, time-saving, and easily discriminates among 4 species (B. napus, B. rapa, Raphanus sativus, and Sinapis alba) based on PCR-product length polymorphism. This marker will be useful to identify maternal lineages and to estimate transgene movement of GM canola.

For the identification of a novel insecticidal protein, a two-dimensional liquid chromatography (PF-2D) system was used in a quantitative proteomic analysis of Xenorhabdus nematophila CBNU strain isolated from entomophagous nematode Steinernema carpocapsae . Protein patterns obtained from minimum and maximum insecticidal activities during cultivation were contrasted, and a novel toxin protein (Txp40) was identified by MALDI-TOF/MS. The DNA sequence of the cloned toxin gene (1089 bp) has an open reading frame encoding 363 amino acids with a predicted molecular mass of 41162 Da. The txp40 identified in this study is most closely related to the known txp40 cloned from X. nematophila EB (ADQ92844) with 94.4% identical sequence residues. Following the expression of the newly identified toxin gene in Escherichia coli , the insecticidal activity of the recombinant toxin protein was determined against Plutella xylostella larvae; a 56.7% mortality rate was observed within 24 h.

Increasing vitamin E activity in economically important oil crops such as soybean will enhance the nutritional value of these crops. An improved soybean transformation system involving pre-culture of soybean seed on medium supplemented with 1 mg/L 6-benzylaminopurine under dark conditions was established. To improve the nutritional value of soybean by increasing the α-tocopherol content, soybeans were transformed with γ-tocopherol methyltransferase (γ-TMT) gene by Agrobacterium-mediated transformation. Frequency of soybean transformation was significantly increased from 0.5 to 4.3% by this modified system, and 13 lines of transgenic soybean plants containing γ-TMT were obtained. The molecular characterization using polymerase chain reaction (PCR), reverse transcriptase PCR, and Southern blot analysis confirmed insertion and inheritance of the transgene in the transgenic plants and their progeny. Morphologically normal and fertile transgenic plants were analyzed, and the majority of transgenic soybean transmitted herbicide resistance at 3:1 or 15:1 ratios to their progeny. Alpha-tocopherol content in transgenic soybean seeds was determined by high-performance liquid chromatography; over-expression of γ-TMT resulted in a 41-fold increase in α-tocopherol over wild-type soybean seeds.

To increase insect resistance in transgenic rice plants, a synthetic truncated cry1Ac gene was linked to the rice rbcS promoter and its transit peptide sequence (tp) for chloroplast-targeted expression. Several transgenic lines were generated by the Agrobacterium-mediated transformation method and the expression levels of the transgene were compared with untargeted expression. Use of the rbcS-tp sequence increased the cry1Ac transcript and protein levels by 25- and 100-fold, respectively, with the accumulated protein in chloroplasts comprising up to 2% of the total soluble proteins. The high level of cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as evidenced by insect feeding assays. Transgenic plants were also evaluated for resistance to natural infestations by rice leaf folder under field conditions. Throughout the entire period of plant growth, the transgenic plants showed no symptoms of damage, whereas nontransgenic control plants were severely damaged by rice leaf folders. Our results demonstrate that the targeting of cry1Ac protein to the chloroplast using the rbcS:tp system confers a high level of plant protection to insects, thus providing an alternative strategy for crop insect management.

Food crisis is one of the most important issues around the world. With a population explosion, Harvesting crops in a classical manner no longer keep up with population explosion. In addition, agricultural pesticide and insect pest give rise to environmental problems. Recent advances in biotechnology ameliorates both issues by inserting productivity amplifying gene and pesticide neutralizing protein encoded gene, maximizing crop productivity and producing pesticide resistant crop, so-called genetically modified (GM) crops. Due to the bioethical issues and potential risk to human, people are concerned about the GM crops, thus many countries make regulations for the GM crops. In order to regulate GM crops, we need fast, accurate and simple methods to the GM crops. In this study we have devised a biosensor to detect phosphinothricin acetyltransferase (PAT) protein that makes GM crops resistant to herbicides. In order to detect PAT protein, anti-PAT antibody was conjugated to polydiacetylene (PDA) vesicles. PDA vesicles have unique colorimetric characteristics, changing their color from blue to red upon external stimuli. Anti-PAT antibody conjugated PDA vesicles are subsequently encapsulated within hydrogel matrix to maximize the intensity of color change, resulting in the sensitivity enhancement of the sensor. Our biosensor could detect as low as the 20nM of PAT protein. Furthermore we developed a microfluidic device to automate the production of uniform sized immunohydrogel beads. Our simple biosensor can be identified by the naked eyes without the aid of special instrument or expertise, widening the potential applications in the area of point-of-care testing (POCT).