환경스트레스에 내성을 갖는 버즈풋 트레포일(Lotus crniculatus L.) 형질전환체를 개발하기 위하여, AtNDPK 유전자가 SWPA2 프로모터에 의해 조절되도록 재조합한 발현벡터 pCAMBIA 2300 /SWPA2::AtNDPK2를 Agrobacterium 형질전환 방법으로 버즈풋 트레포일에 도입하였다. Apgrobacterium과 버즈풋 트레포일 캘러스의 공동배양한 캘러스를 의 kanamycin 및 의 cefotaxim을 첨가한 SH-3-k

엽록제 small HSP의 기능을 조사하기 위하여 항상적으로 발현하는 형질전환 식물체를 작성하였다. 고온 스트레스 하에서의 형질전환 식물체의 고온내성을 chlorophyll 형광으로 측정하였다. Leaf disc를 고온조건에서 5분간 처리한 후, 광화학계 II의 불활성화를 나타내는 Fo 값의 증가 또는 Fv 값의 감소치를 조사하였다. 형질전환 식물체는 고온 스트레스 하에서의 이들 값의 증감율이 현격히 감소하였다. 또한 유식물체를 에서 45분간 처리한 후, 에서 계속적으로 배양하였을 때, 비형질전환 식물체는 전부 고사하였으나, 형질전환 식물체의 약 80%는 생존하였다. 이러한 결과는 엽록체 small HSP가 고온 스트레스 하에서 광합성기구를 보호하는데 있어서 중요한 기능을 담당하고 있음을 나타낸다.

대만산 배추 (Brassica campestris M.)로부터 분리한 BcHSPI7.6 cDNA(내열성 유전자)를 pBKSl-l vector에 subcloning하므로서, NPTII 유전자와 P35S-HSPI7.6 cDNA를 가지는 pBKH4 재조합 플라스미드를 제작하였다. 이들 플라스미드를 갖는 A. tumefaciens LBA4404로서 담배잎 단편을 24시간 동안 공배양하므로서 감염시켰으며, 이들 유전자로 형질전환된 shoot는 의 가나마이신을 첨

Soybean is a crop of importance economically and nutritionally in many parts of the world. Thanks to many new genes brought from genomic research, It is possible to introduce various candidate genes through genetic transformation to see the performance of the genes in field. In our lab, soybean transformations have been tried for last 10 years to probe the possibility of traits improvement by transformation of new gene into soybean. For this purpose, three different genes were transformed into Korean soybean variety, Kwangan. First, the gene that controls early flowering of plant was transformed into Kwangan. Second, a candidate gene for soybean mosaic virus (SMV) resistance was transformed to produce transgenic plants. Third, another candidate gene for drought tolerance was transformed. All the transgenic plants from three genes transformation were produced for their gene insertion and their expression using PCR, qRT-PCR. Further analysis including harvesting seeds is currently undertaken.

To develop transgenic forage crops with enhanced tolerance to abiotic stress, we introduced an alfalfa Hsp23 gene expression vector construct through Agrobacterium-mediated transformation. Integration and expression of the transgene were confirmed by PCR, northern blot, and western blot analyses. Under normal growth conditions, there was no significant difference in the growth of the transgenic plants and the non-transgenic controls. However, when exposed to various stresses such as salt or arsenic, transgenic plants showed a significantly lower accumulation of hydrogen peroxide and thiobarbituric acid reactive substances than control plants. The reduced accumulation of thiobarbituric acid reactive substances indicates that the transgenic plants possessed a more efficient reactive oxygen species-scavenging system. We speculate that the high levels of MsHsp23 proteins in the transgenic plants protect leaves from oxidative damage through chaperon and antioxidant activities. These results suggest that MsHsp23 confers abiotic stress tolerance in transgenic forage crops and may be useful in developing stress tolerance in other crops.

Molecular characterization of crops improved through biotechnology has traditionally been conducted using Southern blot analysis which has been used to determine T-DNA copy number, the presence or absence of backbone (sequence outside of the T-DNA) and to demonstrate generational stability of the T-DNA insert. The advancement of high-throughput DNA sequencing (HTS) technology allows efficient characterization of the transgene incorportated into the genome of the plant by rapidly sequencing the entire plant genome. By combining NGS (Next Generation Sequencing) technologies with bioinformatic methods that identify the T-DNA insert derived from the plasmid vector and genome-T-DNA junction sequences, it has been shown that conclusions equivalent to those of a Southern blot are readily obtained. NGS is done at sufficient coverage depth (>75x) across the entire genome. By mapping the sequence reads to the plasmid vector, and identifying the number of unique junctions, we can confirm insert number, copy number, absence of backbone, across multiple generations. With the widespread availability of NGS and steadily decreasing costs it is likely that academia and industry will fully transition to NGS-based molecular characterizations in the near future.

Serotonin N-acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasmexpressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis.

The world population is projected to reach to 9.6 billion people by 2050. With increasing population and improving living standards, the demand for food is accelerating. In order to meet increasing demand for food while the arable land and other resources are decreasing, agriculture needs all the tools available to sustainably increase crop yields. Development of effective GM traits to protect crops from abiotic and biotic stressors is a critical aspect of sustainable yield improvement. Efficient identification of traits and rapid integration of the traits into commercial elite germplasm requires robust and rapid traits testing. Monsanto have developed numerous high-throughput phenotyping platforms to support rapid trait identification and integration. Selected phenotyping platforms will be reviewed to gain understanding on how they are utilized for trait phenotyping.

The MethioninesulfoxidereductaseB2(MsrB2) gene catalyzes the reduction of free and protein-bound methionine sulfoxide to methionine and is known to provide tolerance to biotic and abiotic environmental stresses. There have yet to be any reports that MsrB2 enhances drought tolerance. Two drought-tolerant transgenic rice lines, L-8 (single copy) and L-23 (two copy), expressing the Capsicum annuum MsrB2 (CaMsrB2) gene were selected for stress tolerance phenotyping under drought stress conditions. CaMsrB2 enhanced relative water content (RWC), maintained substantial quantum yield (Fv/Fm ratio), and subsequently improved photosynthetic pigments. Interestingly, L-23, carrying two-copy T-DNA insertion, showed greater drought tolerance through more effective stomatal regulation, carotenoid concentration, and osmotic potential than the wild type. High-tech infrared technology (FLIR SC620) was used for the selection of stress-tolerant physiotypes. Later, the IR results were correlated with other tested physiological parameters. The IR images, average plant temperature, and physiological parameters of the treated plants were discussed in detail.

We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained GPD, UtrCSP, BrTSR15 and BrTSR53 genes overexpression constructs under the control of the constitutive CaMV 35S promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also FSTs analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 95 FSTs, among which 37 (38.9%) were integrated into genic regions and 58 (61.1%) into intergenic regions. About 27 homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S::BrTSR15 and 35S::BrTSR53 transgenic plants were displayed higher levels of transgene protein than WT plants. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.

Rice is one of the most important cereal crops in the world and a model plant for functional genomics of monocotyledon. Recently, rice crop loss is estimated to be approximately 30% of the total yield due to herbivorous pests, mainly insects. Cry1Ac toxin is a protein produced by the bacterium Bacillus thuringiensis and has insecticidal properties. CryBP1 toxin also is an insecticidal protein produced by the bacterium Bacillus popilliae. These two toxic genes derived bacteria, which were inserted into a binary vector, have been introduced into rice plants by Agrobacterium tumefaciens mediated transformation in order to enhance resistance to insects. Here, we utilized anthers to regenerate transgenic rice plants when it has been plated on the callus induction media as a callus-inducing material. Anther culture has a benefit in terms of being apt to produce doubled haploids in short term in plants breeding compared to seed culture. However, anther culture method in generating transgenic rice still has low productivity of plant regeneration in some genotypes of Japonica rice. Therefore, we examined the efficiency of callus induction and transformation with three different cultivars of Japonica rice, Haiami, Ungwang and Dongjin. In this study, our results showed that Haiami is the best genotype among three cultivars of Japonica rice as callus inducing material in anther culture to produce transgenic rice plants conferring resistance to insects.

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared to seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.

Soybean mosaic virus (SMV), a member of Potyviridae family, is one of the most typical viral diseases and results in yield and quality loss of cultivated soybean. Due to the depletion of genetic resources for resistance breeding, a trial of genetic transformation to improve disease resistance has been performed by introducing SMV-CP and HC-Pro gene by RNA interference (RNAi) method via Agrobacterium-mediated transformation. Transgenic plants were infected with SMV strain G5 and investigated the viral response. As a result, two lines (3 and 4) of SMV-CP(RNAi) transgenic plants and three lines (2, 5 and 6) of HC-Pro(RNAi) transgenic plants showed viral resistance. In genomic Southern blot analysis, most of lines contained at least one T-DNA insertion in both SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. Subsequent investigation confirmed that no viral CP and HC-Pro gene expression was detected in two SMV-resistant lines of SMV-CP(RNAi) and three lines of HC-Pro(RNAi) transgenic plants, respectively. On the other hand, non-transgenic plants and other lines showed viral RNA expression. Viral symptoms affected seed morphology, and clean seeds were harvested from SMV-resistant line of SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. In addition, strong viral gene expression was detected from seeds of SMV-susceptible non-transgenic plants and SMV-susceptible transgenic lines. When compared the viral resistance between SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants, soybean transgenic plants with the HC-Pro gene using RNAi strategy showed much stronger and higher frequency of viral resistance.

The perturbation of the steady state of reactive oxygen species due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 promoter with/without a selection marker, the bar gene. A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol. An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice

Environmental stresses including drought, extreme temperatures, and high salinity are major factors that severely limit crop productivity worldwide. To overcome yield loss due to these environmental stresses, a large number of researches have been conducted to understand how plants respond to and adapt these environmental stresses. Posttranscriptional regulation as well as transcriptional regulation of gene expression is recognized as a key regulatory process in plant stress responses, and these cellular processes are regulated by diverse RNA-binding proteins (RBPs). Over the last years, we have extensively investigated the functional roles of RBPs that harbor an RNA-recognition motif at the N-terminal half and a glycine-rich region at the C-terminal half (glycine-rich RNA-binding proteins, GRPs), zinc finger-containing GRP, and cold shock domain proteins (CSDPs) in Arabidopsis thaliana, rice (Oryza sativa), wheat (Triticum aestivum), and rapeseed (Brasicca napus) under stress conditions. Our comparative analysis demonstrated that certain family members display RNA chaperone function during stress adaptation process in monocotyledonous plants as well as in dicotyledonous plants. These findings point to the importance of the regulation of mRNA metabolism in plant response to environmental stresses and shed new light on the practical application of these RBPs to develop stress-tolerant transgenic crops.

Bacillus thuringiensis(Bt) crystal protein (Cry1Ac) genes encode insecticidal δ-endotoxins that are widely used for the development of insect-resistant crops. Common soybean is a crop of economic and nutritious importance in many parts of the world. Korea soybean variety Kwangan was transformed with Bacillus thuringiensis(Bt) crystal protein genes. We transformed three difference Cry1Ac (Cry1Ac and two modified Cry1Ac) genes into Kwangan using highly efficient soybean transformation system. Transgenic plants with Bt crystal protein genes were confirmed for gene introduction and their expression using PCR, real-time PCR, and RT-PCR. We generated 30 independent lines of transgenic soybean plants. Analysis of the flanking sequences isolated by Inverse PCR revealed complex T-DNA insertion patterns and preferential integration of T-DNA into the intergenic spacer region of the soybean genome. We found 5 different intergenic transgenic soybean lines of soybean genome. Currently, the confirmation of stable gene introduction with Bt genes is also performing by southern blot analysis, physiology test, and agronomic characters are investigating.