1. 본 연구는 GM콩이 비의도적으로 자연에 방출되었을 시 야생콩과의 교잡에 의해 발생 가능한 교잡콩을 모니터링하고 영양학적 측면에서 안전성을 평가하기 위하여 수행하였다. 이를 위해, 베타카로틴 강화 GM콩과 야생콩, 두 품종간 인위적으로 생산한 교잡콩, GM콩의 모본이자 일반 재배 품종인 광안콩 등 4품종을 국내 2개 지역에서 재배하고 콩 종자를 대상으로 주요 영양성분 38종을 분석하였다. 2. 재배 환경에 영향을 받지 않고, GM콩 및 야생콩과 비교해 교잡콩에서 유의적 차이를 보이는 성분은 조단백질, 조섬유, alanine, glycine, leucine, serine, tryptophan, oleic acid, linolenic acid, arachidonic acid 등 10종이었다. 하지만, 분석한 모든 시료의 성분은 국내외 자연 범위에 포함되는 수준이었다. 3. PCA분석 결과, 주요 영양성분 함량 변이는 GM콩은 모본과 유사하였고, 교잡콩은 GM콩 보다는 야생콩과 유사하였다. 즉, 분석한 4품종의 영양성분의 변이는 재배 환경보다는 유전적 요인에 의해 더 크게 영향을 받음을 알았다. 4. 본 연구의 결과는 GM콩과 야생콩간 교잡콩의 영양학적 안전성을 분석한 최초의 보고이며, 이는 향후 GM콩의 비의도적 자연 방출을 모니터링하는데 있어 유용한 기초 자료로써 활용 가치가 있을 것이다.

The vitamin E enhanced transgenic soybean was developed by introducing a perilla γ- tocopherol methyltransferase gene (γ-TMT) under the control of pea vicilin promoter and a selection marker, phosphinothricin acetyltransferase (PAT) gene. With regard to the potential problems of safety, the non-target organism evaluation is required as an essential element for the environmental risk assessment of genetically modified (GM) crops. We studied the effects of the vitamin E enhanced transgenic soybean feeding on survival of Daphnia magna which is commonly used as a model organism in ecotoxicological studies. The Daphnia magna was fed on vitamin E enhanced transgenic soybean and non-genetically modified (non-GM) soybean (Willams 82) at 0, 1,000, 1,800, 3,240, 5,830, 10,500 and 20,000 mg/L concentrations, respectively. The GM soybean used for the test was confirmed to have the γ-TMT/PAT gene expression by the enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) analysis. The results showed that there was no significant differences between cumulative immobilities of Daphnia magna fed on GM soybean and non-GM soybean. The 48hr-EC50 values showed no significant differences between GM soybean (2,416 mg/L) and non-GM soybean (2,408 mg/L). The results of this study suggested that there was no significant differences in toxicity for Daphnia magna between GM soybean and non-GM counterpart.

A large number of transgenic crop varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins have been commercialized in 13 countries since 1996. Although the use of these insect-resistant Bt crops can increase crop quality and yields, concerns remain about the potential negative effects of such crops on ecosystems. Transgenic soybean containing cry1Ac gene have been developed to control Lepidopteran pests of soybean and we aimed to investigate whether this soybean could affect non-target arthropods, which play a major role in ecological functions in agricultural ecosystems. In the present study, we first measured the levels of Cry1Ac protein in Bt soybean at different growth stages of soybean and then we compared the community structure of arthropods occurred in fields of transgenic and wild-type soybean. The levels of Cry1Ac protein in transgenic soybean leaves ranged from 252.9 to 604.5 μg g-1 DW. Multivariate analyses (PerMANOVA and NMDS) showed that the composition of the non-target arthropod community was affected by sampling date but not by soybean genotype. These results suggest that transgenic soybean expressing Cry1Ac protein may not adversely affect such non-target arthropod communities.

This study was carried out to develop of environmental risk assessments and the biosafety guide for Vitamin E enhanced transgenic soybean at LMO (Living Modified Organism) isolation field. In LMO quarantine area of National Institute of Agricultural Sciences, insect species diversities and population densities on vitamin E enhanced transgenic soybean and non-GM soybeans (Willams 82 and Seoritae) were investigated. A total of 17,717 individuals of 77 species from 8 orders were collected in LMO isolation field. In three type soybeans field, total of 5,250 individuals in Vitamin E enhanced transgenic soybean, 5,510 individuals in Willams 82, and 6,957 individuals in Seoritae were collected, respectively. There was no difference between the population densities of insect pests, natural enemies and other insects on Vitamin E enhanced transgenic soybean and Willams 82, while natural enemies density on Seoritae was higher than on Vitamin E enhanced transgenic soybean, but insect pests density on Vitamin E enhanced transgenic soybean was higher. These results provided the insects diversity for risk assessment survey of Vitamin E enhanced transgenic soybean and suggested that the guideline could be useful to detect LMO crops.

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.

The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium -mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium-mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

AtRabG3b and CaMsrB2 genes incorporated into pPZP vetor were transformed to Korean soybean cultivar Kwangan using highly efficient transformation system. AtRabG3b gene plays a positive role in xylem development in Arabidopsis and 64 transgenic plants were produced. CaMsrB2 gene is known to confer drought tolerance in rice and 63 transgenic plants were produced. As a result of PPT leaf painting assay, about 20% of transformation efficiency was observed from 2 times of inoculation. These transgenic plants were confirmed for gene introduction using PCR. Currently, the copy number and the gene expression is investigating using qRT-PCR and RT-PCR. Moreover, 62 lines and 53 lines of T1 seeds from AtRabG3b and CaMsrB2, respectively, were sown in GMO field.

ORE7 gene incorporated into 3 different promoters including pCKLSL-35S, pCKLSL-TP and pCSENIF was transformed to Korean soybean variety Kwangan using highly efficient soybean transformation system. The gene is known to exhibit a delayed leaf senescence phenotype in Arabidopsis. Fourteen, Fifteen and nine transgenic plants were produced from pCKLSL-35S::ORE7, pCKLSL-TP::ORE7 and pCSENIF::ORE7, respectively. Moreover, transgenic plants were confirmed for gene introduction and their expression using PCR, qRT-PCR and RT-PCR. To identify the transgene insertion events, the analysis of flanking sequence was determined. As a results, T-DNA was integrated intergenically in transgenic line 1 of pCKLSL-35S::ORE7 and line 1 of pCSENIF::ORE7. Currently, flanking sequence analysis with pCKLSL-35S::ORE7, pCKLSL-TP::ORE7 and pCSENIF::ORE7 is carrying out to investigate the stable T-DNA insertions.

Insect resistant 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. Korean soybean variety Kwangan was transformed with Insect resistant genes. These genes were transformed into Kwangan using highly efficient soybean transformation system. Transgenic plants harboring Insect resistant genes were confirmed for gene introduction and their expression using PCR, real-time PCR and RT-PCR. The confirmation of stable gene introduction with Insect resistant genes was also performing by Southern blot analysis. In addition, Flanking sequence analysis and agronomic characters were also investigated

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.

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.

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.

Cry1Ac protein is known as one of toxin crystal proteins synthesized from Bacillus thuringenesis that plays a critical role for the insect resistance. Recently, cry1Ac genes have introduced into many plants in general and soybean as well. However, the gene expression of cry1Ac genes in transgenic plants remains low that need to be improved. Several mutations we reintroduced into the cry1Ac genes in order to enhance the insecticidal effect. In this study, the cry1Ac with mutant #2, #11 and #16 were transformed into Kwangan, a Korean soybean variety by using the “half-seed” method. The plant lets carrying modified cry1Ac genes were primarily selected on media containing Phosphinothricine (PPT), a bar selective agent and Basta leaf painting. Then, the presence of introduced genes in T0 plants and the gene expression were investigated by PCR, RT-PCR and Real-time PCR. PCR and RT-PCR analysis showed expression of bar and cry1Ac genes from tested transgenic soybean plants. The number of copy of bar gene ranged from 1 to 3 by Real-time PCR analysis. These results provided a fundamental back ground for our further experiments: Confirmation of the gene expression by Southern blot and identification of the function of modified cry1Ac by insect bioessays.

Bacillus thuringiensis (Bt) crystal protein 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. These genes were transformed 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. Currently, the confirmation of stable gene introduction with Bt genes is also performing by southern blot analysis and physiology test and agronomic characters are investigating.

Korean soybean variety Kwangan was transformed with coat protein (CP), helper component-proteinase (HC-Pro), and ABRE binding factor 3 (ABF3) genes using highly efficient soybean transformation system. Among these genes, CP and HC-Pro were transformed using RNAi technology. Transgenic plants with CP were confirmed for gene introduction and their expression using PCR, real-time PCR, RT-PCR, Southern blot, and Northern blot. To investigate the response of viral infection with CP, T1 plants were inoculated with SMV-infected leaves and confirmed the existence of mosaic symptom in both leaves and seeds. Two transgenic lines with CP were highly resistant to SMV with clear leaves and seeds while SMV-susceptible lines showed mosaic symptom with seed mottling. The transcript levels of T1 plants with CP were also determined by northern blot, suggesting that SMV-resistant T1 plants did not show viral RNA expression whereas SMV-susceptible T1 plants showed viral RNA expression. Currently, the response of viral infection with HC-Pro is investigating to produce SMV-resistant soybean transgenic plants, and the physiological experiment with ABF3 is also carrying out to produce drought-tolerant soybean transgenic plants.

Development of transgenic plant with desirable traits to cultivated plant is one of the important procedures in plant molecular breeding. However, applicable assessment of transgenic plant in laboratorial scale is not much except cultivating transgenic plant for a whole life in field condition. Here, we analyzed chlorophyll fluorescence in three transgenic soybean lines with AtMYB44 transcription factor for assessment of photosynthetic activity under abiotic stresses such as drought. Soybean varieties used in this study were ‘Bert’ and ‘Bert’ derived three transgenic soybeans, ‘AtMYB44 CM35101’, ‘AtMYB44 CM2471’, and ‘AtMYB44 CM4481’. Analyzed five different chlorophyll fluorescence variables are maximum PSII quantum yield (QY_max), steady state PSII quantum yield (QY_Lss), steady state non-photochemical quenching (NPQ_Lss), coefficient of photochemical quenching in steady-state (Qp_Lss), and fluorescence declineratio in steady-state (Rfd_Lss). To determine main chlorophyll fluorescence variable affected by abiotic stress, principal component analysis (PCA) was conducted with five chlorophyll fluorescence variables measured from four varieties. QY_Lss and NPQ_Lss were main chlorophyll fluorescence variables to evaluate abiotic stress, particularly in drought stress. In comparison with transgenic soybean lines based on chlorophyll fluorescence variables, ‘AtMYB44 CM2471’ and ‘AtMYB44 CM4481’ are more tolerant to drought than the others. Interestingly, three transgenic soybean lines which have a same AtMYB44 gene with different regions of chromosome revealed the quite different responses of chlorophyll fluorescence to drought treatment.