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

국내에서는 아직까지 유전자변형 작물이 재배되고 있지 않으나 유전자변형 작물의 환경방출을 위해서는 반드시 환경위해성 평가가 수행되어야 한다. 본 연구에서는 병저항성 유전자변형 벼(OsCK1)로부터 비 형질전환 벼(낙동벼)와 잡초성벼(R55)로의 화분 매개에 의한 유전자 이동성을 평가하였다. 비 형질전환 벼로부터 449,711립의 종자를 얻었으며 잡초성벼로부터는 164,604립의 종자를 수확하였다. 교잡개체는 2회의 제초제 살포와 병저항성 벼에 특이적인 프라이머를 이용한 분자생물학적 방법을 통해 저항성 여부를 검증하였다. 개화기간이 서로 일치하였음에도 불구하고 비 형질전환 벼에서만 교잡개체를 확인할 수 있었으며, 교잡율은 교잡개체가 발생한 거리에 따라 0.013～0.018%로 나타났다. 모든 교잡개체들은 병저항성 유전자변형 벼에 근접한 0.6 m내에서 발견되었다. 화분 매개에 의한 병저항성 유전자변형 벼의 유전자 이동성은 앞서 연구된 결과와 유사한 것으로 나타나 농업환경에 방출되더라도 환경에 미치는 영향은 다른 벼들과 비슷할 것으로 추정되었다. 개화기간 중 온도와 습도 등 기상요인이 벼의 교잡율을 결정하는데 중요한 요소이었다. 따라서 유전자변형 벼와 주변 재배종 및 잡초성벼로의 유전자 이동에 의한 안전관리 대책을 마련하기 위해서 지역의 기상요인과 개화기 중복 등의 요인들이 충분히 고려되어야 할 것이다.

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 assessment of risk associated with the release of GM crops. In this study, we carried out to provide the 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 (CaMsrB2). 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, a selectable marker herbicide resistance unit, and left border in a sequential order. Based on the adaptor-ligation PCR and whole genome sequence database, we confirmed that T-DNA was introduced at the position of 41,737,284 bp of chromosome No. 1. 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. These results will be filed to biosafety assessment document of CaMsrB2 rice.

Rice (Oryza sativa) is the most important staple food of over half the world’s population. This study was conducted to evaluate the possible impact of transgenic rice cultivation on the soil microbial community. Microorganisms were isolated from the rhizosphere of GM and non-GM rice cultivation soils. Microbial community was identified based on the culture-dependent and molecular biology methods. The total numbers of bacteria, fungi, and actinomycete in the rhizosphere soils cultivated with GM and non-GM rice were similar to each other, and there was no significant difference between GM and non-GM rice. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM rice were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in GM and non-GM rice cultivated soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed similar patterns, but didn’t show significant difference to each other. DNAs were isolated from soils cultivating GM and non-GM rice and analyzed for persistence of inserted gene in the soil by using PCR. The PCR analysis revealed that there were no amplified protox gene in soil DNA. These data suggest that transgenic rice does not have a significant impact on soil microbial communities, although continued research may be necessary.

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.

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.

비타민A 강화벼의 복수세대에 대한 후대안정성을 Southern blot과 PCR로 분석한 결과, 비타민A 강화벼의 PAC T3～T6 세대에서는 도입된 모든 유전자들이 안정적으로 도입되어 있으며, backbone DNA는 비타민A 강화벼에 삽입되지 않았음을 확인하였다. 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 PAC T3～T6 복수세대에서 생육시기별 부위별로 안정적으로 발현됨을 입증하였으며, 최종 목적 산물인 카로티노이드 분석 결과에서도 모품종인 낙동벼에 비해 비타민A 강화벼에서 β-carotene은 10.6배 함량이 증가되고, zeaxanthin과 α-carotene는 생성되었음을 확인하였다. 이상의 분석기법을 통해 복수세대에서 비타민A 강화벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

Tocopherols (α-, β-, γ- and δ-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. In the present study, NtTC, which encodes a tobacco tocopherol cyclase ortholog, was cloned and characterized. Compared with control plants, NtTC transgenic rice showed higher tolerance to drought stress, and total tocopherol content increased by 52 % in leaf. Additionally, total antioxitant activity of NtTC transgenic lines was increased significantly by 19%. These results demonstrate that over-expressing NtTC could improve the tolerance to abiotic stress in rice, and tocopherols play a crucial role in the protection of oxidative stress.

An efficient transformation method for soybean [Glycine max (L.) Merr.] using meristematic tissues of germinating seeds has been established. The embryonic axes were excised from germinating seeds of Korean soybean cultivar, Iksannamulkong and 0.5-2 cm long segment containing meristematic tissues were prepared by cutting hypocotyl region. The explants were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector with the bar gene as a selectable marker gene and a β-glucuronidase (GUSINT) reporter gene, and then co-cultured for 7 days on co-cultivation medium (CCM). The meristematic tissues were cultured on shoot induction medium (SIMP6) supplemented with 0.4 mg/l N6-benzylaminopurine (BAP) and 0.1 mg/l indolebutyric acid (IBA) in the presence of 6 mg/l L-phosphinotricin (PPT) for 2 weeks and the surviving explants were transferred to shoot elongation medium (SEMP6). Transformation was confirmed by Southern blot analysis and the transformation efficiencies ranged from 1.48 to 2.07%. The new modified transformation method was successfully implemented for obtaining several transgenic lines with SMV-CP gene. It is expected that this method could efficiently be used for the transformation of recalcitrant soybean cultivars.