This study was conducted to develop environmental risk assessments and biosafety guides for insect-resistant genetically modified rice in an LMO (Living Modified Organism) isolation field. In the LMO quarantine area of Kyungpook National University, the species diversities and population densities of non-target insects found on insect-resistant genetically modified rice (Bt-T), rice resistant to Cnaphalocrocis medinalis, and non-GM rice (Dongjin-byeo and Ilmi-byeo) were investigated. The Bt-T plants were, therefore, evaluated under field conditions to detect possible impacts on above ground insects and spiders. In 2016 and 2017, the study compared transgenic rice and two non-GM reference rice, namely Dongjin-byeo and Ilmi-byeo, at Gunwi. A total of 9,552 individuals from 51 families and 11 orders were collected from the LMO isolation field. From the three types of rice fields, a total of 3,042; 3,212; and 3,297 individuals from the Bt-T, Dongjin-byeo, and Ilmi-byeo were collected, respectively. There was no difference between the population densities of the non-target insect pests, natural enemies, and other insects on the Bt-T compared to non-GM rice. The data on insect species population densities were subjected to principal component analysis (PCA) without distinguishing between the three varieties, namely GM, non-GM, and reference cultivar, in all cultivation years. However, the PCA clearly separated the samples based on the cultivation years. These results suggest that insect species diversities and population densities during plant cultivation are determined by environmental factors (growing condition and seasons) rather than by genetic factors.

Genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by global agricultural companies. Until now, GM crops have not been cultivated commercially in Korea. Commercialization of GM crops requires a compulsory assessment of environmental risk associated with the release of GM crops. This study was conducted to evaluate the frequency of pollen mediated gene flow from Bt transgenic rice (Agb0101) to japonica non-GM rice (Nakdongbyeo), indica non-GM rice (IR36), and weedy rice (R55). A total of 729,917, 596,318 and 230,635 seeds were collected from Nakdongbyeo, IR36, and R55, respectively, which were planted around Agb0101. Selection of the hybrids was determined by repeated spraying of herbicide and Cry1Ac1 immunostrip assay. Finally, the hybrids were confirmed by PCR analysis using specific primer. The hybrids were found in all non-GM rice and out-crossing ranged from 0.0005% at IR36 to 0.0027% at Nakdongbyeo. All of hybrids were located within 1.2 m distance from the Agb0101 rice plot. The meteorological elements including rainfall and temperature during rice flowering time were found to be important factors to determine rice out-crossing rate. Consideration should be taken for many factors like the meteorological elements of field and physiological condition of crop to set up the safety management guideline to prevention of GM crops gene flow.

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 selectable marker-free rice plants containing mcry1Ac insecticidal gene isolated from Bacillus thuringiensis (Bt) were generated using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. The nutritional composition of two lines of transgenic rice plants (RTB5 and RTB11) was compared with that of its non-transgenic counterpart. The results showed that, except for small differences in dietary fiber and some minerals, there was no significant difference between transgenic rice and conventional counterpart variety with respect to their nutrient composition. Most of measured levels of nutrients were within the range of values reported for other commercial cultivars, showing substantial equivalency. Therefore, the insertion of transgenes did not affect the nutritional composition of transgenic RTB5 and RTB11 rice grains.

The β-carotene biofortified transgenic rice was developed by transforming rice cv. Nakdongbyeo with phytoene synthase (Psy) and carotene desaturase (Crt I) genes isolated from Capsicum and Pantoea. The aim of this study was to perform molecular characterization of rice transformants of T5-T7 generation harboring Psy and Ctr I genes driven by endosperm specific globulin promoter for biosafety evaluation of β-carotene biofortified transgenic rice. The structure and sequence of T-DNA in the transformation vector and the insertion sites, flanking sequences and generational stability of inserted T-DNA in transgenic rice lines were analyzed. The transformation vector consisted of right border, MAR gene, carotenogenic genes unit, herbicide resistance selectable marker unit, MAR gene and left border in sequential order. T-DNA was introduced at the position of 30,363,938-30,363,973 bp of chromosome No. 2 by adaptor-ligation PCR. Stable integration of T-DNA and stable expression of bar gene was confirmed in T5 to T7 generations. It was also confirmed that the backbone DNA of transformation vector containing antibacterial gene was not present in the genome of β-carotene biofortified transgenic rice. HPLC analysis confirmed that carotenoids were consistently detected through T5-T7 generations.

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 was not present in Agb0103 rice genome. These results will be filed to biosafety assessment document of Agb0103.

In recent years, novel plant breeding techniques (NPBTs) have emerged, and safety assessment of the novel plant(s) generated using the NPBTs has drawn the attention of many stakeholders. The notable characteristics of the novel plants are as follows: firstly, it is almost impossible to distinguish from the natural mutations in the conventional counterparts, because site-directed nuclease (SDN) and oligonucleotide-directed mutagenesis (ODM) could introduce short indel(s) in the targeted region(s) of the chromosomes. Secondly, the genome constitution of novel plants is almost identical to that of their conventional counterparts, eventually becoming indistinguishable by the introduction of only unmodified gene(s) from sexually compatible species to the target host plant. Thirdly, it is possible to generate new plants that have the desired traits, but without introducing genes. These plants will have some modified bases in their genome by selecting null-segregant(s) from heterozygous transgenic plants or by other epigenetic methods. The Organisation for Economic Co-operation and Development (OECD) and many countries developing genetically modified organisms (GMOs) have concluded that novel plants developed using SDN, ODM, cisgenesis, intragenesis, or null-segregant techniques are treated in the same manner as non-genetically modified (GM) plants or may even have less strict risk assessments depending on the case. Additionally, grafting and agro-infiltration are methods that can be used to avoid or reduce the burden of current strict GMO risk assessment. The risk assessments of some of the novel plants have already been performed and those of commercially important plants are expected to be performed in the near future. Hence, it is necessary to develop a competitive and practical NPBT that can mitigate the concern and revulsion toward GMOs in Korea.