Kijong Lee

Auto-excision of selectable marker genes from transgenic tobacco via a stress inducible FLP/FRT site-specific recombination system

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.

Molecular Analysis of Microbial Community in Soils Cultivating Bt Chinese Cabbage

*(National Academy of Agricultural Science, Suwon, 441-707, Korea) The aim of this study was to investigate the possible impact of Bt Chinese cabbage on the soil microbial community. Microbial communities were isolated from the rhizosphere of one Bt Chinese cabbage variety and four varieties of conventional ones 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 Chinese cabbages were observed to have an insignificant difference. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that the bacterial community structures were very similar to each other and this genetic stability of microbial communities was maintained throughout the culture periods. Analysis of dominant isolates in the rhizosphere of transgenic and conventional Chinese cabbages showed that the dominant isolates from the soil of transgenic Chinese cabbage belonged to the Bacilli and Alphaproteobacteria, while the dominant isolates from the soil of conventional cabbage belonged to the Holophagae and Planctomycetacia, respectively. These results indicate that the Bt transgenic cabbage has no significant impact on the soil microbial communities.

Responses of Misgurnus anguillicaudatus and Cyprinus carpio Fed on Disease Resistant(OsCK1) Rice Variety

BACKGROUND: The disease resistant (OsCK1) rice was generated by inserting choline kinase (CK1) and phosphinothricin acetyltransferase (PAT) genes isolated from Oriza sativa and Streptomyces hygroscopicus into the genome of rice (Nakdongbyeo). With 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. In present study, we studied the effects on survival of Misgurnus anguillicaudatus and Cyprinus carpio, commonly used as a model organism in ecotoxicological studies. METHODS AND RESULTS: The M. anguillicaudatus and C. carpio were fed on disease resistant (OsCK1) rice and non-genetically modified (non-GM) rice (Nakdongbyeo) to 0, 10, 100, 1,000 and 5,000 mg/L, as treatment concentration respectively. The OsCK1 rice used for the test was confirmed to have the OsCK1/PAT gene expression by the PCR and ELISA analysis. Feeding test showed that no significant differences in cumulative immobility and abnormal response of M. anguillicaudatus and C. carpio fed on between OsCK1 rice and non-GM rice. The 96hr- values showed no difference between OsCK1 rice (>5,000 mg/L) and non-GM rice (>5,000 mg/L). CONCLUSION(S): The results of this study suggested that there was no significant difference in toxicity for M. anguillicaudatus and C. carpio between OsCK1 rice and non-GM counterparts.

Acute Toxicity Evaluation to Daphnia magna of Disease Resistant(OsCK1) Rice

BACKGROUND: The disease resistant (OsCK1) rice was generated by inserting choline kinase (CK1) and phosphinothricin acetyltransferase (PAT) genes isolated from Oryza sativa and Streptomyces hygroscopicus into the genome of the rice, Nakdongbyeo. With the potential problems of safeties, the evaluations on non-target organisms are essentially required for the environmental risk assessment of genetically modified (GM) crops. In the present study, we conducted the evaluation of acute toxicity on Daphnia magna that commonly used as a model organism in ecotoxicological studies for non-target organism evaluation. METHODS AND RESULTS: Effect of acute toxicity to Daphnia magna by each concentration were investigated in the disease resistant (OsCK1) rice and non-genetically modified (non-GM) rice, Nakdongbyeo, as concentration (0, 1,000, 1,800, 3,240, 5,830, 10,500 and 20,000 mg/L). The OsCK1 rice used for the test was confirmed to express the OsCK1/PAT gene by the PCR(Polymerase chain reaction) and western blot analysis. Feeding test showed that no significant differences in cumulative immobility and abnormal response of Daphnia magna fed on OsCK1 rice or non-GM rice. The 48hr- values showed no difference between OsCK1 rice (3,147.18 mg/L) and non-GM rice (3,596.27 mg/L). CONCLUSION: This result suggested that there was no significant difference in toxicity to Daphnia magna between OsCK1 rice and non-GM counterpart.

Impact of Virus-resistant Trigonal Cactus Cultivation on Soil Microbial Community

BACKGROUND: Genetically modified(GM) trigonal cactus(Hylocereus trigonus Saff.) contained a coat protein gene of cactus virus X (CVX), which conferred resistance to the virus, phosphinothricin acetyltransferase (bar) gene, which conferred herbicide resistance, and a cauliflower mosaic virus 35S promoter (CaMV 35S). This study was conducted to evaluate the possible impact of GM trigonal cactus cultivation on the soil microbial community. METHODS AND RESULTS: Microorganisms were isolated from the rhizosphere of GM and non-GM trigonal cactus cultivation soils. The total numbers of bacteria, and actinomycete in the rhizosphere soils cultivated GM and non-GM trigonal cactus were similar to each other, and there was no significant difference. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM trigonal cactus were Proteobacteria, Uncultured archaeon, and Uncultured bacterium. The denaturing gradient gel electrophoresis (DGGE) profiles show a similar patterns, significant difference was not observed in each other. DNA was isolated from soil cultivated GM and non-GM trigonal cactus, we analyzed the persistence of the inserted gene by PCR. Amplification of the inserted genes was not observed in the soil DNA, which was collected after harvest. CONCLUSION(S): This result suggests that the GM trigonal cactus cultivation does not change significantly the microbial community.

Effect on Insecticide Susceptibility of Lissorhoptrus oryzophilus Fed on Carotenoid-Biofortified Rice Variety

BACKGROUND: The carotenoid-biofortified (PAC) rice was generated by inserting phytoene synthase (Psy) and carotene desaturase (Crtl) genes isolated from Capsicum annuum cv. Nockwang and Pantoea ananatis into the genome of a conventional variety of rice (Nakdongbyeo). In our present study, we studied the effects on insecticide susceptibility of Rice Water Weevil (Lissorhoptrus oryzophilus). METHODS AND RESULTS: The L. oryzophilus were fed on carotenoid-biofortified (PAC) rice and its near non-genetically modified (GM) counterparts (Nakdongbyeo) under , humidity of , and photoperiod 16L:8D for more than 60 days. Ten adults were soaked in the Clothianidin SC solution for 5 second in different concentrations, and were detected the mortalities after 24, 48 and 72 hours respectively. Every experiment was conducted with three replications. The cumulative mortalities of L. oryzophilus exposed to Clothianidin SC were similar between two types of feed administration. CONCLUSION: The results of this study suggested that carotenoid-biofortified rice might not affect the insecticide susceptibilities of Lissorhoptrus oryzophilus.

The Effects of Genetically Modified Crops on Soil Microbial Community

Abstract BACKGROUND: Genetically modified (GM) crops must receive relevant regulator’s authorization before they can be sold as seed or used food, feed and processing. Before approving any GM crop, the relevant government ministries are required to examine environmental risk assessment to make scientifically sound and socially acceptable decisions. But one of the least studied and understood areas in the environmental risk assessment of GM crops are their impact on soil microbial community. METHODS AND RESULTS: Recently, advanced methods have been developed to characterize the soil microbial community in various environments. In this study, the culture-dependent and culture -independent technical appr-oaches for profiling soil microbial communities are summa-rized and their applicability to assess GM crops are discussed. CONCLUSION(S): We concluded that the effect of GM crops on soil microbial community need to be assessed on a case by case basis. The combination of culture-dependent and culture-independent method was necessary for reliable and detailed assessment of effect of GM crops on soil microbial community.

Effects of Transgenic Soybean Cultivation on Soil Microbial Community in the Rhizosphere

BACKGROUND: Soybean [Glycine max (L.) Merrill] is a legume and an important oil crop worldwide. This study was conducted to evaluate the possible impact of transgenic soybean cultivation on the soil microbial community. METHODS AND RESULTS: Microorganisms were isolated from the rhizosphere 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 transgenic and non-transgenic soybeans were similar to each other, and there was no significant difference between transgenic and non-transgenic soybeans. Dominant bacterial phyla in the rhizosphere soils cultivated with transgenic or non-transgenic soybeans were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in transgenic and non-transgenic soybean soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed the different patterns, but didn’t show significant difference to each other at 0.05 significance level. DNAs were isolated from soils cultivating transgenic or non-transgenic soybeans and analyzed for persistence of transgenes in the soil by using PCR. PCR analysis revealed that there were no amplified γ-tmt and bar gene in soil DNA. CONCLUSION(S): The results of this study suggested that microbial community of soybean field were not significantly affected by cultivation of the transgenic soybeans.

Analysis of right border flanking sequence in transgenic chinese cabbage harboring integrated T-DNA

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.

Effects of Vitamin E enhanced transgenic soybean cultivation on insect diversity

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.