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A simple, rapid and systematic method for the developed GM rice analysis

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Abstract

We generated 383 independent transgenic lines that contained the PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes under the control of a constitutive (CaMV 35S) promoter to generate genetically modified (GM) rice. TaqMan copy number assay was performed to determine the copy numbers of inserted T-DNA. Flanking sequence tags (FSTs) were isolated from 203 single copy T-DNA lines of transgenic plants, and their sequences were mapped to the rice chromosomes. Of the 157 flanking sequence tags that were isolated from single copy lines, transgenes were found to be integrated into genic regions in 58 lines (36 %), whereas 97 lines (62 %) contained transgene insertions in intergenic regions. Approximately 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assays. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNA from leaf tissue of homozygous T1 plants with a single copy and an intergenic insertion of T-DNA. The mRNA expression levels of the examined transgenic rice were significantly increased in all transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants displayed higher levels of transgene protein. Using numerical data for the mass production of transgenic plants can reduce the time required to obtain a genetically modified plant. Moreover, the duration, cost, and efforts required for transformation can be deliberately predicted. These results may be useful for the large-scale production of transgenic plants or T-DNA inserted rice mutants.

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Acknowledgments

This study was supported by a grant from the Next-Generation Biogreen 21 Program (Project No. PJ011257) and a grant from National Academy of Agricultural Science (NAAS) Agenda Program (Project No.PJ010086) in Rural Development Administration, Jeonju, Republic of Korea and National Marine Biodiversity Institute Research Program (2015M00500).

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    Authors and Affiliations

    1. Department of Agricultural Biotechnology, National Academy of Agricultural Science, Jeonju, 560-500, Korea

      Hyemin Lim, A-Ram Kim, Hyeonso Ji, Chang-Kug Kim, Seung Uk Ji, Jung-Il Cho, Soo-Chul Park & Gang-Seob Lee

    2. LMO Technology Development Team, Converging Research Division, National Marin Biodiversity Institute of Korea, Seocheon, 325-902, Korea

      Hyun-Ju Hwang

    3. Graduate School of Biotechnology, Kyung Hee University, Yongin, 446-701, Korea

      Man-Ho Cho

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    1. Hyemin Lim
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    2. Hyun-Ju Hwang
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    3. A-Ram Kim
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    4. Man-Ho Cho
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    5. Hyeonso Ji
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    7. Seung Uk Ji
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    8. Jung-Il Cho
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    9. Soo-Chul Park
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    10. Gang-Seob Lee
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    Corresponding author

    Correspondence to Gang-Seob Lee.

    Additional information

    H. Lim and H.-J. Hwang equally contributed to this work.

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    Lim, H., Hwang, HJ., Kim, AR. et al. A simple, rapid and systematic method for the developed GM rice analysis. Plant Biotechnol Rep 10, 25–33 (2016). https://doi.org/10.1007/s11816-015-0384-1

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    • DOI: https://doi.org/10.1007/s11816-015-0384-1

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