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Development of selectable marker-free transgenic rice plants with enhanced seed tocopherol content through FLP/FRT-mediated spontaneous auto-excision

PLoS ONE, ISSN: 1932-6203, Vol: 10, Issue: 7, Page: e0132667
2015
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Article Description

Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, α-, γ-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice.

Bibliographic Details

http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=84940741702&origin=inward; http://dx.doi.org/10.1371/journal.pone.0132667; http://www.ncbi.nlm.nih.gov/pubmed/26172549; https://dx.plos.org/10.1371/journal.pone.0132667.g002; http://dx.doi.org/10.1371/journal.pone.0132667.g002; https://dx.plos.org/10.1371/journal.pone.0132667.g003; http://dx.doi.org/10.1371/journal.pone.0132667.g003; https://dx.plos.org/10.1371/journal.pone.0132667; https://dx.plos.org/10.1371/journal.pone.0132667.g006; http://dx.doi.org/10.1371/journal.pone.0132667.g006; https://dx.plos.org/10.1371/journal.pone.0132667.g001; http://dx.doi.org/10.1371/journal.pone.0132667.g001; https://dx.plos.org/10.1371/journal.pone.0132667.g005; http://dx.doi.org/10.1371/journal.pone.0132667.g005; https://dx.plos.org/10.1371/journal.pone.0132667.g007; http://dx.doi.org/10.1371/journal.pone.0132667.g007; https://dx.plos.org/10.1371/journal.pone.0132667.g004; http://dx.doi.org/10.1371/journal.pone.0132667.g004; https://dx.doi.org/10.1371/journal.pone.0132667.g006; https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0132667.g006; https://dx.doi.org/10.1371/journal.pone.0132667.g002; https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0132667.g002; https://dx.doi.org/10.1371/journal.pone.0132667.g001; https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0132667.g001; https://dx.doi.org/10.1371/journal.pone.0132667.g004; https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0132667.g004; https://dx.doi.org/10.1371/journal.pone.0132667.g003; https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0132667.g003; https://dx.doi.org/10.1371/journal.pone.0132667; https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132667; https://dx.doi.org/10.1371/journal.pone.0132667.g005; https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0132667.g005; https://dx.doi.org/10.1371/journal.pone.0132667.g007; https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0132667.g007; http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132667; https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0132667&type=printable; http://dx.plos.org/10.1371/journal.pone.0132667.g005; http://dx.plos.org/10.1371/journal.pone.0132667.g002; http://dx.plos.org/10.1371/journal.pone.0132667.g003; http://dx.plos.org/10.1371/journal.pone.0132667; http://dx.plos.org/10.1371/journal.pone.0132667.g006; http://dx.plos.org/10.1371/journal.pone.0132667.g007; http://www.plosone.org/article/metrics/info:doi/10.1371/journal.pone.0132667; http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0132667&type=printable; http://dx.plos.org/10.1371/journal.pone.0132667.g001; http://journals.plos.org/plosone/article/metrics?id=10.1371/journal.pone.0132667; http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0132667; http://dx.plos.org/10.1371/journal.pone.0132667.g004

Hee-Jong Woo; Yang Qin; Soo-Yun Park; Soon Ki Park; Yong-Gu Cho; Kong-Sik Shin; Myung-Ho Lim; Hyun-Suk Cho; Mukesh Jain

Public Library of Science (PLoS)

Biochemistry, Genetics and Molecular Biology; Agricultural and Biological Sciences; Multidisciplinary

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