title: Development of Selectable Marker-Free Transgenic Rice Plants with Enhanced Seed Tocopherol Content through FLP/FRT-Mediated Spontaneous Auto-Excision.
abstract: 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.
authors.affiliation: Biosafety Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Jeonju, Republic of Korea.
authors.forename: Hee-Jong
authors.initials: HJ
authors.lastname: Woo
authors.affiliation: Biosafety Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Jeonju, Republic of Korea.
authors.forename: Yang
authors.initials: Y
authors.lastname: Qin
authors.affiliation: Biosafety Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Jeonju, Republic of Korea.
authors.forename: Soo-Yun
authors.initials: SY
authors.lastname: Park
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea.
authors.forename: Soon Ki
authors.initials: SK
authors.lastname: Park
authors.affiliation: Department of Crop Science, Chungbuk National University, Cheongju, Republic of Korea.
authors.forename: Yong-Gu
authors.initials: YG
authors.lastname: Cho
authors.affiliation: Biosafety Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Jeonju, Republic of Korea.
authors.forename: Kong-Sik
authors.initials: KS
authors.lastname: Shin
authors.affiliation: Biosafety Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Jeonju, Republic of Korea.
authors.forename: Myung-Ho
authors.initials: MH
authors.lastname: Lim
authors.affiliation: Biosafety Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Jeonju, Republic of Korea.
authors.forename: Hyun-Suk
authors.initials: HS
authors.lastname: Cho
mesh_terms: D003062:Codon / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D012275:Oryza / Q000235:genetics* / Q000378:metabolism*; D018384:Oxidative Stress; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D012639:Seeds / Q000378:metabolism; D024505:Tocopherols / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D003062:Codon; D004269:DNA, Bacterial; D005819:Genetic Markers; C036483:T-DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D024505:Tocopherols