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
- 23Citations
- 24Captures
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- Citations23
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- 23
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- 24
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
10.1371/journal.pone.0132667; 10.1371/journal.pone.0132667.g002; 10.1371/journal.pone.0132667.g003; 10.1371/journal.pone.0132667.g006; 10.1371/journal.pone.0132667.g001; 10.1371/journal.pone.0132667.g005; 10.1371/journal.pone.0132667.g007; 10.1371/journal.pone.0132667.g004
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