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.
Highlights
Rice is one of the most important food crops consumed worldwide
We describe the construction of a spontaneous self-excision binary vector using the oxidative-stress inducible marker gene and the rice codon-optimized FLP (mFLP)/FRT system for rice transformation
The region between the left and right T-DNA borders in the binary vector pHWMF is depicted schematically in Fig 1, in which the hygromycin-selectable marker gene and the recombinase gene FLP under the control of the oxidative stress-inducible promoter of the sweet potato were placed between two opposite FRT sites
Summary
Rice is one of the most important food crops consumed worldwide. Rice is a good source of vitamin E, an essential lipid-soluble nutrient that consists of four tocopherols and four tocotrienols. Each of these types of compounds has α-, β-, δ-, and γ-forms determined by the number of methyl groups on the chromanol ring. Tocopherols are important constituents of the human diet and have been shown to aid in immune function [3] and to decrease the risk of a number of degenerative diseases, such as Parkinson’s disease [4] and heart disease [5]. Recent reports have demonstrated that tocopherols can affect important physiological processes in plants, such as germination, photoassimilate export, growth, and leaf senescence; tocopherols have antioxidant functions in photosynthetic membranes and play important roles in plant responses to abiotic stresses [6]
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