title: Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.
abstract: Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Gao
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zou
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhao
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Qingliang
authors.initials: Q
authors.lastname: Li
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Xia
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Ruifang
authors.initials: R
authors.lastname: Yang
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Dekai
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Zhaoxue
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Jumin
authors.initials: J
authors.lastname: Tu
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Yuezhi
authors.initials: Y
authors.lastname: Tao
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Xie
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Zengrong
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China;Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); andInstitute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China squ@mail.zaas.ac.cn.
authors.forename: Shaohong
authors.identifier: http://orcid.org/0000-0003-2072-122X
authors.initials: S
authors.lastname: Qu
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D060467:Disease Resistance / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D007915:Lepidoptera; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic / Q000235:genetics; D019076:Transgenes / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D018744:DNA, Plant; D005819:Genetic Markers; C036483:T-DNA; D049452:Green Fluorescent Proteins