ArticlePlant physiology2015
Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.
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Article in Plant physiology, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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12 citing papers in PubMed, 22 citations in OpenAlex.
- Application of a multiplex CRISPR/Cas9 strategy for elimination of selection markers from transgenic plants.Frontiers in genome editing · 2025Article
- A DEAD box helicase Psp68 positively regulates salt stress responses in marker-free transgenic rice plants.Transgenic research · 2023Article
- Selectable Markers to Marker-Free Selection in Rice.Molecular biotechnology · 2022Review
- A Prospective Review on Selectable Marker-Free Genome Engineered Rice: Past, Present and Future Scientific Realm.Frontiers in genetics · 2022Review
- Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease UsingFrontiers in plant science · 2021Article
- Low Long Terminal Repeat (LTR)-Retrotransposon Expression in Leaves of the Marine PhanerogamLife (Basel, Switzerland) · 2020Article
- A computational genome-wide analysis of long terminal repeats retrotransposon expression in sunflower roots (Helianthus annuus L.).Genetica · 2020Article
- An Efficient Gene Excision System in Maize.Frontiers in plant science · 2020Article
- Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector.Genes · 2019Article
- Production of Marker-free Transgenic Rice (Iranian journal of biotechnology · 2017Article
- Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis.Plant cell reports · 2016Article
- Development of Marker-Free Insect-Resistant Indica Rice byFrontiers in plant science · 2016Article
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Authors and funding
16 authors at 2 institutions in 3 countries.
Xiaoqing GaoState 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.
Jie ZhouState 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.
Jun LiState 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.
Xiaowei ZouState 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.
Jianhua ZhaoState 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.
Qingliang LiState 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.
Ran XiaState 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.
Ruifang YangState 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.
Dekai WangState 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.
Zhaoxue ZuoState 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.
Jumin TuState 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.
Yuezhi TaoState 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.
Xiaoyun ChenState 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.
Qi XieState 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.
Zengrong ZhuState 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.
Shaohong QuState 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.ORCID http://orcid.org/0000-0003-2072-122X
ZheJiang Academy of Agricultural Sciences · CNChinese Academy of Sciences · CN
Funding
No grant is acknowledged in the PubMed record.
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.
Indexed as
AnimalsBacillus thuringiensisDisease ResistanceDNA, BacterialDNA, PlantDNA Transposable ElementsGenetic MarkersGenetic VectorsGreen Fluorescent ProteinsLepidopteraOryzaPlants, Genetically ModifiedReverse Transcriptase Polymerase Chain ReactionTransformation, GeneticTransgenesDNA, BacterialDNA, PlantDNA Transposable ElementsGenetic MarkersGreen Fluorescent ProteinsT-DNA
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