Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)
Since maize is one of the most important cereal crops in the world, establishment of an efficient genetic transformation system is critical for its improvement. In the current study, several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using im...
Saved in:
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
KeAi Communications Co., Ltd.
2013-12-01
|
| Series: | Journal of Integrative Agriculture |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2095311913605675 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849248269482328064 |
|---|---|
| author | Gui-rong YU Yan LIU Wen-ping DU Jun SONG Min LIN Li-yuan XU Fang-ming XIAO Yong-sheng LIU |
| author_facet | Gui-rong YU Yan LIU Wen-ping DU Jun SONG Min LIN Li-yuan XU Fang-ming XIAO Yong-sheng LIU |
| author_sort | Gui-rong YU |
| collection | DOAJ |
| description | Since maize is one of the most important cereal crops in the world, establishment of an efficient genetic transformation system is critical for its improvement. In the current study, several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants. Infection ability and efficiency of transformation of A. tumefaciens sp. strains EHA105 and LBA4404, different heat treatment times of immature embryos before infection, influence of L-cysteine addition in co-cultivation medium after transformation, and how different ways of selection and cultivation influence the efficiency of transformation were compared. Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599, Zong 31 and BA, under the optimum conditions. Results showed that the hypervirulent Agrobacterium tumefaciens sp. strain EHA105 was more infectious than LBA4404. Inclusion of L-cysteine (100 mg L−1) in co-cultivation medium, and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency. Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli. During induction of germination, adding a high concentration of 6-BA (5 mg L−1) and a low concentration of 2,4-D (0.2 mg L−1) to regeneration medium significantly enhanced germination percentage. Using the optimized transformation procedure, more than 800 transgenic plants were obtained from 78599, Zong 31 and BA. By spraying herbicide glyphosate on leaves of transgenic lines, we identified 66 primary glyphosate-resistant plants. The transformation efficiency was 8.2%. PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome. |
| format | Article |
| id | doaj-art-efbbe12a9f474fcfbc4a94482bfe4de8 |
| institution | Kabale University |
| issn | 2095-3119 |
| language | English |
| publishDate | 2013-12-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Journal of Integrative Agriculture |
| spelling | doaj-art-efbbe12a9f474fcfbc4a94482bfe4de82025-08-20T03:57:55ZengKeAi Communications Co., Ltd.Journal of Integrative Agriculture2095-31192013-12-0112122134214210.1016/S2095-3119(13)60567-5Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)Gui-rong YU0Yan LIU1Wen-ping DU2Jun SONG3Min LIN4Li-yuan XU5Fang-ming XIAO6Yong-sheng LIU7Key Laboratory for Bio-Resource and Eco-Environment, Ministry of Education/State Key Laboratory of Hydraulics and Mountain River Engineering, College of Life Science, Sichuan University, Chengdu 610064, P.R. China; Institute of Biotechnology & Nuclear Techniques, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. China; YU Gui-rongCrop Sciences Research Institute, China Academy of Agricultural Sciences, Beijing 100081, P.R. ChinaInstitute of Biotechnology & Nuclear Techniques, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. ChinaInstitute of Biotechnology & Nuclear Techniques, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. ChinaBiotechnology Research Institute, China Academy of Agricultural Sciences, Beijing 100081, P.R. ChinaInstitute of Biotechnology & Nuclear Techniques, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. ChinaDepartment of Plant, Soil and Entomological Sciences, University of Idaho Moscow, ID 83844-2339, USAKey Laboratory for Bio-Resource and Eco-Environment, Ministry of Education/State Key Laboratory of Hydraulics and Mountain River Engineering, College of Life Science, Sichuan University, Chengdu 610064, P.R. China; Correspondence LIU Yong-shengSince maize is one of the most important cereal crops in the world, establishment of an efficient genetic transformation system is critical for its improvement. In the current study, several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants. Infection ability and efficiency of transformation of A. tumefaciens sp. strains EHA105 and LBA4404, different heat treatment times of immature embryos before infection, influence of L-cysteine addition in co-cultivation medium after transformation, and how different ways of selection and cultivation influence the efficiency of transformation were compared. Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599, Zong 31 and BA, under the optimum conditions. Results showed that the hypervirulent Agrobacterium tumefaciens sp. strain EHA105 was more infectious than LBA4404. Inclusion of L-cysteine (100 mg L−1) in co-cultivation medium, and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency. Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli. During induction of germination, adding a high concentration of 6-BA (5 mg L−1) and a low concentration of 2,4-D (0.2 mg L−1) to regeneration medium significantly enhanced germination percentage. Using the optimized transformation procedure, more than 800 transgenic plants were obtained from 78599, Zong 31 and BA. By spraying herbicide glyphosate on leaves of transgenic lines, we identified 66 primary glyphosate-resistant plants. The transformation efficiency was 8.2%. PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.http://www.sciencedirect.com/science/article/pii/S2095311913605675maizeimmature embryoAgrobacterium tumefaciens-mediated transformationtransgenic approachglyphosate resistance |
| spellingShingle | Gui-rong YU Yan LIU Wen-ping DU Jun SONG Min LIN Li-yuan XU Fang-ming XIAO Yong-sheng LIU Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.) Journal of Integrative Agriculture maize immature embryo Agrobacterium tumefaciens-mediated transformation transgenic approach glyphosate resistance |
| title | Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.) |
| title_full | Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.) |
| title_fullStr | Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.) |
| title_full_unstemmed | Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.) |
| title_short | Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.) |
| title_sort | optimization of agrobacterium tumefaciens mediated immature embryo transformation system and transformation of glyphosate resistant gene 2mg2 epsps in maize zea mays l |
| topic | maize immature embryo Agrobacterium tumefaciens-mediated transformation transgenic approach glyphosate resistance |
| url | http://www.sciencedirect.com/science/article/pii/S2095311913605675 |
| work_keys_str_mv | AT guirongyu optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl AT yanliu optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl AT wenpingdu optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl AT junsong optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl AT minlin optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl AT liyuanxu optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl AT fangmingxiao optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl AT yongshengliu optimizationofagrobacteriumtumefaciensmediatedimmatureembryotransformationsystemandtransformationofglyphosateresistantgene2mg2epspsinmaizezeamaysl |