Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)

doi:10.1016/S2095-3119(13)60567-5

Journal of Integrative Agriculture ›› 2013, Vol. 12 ›› Issue (12): 2134-2142.DOI: 10.1016/S2095-3119(13)60567-5

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)

YUGui-rong12, LIUYan3, DUWen-ping2, SONGJun2, LINMin4, XULi-yuan2, XIAOFang-ming5

YUGui-rong12, LIUYan3, DUWen-ping2, SONGJun2, LINMin4, XULi-yuan2, XIAOFang-ming5

收稿日期:2012-11-12 出版日期:2013-12-01 发布日期:2013-12-17
通讯作者: LIU Yong-sheng, E-mail: liuyongsheng1122@hfut.edu.cn
作者简介:YU Gui-rong, E-mail: yuguirong@163.com
基金资助:
The study was supported by the National Key Project of transgenic varieties breeding (2009ZX08003-003B), the Light of West Talent Training Project of China (2010- 2011) and the Project of Sichuan Province Finance Genetic Engineering, China (2011JYGC01-002).

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)

YUGui-rong12, LIUYan3, DUWen-ping2, SONGJun2, LINMin4, XULi-yuan2, XIAOFang-ming5

YUGui-rong12, LIUYan3, DUWen-ping2, SONGJun2, LINMin4, XULi-yuan2, XIAOFang-ming5

Received:2012-11-12 Online:2013-12-01 Published:2013-12-17
Contact: LIU Yong-sheng, E-mail: liuyongsheng1122@hfut.edu.cn
About author:YU Gui-rong, E-mail: yuguirong@163.com
Supported by:
The study was supported by the National Key Project of transgenic varieties breeding (2009ZX08003-003B), the Light of West Talent Training Project of China (2010- 2011) and the Project of Sichuan Province Finance Genetic Engineering, China (2011JYGC01-002).

摘要/Abstract

摘要： 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.

关键词: maize , immature embryo , Agrobacterium tumefaciens-mediated transformation , transgenic approach , glyphosate resistance

Abstract: 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.

Key words: maize , immature embryo , Agrobacterium tumefaciens-mediated transformation , transgenic approach , glyphosate resistance

YUGui-rong12, LIUYan3, DUWen-ping2, SONGJun2, LINMin4, XULi-yuan2, XIAOFang-ming5. Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)[J]. Journal of Integrative Agriculture, 2013, 12(12): 2134-2142.

YUGui-rong12 , LIUYan3 , DUWen-ping2 , SONGJun2 , LINMin4 , XULi-yuan2 , XIAOFang-ming5 . Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)[J]. Journal of Integrative Agriculture, 2013, 12(12): 2134-2142.

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Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea mays L.)

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