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Journal of Integrative Agriculture  2017, Vol. 16 Issue (03): 551-558    DOI: 10.1016/S2095-3119(16)61458-2
Crop Genetics · Breeding · Germplasm Resources Advanced Online Publication | Current Issue | Archive | Adv Search |
Development of glyphosate-tolerant transgenic cotton plants harboring the G2-aroA gene
ZHANG Xiao-bing1, 2*, TANG Qiao-ling1*, WANG Xu-jing1, WANG Zhi-xing1

1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

2 Biology Institute, Hebei Academy of Sciences, Shijiazhuang 050051, P.R.China

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Abstract  Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China, the development of glyphosate-resistant cotton holds great promise.  Using an Agrobacterium-mediated transformation method, a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was transformed into cotton cultivar K312.  The transgenic cotton plants were regenerated from a callus tissue culture via kanamycin selection.  Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit.  The results from polymerase chain reaction (PCR) and Southern and Western blot analyses indicated that the target gene was integrated into the cotton chromosome and was expressed effectively at the protein level.  The glyphosate tolerance analysis showed that the transgenic cotton had a high resistance to glyphosate.  Further, even cotton treated with 45.0 mmol L–1 of glyphosate was able to slowly grow, bloom and seed.  The transgenic cotton may be used for cotton breeding research of glyphosate-tolerant cotton.
Keywords:   cotton (Gossypium hirsutum L.)      Agrobacterium-mediated method      glyphosate, G2-aroA      genetic transformation  
Received: 15 March 2016   Accepted: 07 March 2017

This work was supported by the Genetically Modified Major Projects, China (2012ZX08011-003 and 2014ZX08011-004B).

Corresponding Authors:  WANG Xu-jing, Tel: +86-10-82106124, E-mail:; WANG Zhi-xing, Tel: +86-10-82106102, E-mail:   
About author:  ZHANG Xiao-bing, E-mail:

Cite this article: 

ZHANG Xiao-bing, TANG Qiao-ling, WANG Xu-jing, WANG Zhi-xing. 2017. Development of glyphosate-tolerant transgenic cotton plants harboring the G2-aroA gene. Journal of Integrative Agriculture, 16(03): 551-558.

Awan M F, Abass A, Muzaffar A, Ali A, Tabassum B, Rao A Q, Nasir I A, Husnain T. 2015. Transformation of insect and herbicide resistance genes in cotton (Gossypium hirsutum L.). Journal of Agricultural Science and Technology, 17, 287–298.
Bubner B, Baldwin I T. 2004. Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Reports, 23, 263–271.
Castle L A, Siehl D L, Gorton R, Patten P A, Chen Y H, Bertain S, Cho H J, Duck N, Wong J, Liu D, Lassner M W. 2004. Discovery and directed evolution of a glyphosate tolerance gene. Science, 304, 1151–1154.
Chakravarthy V S, Reddy T P, Reddy V D, Rao K V. 2014. Current status of genetic engineering in cotton (Gossypium hirsutum L.): An assessment. Critical Reviews in Biotechnology, 34, 144–160.
Chaudhry B, Yasmin A, Husnain T, Riazuddin S. 1999. Mini-scale genomic DNA extraction from cotton. Plant Molecular Biology Reporter, 17, 280.
Dun B Q, Wang X J, Lu W, Chen M, Zhang W, Ping S Z, Wang Z X, Zhang B M, Lin M. 2014. Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes. The Crop Journal, 2, 164–169.
Funke T, Han H, Healy-Fried M L, Fischer M, Schonbrunn E. 2006. Molecular basis for the herbicide resistance of Roundup Ready crops. Proceedings of the National Academy of Sciences of the United States of America, 103, 13010–13015.
Gaines T A, Zhang W, Wang D, Bukun B, Chisholm S T, Shaner D L, Nissen S J, Patzoldt W L, Tranel P J, Culpepper A S, Grey T L, Webster T M, Vencill W K, Sammons R D, Jiang J, Preston C, Leach J E , Westra P. 2010. Gene amplification confers glyphosate resistance in Amaranthus palmeri. Proceedings of the National Academy of Sciences of the United States of America, 107, 1029–1034.
Gong Y Y, Xu Z Z, Guo S Q, Shu H M, Jiang L, Ni W C. 2016. Genome-wide analysis of the EPSPS genes in upland cotton. Acta Agriculturae Boreali-Sinica, 31, 15–21. (in Chinese)
Guo S D, Wang Y, Sun G Q, JIin S Q, Zhou T, Meng Z G, Zhang R. 2015. Twenty years of research and application of transgenic cotton in China. Scientia Agricultura Sinica, 48, 3372–3387. (in Chinese)
Leelavathi S, Sunnichan V G, Kumria R, Vijaykanth G P, Bhatnagar R K, Reddy V S. 2004. A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Reports, 22, 465–470.
Li J. 2013. Research on the novel glyphosate-resistant corn and the selectively terminable strategy. Ph D thesis, Zhejiang University, China. (in Chinese)
Ma X Y, Wu H W, Jiang W L, Ma Y J, Ma Y. 2016. Weed and insect control affected by mixing insecticides with glyphosate in cotton. Journal of Integrative Agriculture, 15, 373–380.
Malherbe D F, du Toit M, Cordero Otero R R, van Rensburg P, Pretorius I S. 2003. Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production. Applied Microbiology and Biotechnology, 61, 502–511.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.
Padgette S R, Kolacz K, Delannay X, Re D, LaVallee B, Tinius C, Rhodes W, Otero Y, Barry G, Eichholtz D. 1995. Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Science, 35, 1451–1461.
Paterson A H, Brubaker C L, Wendel J F. 1993. A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Molecular Biology Reporter, 11, 122–127.
Pline W A, Wilcut J W, Duke S O, Edmisten K L, Wells R. 2002. Tolerance and accumulation of shikimic acid in response to glyphosate applications in glyphosate-resistant and nonglyphosate-resistant cotton (Gossypium hirsutum L.). Journal of Agricultural and Food Chemistry, 50, 506–512.
Steinrucken H C , Amrhein N. 1980. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochemical and Biophysical Research Communications, 94, 1207–1212.
Tian Y S, Xiong A S, Xu J, Zhao W, Gao F, Fu X Y, Xu H, Zheng J L, Peng R H , Yao Q H. 2010. Isolation from Ochrobactrum anthropi of a novel class II 5-enopyruvylshikimate-

3-phosphate synthase with high tolerance to glyphosate. Applied and Environmental Microbiology, 76, 6001–6005.
Vats S. 2015. Herbicides: History, classification and genetic manipulation of plants for herbicide resistance. Sustainable Agriculture Reviews, 15, 153–192.
Wang X J, Jin X, Dun B Q, Kong N, Jia S R, Tang Q L, Wang Z X. 2014. Gene-splitting technology: A novel approach for the containment of transgene flow in Nicotiana tabacum. PLOS ONE, 9, doi: 10.1371/journal.pone.0099651
Wang Y Y. 2015. Identification of natural resistance to glyphosate in Gossypium and the excavation of glyphosate-resistant gene resources in Gossypium hirsutum races. Ph D thesis, Chinese Academy of Agricultural Sciences, China. (in Chinese)
Weng H B, Pan A H, Yang L T, Zhang C M, Liu Z L, Zhang D B. 2004. Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Molecular Biology Reporter, 22, 289–300.
Xie L X, Li Y F, Xu P L. 2004. Glyphosate-resistant cotton (Gossypium hirsutum L.) transformed with aroAM12 gene via Agrobacterium tumefaciens. Journal of Plant Physiology and Molecular Biology, 30, 173–178. (in Chinese)
Yan H Q, Chang S H, Tian Z X, Zhang L, Sun Y C, Li Y, Wang J, Wang Y P. 2011. Novel AroA from Pseudomonas putida confers tobacco plant with high tolerance to glyphosate. PLoS ONE, 6, doi: 10.1371/journal.pone.0019732
Yan S F, Zhu S J, Liu H F, Lu C X, Tie S G. 2015. Genetic analysis of transgenic glyphosate-resistance cotton with EPSPS gene. Acta Agriculturae Boreali-Sinica, 30, 54–57. (in Chinese)
Yang L T, Chen J X, Huang C, Liu Y H, Jia S R, Pan L W, Zhang D B. 2005. Validation of a cotton-specific gene, Sadl, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons. Plant Cell Reports, 24, 237–245.
Zhang B. 2013. Transgenic cotton: From biotransformation methods to agricultural application. Methods in Molecular Biology, 958, 3–15.
Zhang X B. 2015. Development of glyphosate-tolerant transgenic cotton plants harboring G2-aroA gene and event-specific testing. Ph D thesis, Chinese Academy of Agricultural Sciences, China. (in Chinese)
Zhang X B, Tang Q L, Wang X J, Wang Z X. 2016. Structure of exogenous gene integration and event-specific detection in the glyphosate-tolerant transgenic cotton line BG2-7. PLOS ONE, 11, e0158384.
Zhang Z Z, Tang L P, Wang Y, He J G, Cai W L, Rong J K. 2014. Study on transformation of glyphosate-resistant gene aroA into embryogenic calli of upland cotton via agrobacterium tumefaciens. Cotton Science, 26, 237–243. (in Chinese)
Zhao F Y, Li Y F, Xu P. 2006. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L. cv. Zhongmian 35) using glyphosate as a selectable marker. Biotechnology Letters, 28, 1199–1207.
Zhao T. 2008. Discovered of a glyphosate resistant gene and bred of transgenic rice with glyphosate resistance. Ph D thesis, Zhejiang University, China. (in Chinese)
Zhu Y. 2002. Identification of glyphosate-tolerant Psedomonas fluorescens strain G2 from extremely polluted environment and cloning of its EPSP synthase gene. MSc thesis, Chinese Academy of Agricultural Sciences, China. (in Chinese)
Zhu Y, Yu Z L, Lin M. 2003. Bioresistane or biodegradation of glyphosate and construction of transgenic platns. Molecular Plant Breeding, 1, 435–441. (in Chinese)
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