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Physiol Mol Biol Plants. 2017 Apr;23(2):453-460. doi: 10.1007/s12298-017-0424-0. Epub 2017 Mar 14.

Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide.

Author information

1
Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, P.O.Box 577, Jhang Road, Faisalabad, Pakistan.
2
Pakistan Institute of Engineering and Applied Sciences, Nilore, Islamabad, Pakistan.
3
Departamento de CiênciasBiológicas, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Av. Pádua Dias, 11, Piracicaba, SP 13418-900 Brazil.

Abstract

Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.

KEYWORDS:

CP4-EPSPS; Gene expression; Herbicide resistance; Southern hybridization; Transgenic crops; qRT-PCR

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