Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

Niloofar Rostamkhani, Ali Haghnazari, Masoud Tohidfar and Aboubakr Moradi
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Niloofar Rostamkhani: Department of Agricultural Biotechnology, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
Ali Haghnazari: Department of Agricultural Biotechnology, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
Masoud Tohidfar: Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran
Aboubakr Moradi: Department of Agricultural Biotechnology, Faculty of Agriculture, University of Zanjan, Zanjan, Iran

Czech Journal of Genetics and Plant Breeding, 2011, vol. 47, issue 4, 140-148

Abstract: In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T2 transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65°C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10-1 to 10-8) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

Keywords: Bt; chitinase; CryIA(b); detection; loop-mediated isothermal amplification; transgenic cotton (search for similar items in EconPapers)
Date: 2011
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Persistent link: https://EconPapers.repec.org/RePEc:caa:jnlcjg:v:47:y:2011:i:4:id:7-2011-cjgpb

DOI: 10.17221/7/2011-CJGPB

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