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Validation of ST-LS1 as an Endogenous Reference Gene for Detection of AmA1 and cry1Ab Genes in Genetically Modified Potatoes using Multiplex and Real Time PCR

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Abstract

Solanum tuberosum L. belonging to family Solanaceae being the most important tuberous vegetable crop, the development of genetically modified (GM) potato with improved traits is the need of the hour. PCR (polymerase chain reaction) assays are being widely used in GM detection to meet the regulatory and legislative requirements. Detection of target sequences along with plant species specific endogenous reference genes will help in developing reliable and precise PCR assays. In the present study, ST-LS1, a single copy gene from S. tuberosum, was validated as an endogenous reference gene for potato with the PCR assays after testing on different members of the Solanaceae family, viz., Solanum melongena, Solanum lycopersicon, Capsicum annum, Datura stramonium, Petunia hybrida and other GM crops that are under different stages of testing in field trials in India, viz., Gossypium hirsutum, Oryza sativa, Brassica oleracea var. botrytis, Abelmoschus esculentus and Zea mays. The primer pair for ST-LS1 gene of potato was designed and validated by amplifying 223 bp desired fragment of ST-LS1 in potato only, whereas no amplicon was detected in other crops taken up for the study. The specificity of designed primer pair was further validated on real time PCR using SYBR Green I system showing no fluorescent signals with any of the crops tested other than potato. Furthermore, multiplex PCR employing the validated ST-LS1 gene as an internal control was performed to detect AmA1 and cry1Ab genes in GM potatoes. The validated qualitative and quantitative PCR protocols are specific for identification of GM potato and further can be used for quantitative detection of transgenes. The developed protocols for amplification of ST-LS1 gene can also detect as low as 0.01 ng/μl potato DNA, which meets the regulatory requirements for establishment of a GM detection protocol for testing 0.01% contamination as per the Supreme Court of India’s directions.

Resumen

Se necesita en estos momentos el desarrollo de organismos genéticamente modificados (GM) de papa con mejores características debido a que Solanum tuberosum L., perteneciente a la familia Solanaceae, es el cultivo tubérculo hortícola más importante. Los ensayos de PCR (reacción en cadena de la polimerasa) están siendo ampliamente utilizados en la detección de GM para responder a las exigencias reglamentarias y legislativas. La detección de secuencias meta junto con genes endógenos de referencia de especies de plantas específicas ayudará al desarrollo confiable y preciso de ensayos de PCR. En el presente estudio, ST-LS1, un gen de una sola copia de S. tuberosum, fue validado como un gen endógeno de referencia para la papa en ensayos de PCR después de la prueba en diferentes miembros de la familia de las solanáceas, es decir, Solanum melongena, Solanum lycopersicon, Capsicum annum, Datura stramonium, Petunia hybrida y otros cultivos GM que están en diferentes etapas de prueba en ensayos de campo en la India, a saber Gossypium hirsutum, Oryza sativa, Brassica oleracea var. botrytis, Abelmoschus esculentus y Zea mays. El par cebador para el gen ST-LS1 de la papa fue diseñado y validado mediante la amplificación del deseado fragmento 223 pb de ST-LS1 en la papa sólo, mientras que no se detectó amplicón en otros cultivos tomados para el estudio. La especificidad del par de cebadores diseñados fue validado con PCR en tiempo real utilizando el sistema SYBR Green I que no mostró señales fluorescentes con ninguno de los cultivos de prueba distintos de la papa. Además, PCR múltiplex que empleó el gen validado ST-LS1 como un control interno fue usado para detectar los genes AmA1 y cry1Ab en papas GM. Los protocolos PCR cualitativo y cuantitativo validados fueron específicos para la identificación de papa GM y pueden ser utilizados para la detección cuantitativa de los transgenes. Los protocolos desarrollados para la amplificación del gen ST-LS1 también pueden detectar tan poco como 0.01 ng/μl de ADN de papa, los que reúnen los requisitos reglamentarios para el establecimiento de un protocolo de detección de transgénicos para la prueba del 0.01% de contaminación según las instrucciones de la Corte Suprema de la India.

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Abbreviations

ST-LS1 :

Solanum tuberosum gene with leaf/stem-specific expression

PCR:

polymerase chain reaction

GM:

genetically modified

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Acknowledgements

The authors duly acknowledge the financial support provided by the Department of Biotechnology, Government of India. The authors are thankful to Director, Central Potato Research Institute, Shimla and Director, National Institute of Plant Genome Research, New Delhi for providing the plantlets of GM potato lines with AmA1 and cry1Ab genes; Maharashtra Hybrid Seeds Company, Mumbai for providing the lyophilized leaf samples of Bt cotton, Bt rice, Bt Okra; and Sungro Seeds Pvt. Ltd., New Delhi for providing leaf samples of Bt cauliflower. Authors are also thankful to Director, National Bureau of Plant Genetic Resources, New Delhi, for providing the necessary facilities.

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  1. Referral Centre for Molecular Diagnosis of Transgenic Planting Materials, National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, 110012, India

    Gurinder Jit Randhawa, Monika Singh & Ruchi Sharma

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  1. Gurinder Jit Randhawa
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  2. Monika Singh
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  3. Ruchi Sharma
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Correspondence to Gurinder Jit Randhawa.

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Randhawa, G.J., Singh, M. & Sharma, R. Validation of ST-LS1 as an Endogenous Reference Gene for Detection of AmA1 and cry1Ab Genes in Genetically Modified Potatoes using Multiplex and Real Time PCR. Am. J. Pot Res 86, 398–405 (2009). https://doi.org/10.1007/s12230-009-9095-x

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  • DOI: https://doi.org/10.1007/s12230-009-9095-x

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