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Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker

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Abstract

A new method for obtaining transgenic cowpea was developed using positive selection based on the Escherichia coli 6-phosphomannose isomerase gene as the selectable marker and mannose as the selective agent. Only transformed cells were capable of utilizing mannose as a carbon source. Cotyledonary node explants from 4-day-old in vitro-germinated seedlings of cultivar Pusa Komal were inoculated with Agrobacterium tumefaciens strain EHA105 carrying the vector pNOV2819. Regenerating transformed shoots were selected on medium supplemented with a combination of 20 g/l mannose and 5 g/l sucrose as carbon source. The transformed shoots were rooted on medium devoid of mannose. Transformation efficiency based on PCR analysis of individual putative transformed shoots was 3.6%. Southern blot analysis on five randomly chosen PCR-positive plants confirmed the integration of the pmi transgene. Qualitative reverse transcription (qRT-PCR) analysis demonstrated the expression of pmi in T0 transgenic plants. Chlorophenol red (CPR) assays confirmed the activity of PMI in transgenic plants, and the gene was transmitted to progeny in a Mendelian fashion. The transformation method presented here for cowpea using mannose selection is efficient and reproducible, and could be used to introduce a desirable gene(s) into cowpea for biotic and abiotic stress tolerance.

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Abbreviations

BAP:

6-Benzylaminopurine

TDZ:

Thidiazuron

pmi :

Phosphomannose isomerase

CPR:

Chlorophenol red

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Acknowledgments

We thank Syngenta, Switzerland, for providing the plasmid pNOV2819 and Prof. K. Veluthambi, MKU, Madurai, India for the Agrobacterium strain. This work was supported by the grain legume improvement grant and program support grant from the Department of Biotechnology, Government of India. Souvika Bakshi and Nand Kishor Roy are grateful to the Council of Scientific and Industrial Research (CSIR) for Senior and Junior Research Fellowships respectively. Authors are also thankful to the editor for useful suggestions that helped in improving the clarity of the manuscript.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, India

    Souvika Bakshi, Bedabrata Saha, Nand Kishor Roy, Sagarika Mishra & Lingaraj Sahoo

  2. Department of Life Sciences and Bioinformatics, Assam University, Silchar, 788011, India

    Sanjib Kumar Panda

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  1. Souvika Bakshi
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  2. Bedabrata Saha
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  3. Nand Kishor Roy
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  4. Sagarika Mishra
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  5. Sanjib Kumar Panda
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  6. Lingaraj Sahoo
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Correspondence to Lingaraj Sahoo.

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Communicated by D. Zaitlin.

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Bakshi, S., Saha, B., Roy, N.K. et al. Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker. Plant Cell Rep 31, 1093–1103 (2012). https://doi.org/10.1007/s00299-012-1230-3

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