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Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration

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Abstract

An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens resulted in highest transient GUS expression efficiency (93% explants expressing GUS at regenerating sites). After 3 days of co-cultivation, the explants were cultured in 150 mg/l kanamycin-containing selection medium and putative transformed plants were recovered. The presence, integration and expression of nptII and cry1Ac genes in T0 transgenic plants were confirmed by polymerase chain reaction (PCR), genomic Southern and qualitative reverse transcription (RT)-PCR analysis. Western blot hybridization and enzyme-linked immunosorbent assay (ELISA) detected and demonstrated the accumulation of Cry1Ac protein in transgenic plants. The cry1Ac gene transmitted in a Mendelian fashion. The stable transformation efficiency increased by 88.4% using both sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration than simple Agrobacterium-mediated transformation in cowpea.

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Abbreviations

BAP:

6-Benzylaminopurine

TDZ:

Thidiazuron

GUS:

β-Glucuronidase

nptII :

Neomycin phosphotransferase II

SAAT:

Sonication-assisted Agrobacterium-mediated transformation

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Acknowledgments

We thank Prof. I. Altossar, University of Ottawa, Canada for providing p1AcPRD, Prof. K. Veluthambi, MKU, Madurai, India for Agrobacterium strain and Center for Application of Molecular Biology to International Agriculture (CAMBIA), Australia for pCAMBIA2301. The research was supported by the grants from Department of Biotechnology, Government of India. Souvika Bakshi is grateful to Council of Scientific and Industrial Research (CSIR) for Senior Research Fellowship, and Ayan Sadhukhan and Sagarika Mishra to Indian Institute of Technology Guwahati for doctoral fellowship.

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

    Souvika Bakshi, Ayan Sadhukhan, Sagarika Mishra & Lingaraj Sahoo

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  1. Souvika Bakshi
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  2. Ayan Sadhukhan
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Correspondence to Lingaraj Sahoo.

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Communicated by K. Toriyama.

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Bakshi, S., Sadhukhan, A., Mishra, S. et al. Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration. Plant Cell Rep 30, 2281–2292 (2011). https://doi.org/10.1007/s00299-011-1133-8

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