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High-efficient Agrobacterium-mediated in planta transformation in black gram (Vigna mungo (L.) Hepper)

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Abstract

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA® (4 mg l−1) containing MS medium followed by BASTA® foliar spray on 15-day-old black gram plants (35 mg l−1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.

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Abbreviations

bar :

Bialaphos resistant gene

CaMV35S:

Cauliflower Mosaic Virus 35S

GUS:

β-Glucuronidase

MIC:

Minimum inhibitory concentration

MS:

Murashige and Skoog medium

PPT:

Phosphinothricin

YEP:

Yeast extract and peptone

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Acknowledgments

Prof. A. Ganapathi is thankful to the University Grants Commission (UGC), Government of India, for the award of UGC–Emeritus Fellowship scheme (No.F.6-6/2014-15/EMERITUS-2014-15-OBC-3703/(SA-II)). The first author is thankful to University Grants Commission (UGC), Government of India for the award of Project Fellow. V. Vasudevan acknowledges BharathidasanUniversity, Tiruchirappalli, for the award of the University Research Fellowship. Arunachalam Chinnathambi and Sulaiman Ali Alharbi would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research group no. (RG-1435-081).

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Authors and Affiliations

  1. Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India

    Gnanajothi Kapildev, Venkatachalam Vasudevan & Andy Ganapathi

  2. Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Kingdom of Saudi Arabia

    Arunachalam Chinnathambi & Sulaiman Ali Alharbi

  3. Genetics and Genomics Laboratory, Chungnam National University, Daejeon, South Korea

    Ganeshan Sivanandhan

  4. International Institute of Tropical Agriculture (IITA), Nairobi, Kenya

    Manoharan Rajesh

  5. Molecular Genetics and Epigenomics Lab, CIBER, Department of Agriculture and Natural Research, Delaware State University, Dover, DE, 19901, USA

    Subramanian Mayavan

  6. Department of Horticultural Science, Kyungpook National University, Daegu, 702-701, Republic of Korea

    Muthukrishnan Arun

  7. Centre for Nanoscience and Nanotechnology, University of Madras, Chennai, 600005, India

    Murugaraj Jeyaraj

  8. Periyar E.V.R College, Tiruchirappalli, 620 023, Tamil Nadu, India

    Natesan Selvaraj

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  1. Gnanajothi Kapildev
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  2. Arunachalam Chinnathambi
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  3. Ganeshan Sivanandhan
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  4. Manoharan Rajesh
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  5. Venkatachalam Vasudevan
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  6. Subramanian Mayavan
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  7. Muthukrishnan Arun
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  8. Murugaraj Jeyaraj
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  9. Sulaiman Ali Alharbi
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  10. Natesan Selvaraj
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  11. Andy Ganapathi
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Correspondence to Andy Ganapathi.

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Kapildev, G., Chinnathambi, A., Sivanandhan, G. et al. High-efficient Agrobacterium-mediated in planta transformation in black gram (Vigna mungo (L.) Hepper). Acta Physiol Plant 38, 205 (2016). https://doi.org/10.1007/s11738-016-2215-6

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  • DOI: https://doi.org/10.1007/s11738-016-2215-6

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