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Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.)

  • Genetic Transformation
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Abstract

A synthetic gene encoding the antimicrobial peptide magainin has been designed, cloned, and engineered for regulation by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (nos) terminator. The plant expression cassette was introduced into the vector pSB11-bar (with the glyphosate [Basta®] resistance gene, bar), and the recombinant plasmid was mobilized into Agrobacterium tumefaciens strain LBA4404 for the generation of a super-binary vector pSB111-bar-mag. Magainins, positively charged amphipathic antimicrobial peptides of 21–26 amino acid residues, are potential candidates for the development of disease resistant transgenic plants. Six-wk-old pearl millet (Pennisetum glaucum [L.] R. Br.) calli and A. tumefaciens harboring pSB111-bar-mag were cocultivated in a medium supplemented with 400 μM acetosyringone and 3.3 mM l-cysteine. Out of 3,000 infected calli subjected to selection on phosphinothricin medium, 82 calli showed sectors of healthy growth, resulting in a transformation frequency of 2.73%. Among 13 Basta-tolerant putative transformed plants, eight were fertile and their transgenic nature and expression of the transgene was characterized by Southern and Northern blot analyses, respectively. Subsequent T1 progenies co-segregated for bar and magainin genes in a 3:1 ratio. Bioassays that challenged the eight transgenic T1 plant progenies against three highly virulent strains of Sclerospora graminicola, viz., Sg 384, Sg 445, and Sg 492 failed to show resistance. The failure of synthetic magainin gene to confer resistance against downy mildew in pearl millet may be attributed to the complexity of the cell wall and cell membrane of the pathogen.

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Acknowledgments

The authors acknowledge financial support from the Department of Biotechnology, Government of India, New Delhi. The senior author acknowledges the Council of Scientific and Industrial Research for awarding the research fellowship. Critical reading of the manuscript by Prof. T. Papi Reddy and Prof. C. C. Giri is gratefully acknowledged. We also thank Mr. M. Suresh Reddy for his support. Thanks are due to Dr. R. P Thakur, Dr. Rajan Sharma, and Sri. V. Pandu Ranga Rao of ICRISAT, Hyderabad for their expert help in carrying out bioassays.

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  1. Centre for Plant Molecular Biology, Osmania University, Hyderabad, 500 007, India

    R. Ramadevi, K. V. Rao & V. D. Reddy

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  1. R. Ramadevi
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Ramadevi, R., Rao, K.V. & Reddy, V.D. Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.). In Vitro Cell.Dev.Biol.-Plant 50, 392–400 (2014). https://doi.org/10.1007/s11627-013-9592-y

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