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Agrobacterium-mediated transformation of chitinase gene from the actinorhizal tree Casuarina equisetifolia in Nicotiana tabacum

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Abstract

Genetic transformation of plants offers the possibility of testing hypotheses on the function of individual genes and enables exploration of transgenes for targeted trait improvement. Cloning of the full-length class I chitinase from the Casuarina equisetifolia (CeChi1) was earlier reported by our team. In the present study, tobacco was used as a model system to functionally evaluate the potential of CeChi1 driven by ubiquitin promoter. The pUH-CeChi1 construct was introduced into tobacco by Agrobacterium-mediated transformation and the putative transformants were confirmed for stable gene integration, transgene expression and recombinant protein production using PCR, RT-qPCR, antifungal assays and in planta analysis. The in vitro antifungal bioassay using the total proteins from leaves of transformed plantlets revealed the characteristic lysis of hyphal tips of pathogenic fungi including Trichosporium vesiculosum, Fusarium oxysporum and Rhizoctonia solani. The in planta bioassay of transformed tobacco showed reduced disease symptoms when compared to untransformed wild plants. The study revealed that the class I chitinase isolated from C. equisetifolia can act as a potential gene resource in future transformation programs for incorporating disease tolerance.

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Abbreviations

BAP:

benzylaminopurine

hph :

hygromycin phosphotransferase

IBA:

indole butyric acid

MS:

Murashige and Skoog

NAA:

naphthalene acetic acid

PMSF:

phenylmethyl sulfonyl fluoride

PR:

pathogenesis-related

RT-qPCR:

quantitative real-time PCR

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Acknowledgements

The authors acknowledge Dr. R. Viswanathan, Principal Scientist and Head (Plant Protection), Sugarcane Breeding Institute, Coimbatore, India, for providing facilities to conduct RT-qPCR and Dr. V.Mohan, Scientist, Division of Forest Protection, Institute of Forest Genetics and Tree Breeding, Coimbatore, India, for providing the fungal strains. The authors are grateful to Dr. K. Ulaganathan, Professor, Centre for Plant Molecular Biology, Osmania University, Hyderabad, Andhra Pradesh, India, for providing the seeds of Nicotiana tabacum. The authors also thank Dr. D. Sudhakar, Professor, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore, India, for providing pUH vector for transformation studies. The authors acknowledge Dr. V. Sivakumar, Institute of Forest Genetics and Tree Breeding, Coimbatore, India, for conducting the digital analysis of fungal hyphae. Finally, the authors thank the Department of Biotechnology, Ministry of Science and Technology, Government of India, for the financial support.

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  1. Division of Plant Biotechnology, Institute of Forest Genetics and Tree Breeding, P.B. No. 1061, R.S. Puram P.O., Coimbatore, 641002, India

    Radha Veluthakkal & Modhumita Ghosh Dasgupta

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  1. Radha Veluthakkal
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  2. Modhumita Ghosh Dasgupta
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Correspondence to Modhumita Ghosh Dasgupta.

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Veluthakkal, R., Dasgupta, M.G. Agrobacterium-mediated transformation of chitinase gene from the actinorhizal tree Casuarina equisetifolia in Nicotiana tabacum. Biologia 70, 905–914 (2015). https://doi.org/10.1515/biolog-2015-0114

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