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An Improvised Hairy Root Transformation Method for Efficient Gene Silencing in Roots and Nodules of Arachis hypogaea

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Plant Gene Silencing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2408))

Abstract

Peanut (Arachis hypogaea) is a major oilseed crop and is widely cultivated in tropical and subtropical climate zone worldwide. Peanut belongs to the Papilionoid family with an atypical nodule developmental program. In particular, rhizobia enter through developmental cracks and lead to the formation of aeschynomenoid subtype determinate nodules. Peanut nodules are efficient nitrogen-fixers and form swollen bacteroid containing symbiosomes. The allotetraploid genome and recalcitrance to stable transformation used to be the major bottleneck for peanut biologists. Recent genome sequencing of peanut cultivar Tifrunner has opened up a huge opportunity for molecular research. A composite plant contains transformed roots with a non-transformed shoot. The composite plant-based approach has already proven to be a tool of choice for high throughput studies in root biology. The available protocols failed to generate efficient hairy root transformation in the genome sequenced cultivar Tifrunner. Here we describe an efficient hairy root transformation and composite plant generation protocol for the peanut cultivar Tifrunner. Our protocol generated ~92% plant regeneration efficiency with between 21.8% and 58.6% co-transformed root regeneration. We also show that this protocol can be efficiently used for protein localization, promoter GUS analysis, monitoring hormone response, and RNAi mediated knockdown of the genes using genome sequenced cultivar Tifrunner.

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Acknowledgments

We thank Janila Pasupuleti and Vania C. R. Azevedo, ICRISAT, India for providing A. hypogaea cultivar Tifrunner seeds, Michael Udvardi, Nobel Research Institute, Oklahoma, USA for A. rhizogenes ARqua1 strain, M. DasGupta, Department of Biochemistry, University of Calcutta, for providing A. rhizogenes R1000 strain, Fernando Ibáñez, Departamento de Ciencias Naturales, Universidad Nacional de Río Cuarto, Argentina, for Bradyrhizobium SEMIA 6144, S. Takuya, University of Tsukuba, Tsukuba, Japan, for providing the DR5-GFP-NLS construct, NIPGR for their confocal facilities; CIF-NIPGR; NIPGR-DELCON for their support. This work is supported by core research grant from National Institute of Plant Genome Research, Ramalingaswami Re-entry grant, DBT (BT/RLF/Re-entry/41/2013) and SERB ECR grant (ECR/2018/001215) and Mr. Bikash Raul’s fellowship is supported by CSIR (File No. 09/803(0141)/2017-EMR-I).

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Authors and Affiliations

  1. National Institute of Plant Genome Research, New Delhi, India

    Bikash Raul & Senjuti Sinharoy

Authors
  1. Bikash Raul
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  2. Senjuti Sinharoy
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Correspondence to Senjuti Sinharoy .

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Editors and Affiliations

  1. Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, OK, USA

    Kirankumar S. Mysore

  2. National Institute of Plant Genome Research, New Delhi, India

    Muthappa Senthil-Kumar

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Raul, B., Sinharoy, S. (2022). An Improvised Hairy Root Transformation Method for Efficient Gene Silencing in Roots and Nodules of Arachis hypogaea. In: Mysore, K.S., Senthil-Kumar, M. (eds) Plant Gene Silencing. Methods in Molecular Biology, vol 2408. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1875-2_20

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  • DOI: https://doi.org/10.1007/978-1-0716-1875-2_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1874-5

  • Online ISBN: 978-1-0716-1875-2

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