Abstract
Agrobacterium rhizogenes has the ability to transform plant cells by transferring the T-DNA from the Ri plasmid to the plant cell genome. These infected plant cells divide and organize the formation of adventitious roots, called hairy roots. When the A. rhizogenes is additionally transformed with a binary vector, the cells infected can indeed be transformed with this second T-DNA producing transgenic hairy roots. In this chapter, we present the protocol to produce transgenic hairy roots from in vitro potato (Solanum tuberosum) plants injected with transformed A. rhizogenes, generating plants with a wild-type shoot and a transgenic root system. Specifically, we detail the procedure to obtain in vitro-cultured hairy roots with a downregulated gene of interest, by using a Gateway-based binary vector able to produce a RNA hairpin triggering the RNA interference mechanism (hpRNAi). We also present the protocol to analyze the downregulation of the target gene in hairy roots by means of reverse-transcription reaction followed by real-time PCR (qPCR).
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Acknowledgments
This work was supported by the Ministerio de Economía y Competitividad and Ministerio de Ciencia e Innovación and European Regional Development Fund (AGL2015-67495-C2-1-R and PID2019-110330GB-C21), and the University of Girona (MPCUdG2016/078 and PhD contracts to SF and CS). The authors are grateful to Dr. Inge Broer (Institute for Land Use, University of Rostock, Germany) for providing the A. rhizogenes strain, and Dr. Marçal Soler and Dr. Anna Plasencia for the help and support received in initial stages of the A. rhizogenes transformation experiments (Toulouse III Paul Sabatier University—CNRS, Plant Research Laboratory (LRSV), France). The authors thank Ferran Fontdecaba and Jennifer López who assisted with some of the experiments while they were doing their final degree projects.
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Fernández-Piñán, S., Sànchez-Guirado, C., Figueras, M., Serra, O. (2021). Gene Downregulation in Potato Roots Using Agrobacterium rhizogenes-Mediated Transformation. In: Dobnik, D., Gruden, K., Ramšak, Ž., Coll, A. (eds) Solanum tuberosum. Methods in Molecular Biology, vol 2354. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1609-3_17
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DOI: https://doi.org/10.1007/978-1-0716-1609-3_17
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