Abstract
Legumes, the second most important crop to humans possess unique ability to fix atmospheric nitrogen, making them one of the major contributors to sustainable agriculture. In legumes, molecular characterization of genes by stable transformation is difficult due to their recalcitrant nature to the whole-plant regeneration in desired varieties. The Agrobacterium rhizogenes-mediated generation of transgenic hairy roots or composite plants may facilitate a rapid and convenient alternative to study nodule biology. Functional analysis of genes involved in legume nodulation has been proven as successful for model legumes, viz., Medicago truncatula and Lotus japonicus, using transgenic hairy roots. Besides sharing some common features of nodulation among legumes, the symbiotic signaling is a complex and specific process. Here, we describe an improved protocol for hairy root transformation of a legume crop chickpea (Cicer arietinum L.) and the method to study nodulation to uncover the signaling components. Using the described protocol, transgenic hairy roots were generated in chickpea and selected based on the red fluorescence protein (RFP) microscopy. This protocol can be extended to other underutilized legumes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Atlas RM (1997) In: Parks LC (ed) Handbook of microbial media, 2nd edn. CRC Press Inc, Boca Raton, 401 p
Boisson A, Chabaud M, Garcia F, Bécard G, Rosenberg C, Barker DG (2001) Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant Microb Interact 14:695–700
BrĂgido C, Robledo M, MenĂ©ndez E, Mateos PF, Oliveira S (2012) A ClpB chaperone knockout mutant of Mesorhizobium ciceri shows a delay in the root nodulation of chickpea plants. Mol Plant Microbe Interact 25(12):1594–1604
Broughton WJ, Dilworth MJ (1971) Control of leghemoglobin in snake beans. Biochem J 125:1075–1080
Chakrabarty R, Banerjee R, Chung MS, Farman M, Citovsky V, Hogenhout SA, Tzfira T, Goodin M (2007) pSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana–virus interactions. Mol Plant Microbe Interact 20:740–750
Häkkinen ST, Oksman-Caldentey K (2018) Progress and prospects of hairy root research. In: Srivastava V, Mehrotra S, Mishra S (eds) Hairy roots: an effective tool of plant biotechnology. Springer, Singapore, pp 3–9
Healey A, Furtado A, Cooper T, Henry R (2014) Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods 10:21
Jha U (2018) Current advances in chickpea genomics: applications and future perspectives. Plant Cell Rep 37(7):947–965
Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340
Xu Y, Liu F, Han G, Wang W, Zhu S, Li X (2018) Improvement of Lotus japonicus hairy root induction and development of a mycorrhizal symbiosis system. Appl Plant Sci 6(4):e1141
Acknowledgments
The authors thank Dr. Jean-Michel Ané, University of Wisconsin, Madison, USA for providing the Agrobacterium rhizogenes ARqua1strain; Dr. Saikat Bhattacharjee, Regional Centre for Biotechnology (RCB), India for providing pSITE-4NB destination vector; and Dr. Solange Oliveira, Universidade de Evora, Evora, Portugal for providing the Mesorhizobium ciceri LMS-1. This work is partially supported by core grant of National Institute of Plant Genome Research and DBT-CPCFG project grant (BT/AGR/CG-Phase II/01/2014). JS acknowledge Council of Scientific and Industrial Research, India for a fellowship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Nature Singapore Pte Ltd.
About this chapter
Cite this chapter
Singh, J., Kumar, K., Verma, P.K. (2020). Functional Characterization of Genes Involved in Legume Nodulation Using Hairy Root Cultures. In: Srivastava, V., Mehrotra, S., Mishra, S. (eds) Hairy Root Cultures Based Applications. Rhizosphere Biology. Springer, Singapore. https://doi.org/10.1007/978-981-15-4055-4_14
Download citation
DOI: https://doi.org/10.1007/978-981-15-4055-4_14
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-15-4054-7
Online ISBN: 978-981-15-4055-4
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)