Skip to main content
SpringerLink
Log in

Optimization of Hairy Root Transformation for the Functional Genomics in Chickpea: A Platform for Nodule Developmental Studies

  • Protocol
  • First Online:
Legume Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2107))

Abstract

Chickpea is a major protein source in low socio-economic classes and cultivated in marginal soil without fertilizer or irrigation. As a result of its root nodule formation capacity chickpea can directly use atmospheric nitrogen. Chickpea is recalcitrant to stable transformation, particularly root regeneration efficiency of chickpea is low. The composite plant-based system with a non-transformed shoot and transformed root is particularly important for root biologist and this approach has already been used successfully for root nodule symbiosis, arbuscular mycorrhizal symbiosis, and other root-related studies. Use of fluorescent marker-based approach can accurately identify the transformed root from its non-transgenic counterpart. RNAi-based gene knockout, overexpression of genes, promoter GUS analysis to understand tissue specific expression and localization of protein can be achieved using the hairy root-based system. We have already published a hairy root-based transformation and composite plant regeneration protocol of chickpea. Here we are describing the recent modification that we have made to increase the transformation frequency and nodule morphology. Further, we have developed a pouch based artificial system, large number of plants can be scored for its nodule developmental phenotype, by using this system.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Jain M, Misra G, Patel RK, Priya P, Jhanwar S, Khan AW et al (2013) A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.). Plant J 74:715–729

    Article  CAS  Google Scholar 

  2. Mandal D, Sinharoy S (2019) A toolbox for nodule development studies in chickpea: a hairy-root transformation protocol and an efficient laboratory strain of Mesorhizobium sp. Mol Plant-Microbe Interact 32:367–378

    Article  Google Scholar 

  3. Montiel J, Downie JA, Farkas A, Bihari P, Herczeg R, Balint B et al (2017) Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides. Proc Natl Acad Sci U S A 114:5041–5046

    Article  CAS  Google Scholar 

  4. Varshney RK, Thudi M, Roorkiwal M, He W, Upadhyaya HD, Yang W et al (2019) Resequencing of 429 chickpea accessions from 45 countries provides insights into genome diversity, domestication and agronomic traits. Nat Genet 51:857–864

    Article  CAS  Google Scholar 

  5. Pandey A, Chakraborty S, Chakraborty N (2018) Nuclear proteome: isolation of intact nuclei, extraction of nuclear proteins, and 2-DE analysis. Methods Mol Biol 1696:41–55

    Article  CAS  Google Scholar 

  6. Ramalingam A, Kudapa H, Pazhamala LT, Weckwerth W, Varshney RK (2015) Proteomics and metabolomics: two emerging areas for legume improvement. Front Plant Sci 6:1116

    PubMed  PubMed Central  Google Scholar 

  7. Varshney RK, Song C, Saxena RK, Azam S, Yu S, Sharpe AG et al (2013) Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nat Biotechnol 31:240–246

    Article  CAS  Google Scholar 

  8. Georgiev MI, Agostini E, Ludwig-Muller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol 30:528–537

    Article  CAS  Google Scholar 

  9. Stougaard J, Petersen TE, Marcker KA (1987) Expression of a complete soybean leghemoglobin gene in root nodules of transgenic Lotus corniculatus. Proc Natl Acad Sci U S A 84:5754–5757

    Article  CAS  Google Scholar 

  10. Limpens E, Ramos J, Franken C, Raz V, Compaan B, Franssen H et al (2004) RNA interference in agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. J Exp Bot 55:983–992

    Article  CAS  Google Scholar 

  11. Sinharoy S, Pislariu CI, Udvardi MK (2015) A high-throughput RNA interference (RNAi)-based approach using hairy roots for the study of plant-rhizobia interactions. Methods Mol Biol 1287:159–178

    Article  CAS  Google Scholar 

  12. Kumagai H, Kouchi H (2003) Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Mol Plant-Microbe Interact 16:663–668

    Article  CAS  Google Scholar 

  13. Kereszt A, Li D, Indrasumunar A, Nguyen CD, Nontachaiyapoom S, Kinkema M et al (2007) Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat Protoc 2:948–952

    Article  CAS  Google Scholar 

  14. Boisson-Dernier A, Chabaud M, Garcia F, Becard 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-Microbe Interact 14:695–700

    Article  CAS  Google Scholar 

  15. Diaz CL, Spaink HP, Kijne JW (2000) Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene. Mol Plant-Microbe Interact 13:268–276

    Article  CAS  Google Scholar 

  16. Stiller J, Martirani L, Tuppale S, Chian R-J, Chiurazzi M, Gresshoff PM (1997) High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus. J Exp Bot 48:1357–1365

    Article  CAS  Google Scholar 

  17. Diouf D, Gherbi H, Prin Y, Franche C, Duhoux E, Bogusz D (1995) Hairy root nodulation of Casuarina glauca: a system for the study of symbiotic gene expression in an actinorhizal tree. Mol Plant-Microbe Interact 8:532–537

    Article  CAS  Google Scholar 

  18. Quandt HJ, Puhler A, Broer I (1993) Transgenic root nodules of Vicia hirsuta. A fast and efficient system for the study of gene expression in indeterminate-type nodules. Mol Plant-Microbe Interact 6:699–703

    Article  Google Scholar 

  19. Lee NG, Stein B, Suzuki H, Verma DP (1993) Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant. Plant J 3:599–606

    Article  CAS  Google Scholar 

  20. Cheon CI, Lee NG, Siddique ABM, Bal AK, Verma DPS (1993) Roles of plant homologs of Rabi p and Rab7p in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de novo during root nodule symbiosis. EMBO J 12:4125–4135

    Article  CAS  Google Scholar 

  21. Díaz C, Melchers LS, Hooykaas PJJ, Lugtenberg BJJ, Kijne JW (1989) Root lectin as a determinant of host-plant specificity in the rhizobium-legume symbiosis. Nature 338:579–558

    Article  Google Scholar 

  22. Mellor KE, Hoffman AM, Timko MP (2012) Use of ex vitro composite plants to study the interaction of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides. Plant Methods 8:22

    Article  CAS  Google Scholar 

  23. Imanishi L, Vayssieres A, Franche C, Bogusz D, Wall L, Svistoonoff S (2011) Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection. Mol Plant-Microbe Interact 24:1317–1324

    Article  CAS  Google Scholar 

  24. Clemow SR, Clairmont L, Madsen LH, Guinel FC (2011) Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea. Plant Methods 7:46

    Article  Google Scholar 

  25. Bonaldi K, Gherbi H, Franche C, Bastien G, Fardoux J, Barker D et al (2010) The nod factor-independent symbiotic signaling pathway: development of agrobacterium rhizogenes-mediated transformation for the legume Aeschynomene indica. Mol Plant-Microbe Interact 23:1537–1544

    Article  CAS  Google Scholar 

  26. Sinharoy S, Saha S, Chaudhury SR, Dasgupta M (2009) Transformed hairy roots of Arachis hypogea: a tool for studying root nodule symbiosis in a non-infection thread legume of the Aeschynomeneae tribe. Mol Plant-Microbe Interact 22:132–142

    Article  CAS  Google Scholar 

  27. Estrada-Navarrete G, Alvarado-Affantranger X, Olivares JE, Diaz-Camino C, Santana O, Murillo E et al (2006) Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics. Mol Plant-Microbe Interact 19:1385–1393

    Article  CAS  Google Scholar 

  28. Van-de-Velde W, Mergeay J, Holsters M, Goormachtig S (2003) Agrobacterium rhizogenes-mediated transformation of Sesbania rostrata. Plant Sci 165:1281–1288

    Article  CAS  Google Scholar 

  29. Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K et al (2014) Hairy root transformation using agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166:455–469

    Article  CAS  Google Scholar 

  30. Hofgen R, Willmitzer L (1988) Storage of competent cells for agrobacterium transformation. Nucleic Acids Res 16:9877

    Article  CAS  Google Scholar 

  31. Auriac MC, Timmers AC (2007) Nodulation studies in the model legume Medicago truncatula: advantages of using the constitutive EF1alpha promoter and limitations in detecting fluorescent reporter proteins in nodule tissues. Mol Plant-Microbe Interact 20:1040–1047

    Article  CAS  Google Scholar 

  32. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  CAS  Google Scholar 

  33. Ivanov S, Harrison MJ (2014) A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula. Plant J 80:1151–1163

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank J. Terpolilli, Murdoch University, Australia for providing M. ciceri CC1192; Senthil-Kumar Muthappa, NIPGR, New Delhi for providing chickpea seeds (BGD 112, PUSA 372, GNG 1958, GNG 1488, GNG 1581, RSG 931); Michael Udvardi, Nobel Research Institute, Oklahoma, USA for A. rhizogenes ARqua1 strain; Maria J. Harrison, Boyce Thompson Institute for Plant Research Ithaca, USA for providing pAtUb driven subcellular marker construct; M. DasGupta, Department of Biochemistry, University of Calcutta, for providing pCAMBIA2301 vector; NIPGR for their confocal facilities; and CIF-NIPGR; NIPGR-DELCON for their support. This work is supported by core research grant from National Institute of Plant Genome Research, Ramalingwaswami Re-entry grant, DBT (BT/RLF/Re-entry/41/2013).

Author information

Authors and Affiliations

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

    Drishti Mandal, Deevita Srivastava & Senjuti Sinharoy

Authors
  1. Drishti Mandal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deevita Srivastava
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Senjuti Sinharoy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Senjuti Sinharoy .

Editor information

Editors and Affiliations

  1. School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India

    Mukesh Jain

  2. Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, Gautam Buddha Nagar, Uttar Pradesh, India

    Rohini Garg

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Mandal, D., Srivastava, D., Sinharoy, S. (2020). Optimization of Hairy Root Transformation for the Functional Genomics in Chickpea: A Platform for Nodule Developmental Studies. In: Jain, M., Garg, R. (eds) Legume Genomics. Methods in Molecular Biology, vol 2107. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0235-5_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0235-5_18

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0234-8

  • Online ISBN: 978-1-0716-0235-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation