Skip to main content
SpringerLink
Log in
SpringerLink
Log in

Screening Stress Tolerance Traits in Arabidopsis Cell Cultures

  • Protocol
  • First Online:
Environmental Responses in Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1398))

  • 3932 Accesses

Abstract

Screening for tolerance traits in plant cell cultures can combine the efficiency of microbial selection and plant genetics. Agrobacterium-mediated transformation can efficiently introduce cDNA library to cell suspension cultures generating population of randomly transformed microcolonies. Transformed cultures can subsequently be screened for tolerance to different stress conditions such as salinity, high osmotic, or oxidative stress conditions. cDNA inserts in tolerant cell lines can be easily identified by PCR amplification and homology search of the determined nucleotide sequences. The described methods have been tested and used to identify regulatory genes controlling salt tolerance in Arabidopsis. As cDNA libraries can be prepared from any plants, natural diversity can be explored by using extremophile plants as gene source.

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
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

References

  1. Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55:307–319

    Article  CAS  PubMed  Google Scholar 

  2. Chinnusamy V et al (2002) Screening for gene regulation mutants by bioluminescence imaging. Sci STKE 2002:1–10

    Google Scholar 

  3. Alonso JM, Ecker JR (2006) Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7:524–536

    Article  CAS  PubMed  Google Scholar 

  4. Koiwa H et al (2006) Identification of plant stress-responsive determinants in Arabidopsis by large-scale forward genetic screens. J Exp Bot 57:1119–1128

    Article  CAS  PubMed  Google Scholar 

  5. Papdi C et al (2010) Genetic screens to identify plant stress genes. Methods Mol Biol 639:121–139

    Article  CAS  PubMed  Google Scholar 

  6. Kushnir S et al (1995) Characterization of Arabidopsis thaliana cDNAs that render yeasts tolerant toward the thiol-oxidizing drug diamide. Proc Natl Acad Sci U S A 92:10580–10584

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Eswaran N et al (2010) Yeast functional screen to identify genetic determinants capable of conferring abiotic stress tolerance in Jatropha curcas. BMC Biotechnol 10:23

    Article  PubMed Central  PubMed  Google Scholar 

  8. Kumar R et al (2012) Functional screening of cDNA library from a salt tolerant rice genotype Pokkali identifies mannose-1-phosphate guanyl transferase gene (OsMPG1) as a key member of salinity stress response. Plant Mol Biol 79:555–568

    Article  CAS  PubMed  Google Scholar 

  9. Shi J et al (1995) Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean. Plant J 8:381–390

    Article  CAS  PubMed  Google Scholar 

  10. Kleinow T et al (2000) Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast. Plant J 23:115–122

    Article  CAS  PubMed  Google Scholar 

  11. McCoy TJ (1987) Characterization of alfalfa (Medicago sativa L.) plants regenerated from selected NaCl tolerant cell lines. Plant Cell Rep 6:417–422

    Article  CAS  PubMed  Google Scholar 

  12. Rout GR et al (2008) Selection of salt tolerant plants of Nicotiana tabacum L. through in vitro and its biochemical characterization. Acta Biol Hung 59:77–92

    Article  CAS  PubMed  Google Scholar 

  13. Janardhan Reddy P, Vaidyanath K (1986) In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.). Theor Appl Genet 71:757–760

    Article  CAS  PubMed  Google Scholar 

  14. Verma D et al (2013) In vitro selection and field responses of somaclonal variant plants of rice cv PR113 for drought tolerance. Plant Signal Behav 8:e23519

    Article  PubMed Central  PubMed  Google Scholar 

  15. Matheka JM et al (2008) In vitro Selection and characterization of drought tolerant somaclones of tropical maize (Zea mays L). Biotechnol Adv 7:641–650

    CAS  Google Scholar 

  16. Pistelli L et al (2012) Novel Prunus rootstock somaclonal variants with divergent ability to tolerate waterlogging. Tree Physiol 32:355–368

    Article  CAS  PubMed  Google Scholar 

  17. Lu S et al (2007) In vitro selection of salinity tolerant variants from triploid bermudagrass (Cynodon transvaalensis x C. dactylon) and their physiological responses to salt and drought stress. Plant Cell Rep 26:1413–1420

    Article  CAS  PubMed  Google Scholar 

  18. Wenzel G, Foroughi-Wehr B (1990) Progeny tests of barley, wheat, and potato regenerated from cell cultures after in vitro selection for disease resistance. Theor Appl Genet 80:359–365

    Article  CAS  PubMed  Google Scholar 

  19. Guenzi AC et al (1992) Genetic analysis of a grass dwarf mutation induced by wheat callus culture. Theor Appl Genet 84:952–957

    CAS  PubMed  Google Scholar 

  20. Kaeppler SM et al (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188

    Article  CAS  PubMed  Google Scholar 

  21. Wang QM, Wang L (2012) An evolutionary view of plant tissue culture: somaclonal variation and selection. Plant Cell Rep 31:1535–1547

    Article  CAS  PubMed  Google Scholar 

  22. Koncz C et al (1992) T-DNA insertional mutagenesis in Arabidopsis. Plant Mol Biol 20:963–976

    Article  CAS  PubMed  Google Scholar 

  23. Azpiroz-Leehan R, Feldmann KA (1997) T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends Genet 13:152–156

    Article  CAS  PubMed  Google Scholar 

  24. Szabados L, Koncz C (2003) Identification of T-DNA insertions in Arabidopsis genes. In: Prade RA, Bohnert HJ (eds) Genomics of plants and fungi. Marcel Dekker Inc., New York, pp 255–277

    Google Scholar 

  25. Rosso MG et al (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53:247–259

    Article  CAS  PubMed  Google Scholar 

  26. Weigel D et al (2000) Activation tagging in Arabidopsis. Plant Physiol 122:1003–1013

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Jeong DH et al (2002) T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol 130:1636–1644

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Nakazawa M et al (2003) Activation tagging, a novel tool to dissect the functions of a gene family. Plant J 34:741–750

    Article  CAS  PubMed  Google Scholar 

  29. Wan S et al (2009) Activation tagging, an efficient tool for functional analysis of the rice genome. Plant Mol Biol 69:69–80

    Article  CAS  PubMed  Google Scholar 

  30. LeClere S, Bartel B (2001) A library of Arabidopsis 35S-cDNA lines for identifying novel mutants. Plant Mol Biol 46:695–703

    Article  CAS  PubMed  Google Scholar 

  31. Ichikawa T et al (2006) The FOX hunting system: an alternative gain-of-function gene hunting technique. Plant J 48:974–985

    Article  CAS  PubMed  Google Scholar 

  32. Nakamura H et al (2007) A genome-wide gain-of function analysis of rice genes using the FOX-hunting system. Plant Mol Biol 65:357–371

    Article  CAS  PubMed  Google Scholar 

  33. Fujita M et al (2007) Identification of stress-tolerance-related transcription-factor genes via mini-scale Full-length cDNA Over-eXpressor (FOX) gene hunting system. Biochem Biophys Res Commun 364:250–257

    Article  CAS  PubMed  Google Scholar 

  34. Papdi C et al (2008) Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system. Plant Physiol 147:528–542

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Perez-Salamo I et al (2014) The heat shock factor A4A confers salt tolerance and is regulated by oxidative stress and the mitogen-activated protein kinases MPK3 and MPK6. Plant Physiol 165:319–334

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Rigo G et al (2012) Transformation using controlled cDNA overexpression system. Methods Mol Biol 913:277–290

    CAS  PubMed  Google Scholar 

  37. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  38. Mathur J et al (1995) A simple method for isolation, liquid culture, transformation and regeneration of Arabidopsis thaliana protoplasts. Plant Cell Rep 14:221–226

    CAS  PubMed  Google Scholar 

  39. Koncz C et al. (1994) Specialized vectors for gene tagging and expression studies. In: Gelvin SB (ed) Plant Molecular Biology Manual, Vol B2. Kluwer Academic Publishers, pp. 53-74

    Google Scholar 

  40. Ozawa K, Komamine A (1989) Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.). Theor Appl Genet 77:205–211

    Article  CAS  PubMed  Google Scholar 

  41. Ahmed KZ, Sagi F (1993) Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.). Plant Cell Rep 12:175–179

    Article  CAS  PubMed  Google Scholar 

  42. Kieran PM et al (1997) Plant cell suspension cultures: some engineering considerations. J Biotechnol 59:39–52

    Article  CAS  PubMed  Google Scholar 

  43. Zhong JJ (2001) Biochemical engineering of the production of plant-specific secondary metabolites by cell suspension cultures. Adv Biochem Eng Biotechnol 72:1–26

    Article  CAS  PubMed  Google Scholar 

  44. KimSW OMJ (2009) Establishment of plant regeneration and cryopreservation system from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis. Methods Mol Biol 547:107–115

    Article  Google Scholar 

  45. Mustafa NR (2011) Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protoc 6:715–742

    Article  CAS  PubMed  Google Scholar 

  46. Moscatiello R et al (2013) Plant cell suspension cultures. Methods Mol Biol 953:77–93

    CAS  PubMed  Google Scholar 

  47. Offringa R, van der Lee F (1995) Isolation and characterization of plant genomic DNA sequences via (inverse) PCR amplification. Methods Mol Biol 49:181–195

    CAS  PubMed  Google Scholar 

  48. Springer NM (2010). Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB. Cold Spring Harb Protoc 2010: pdb prot5515

    Google Scholar 

  49. Berendzen K et al (2005) A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta. Plant Methods 1:4

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgements

This research was supported by Hungarian Scientific Research Fund (OTKA Grants no. K81765, NN110962), MTA-CNR collaboration grant (2013–2015), and IPA project no. HUSRB/1002/214/036. Authors acknowledge the assistance of Annamária Király.

Author information

Authors and Affiliations

  1. Institute of Plant Biology, Biological Research Centre, Temesvari krt. 62, 6726, Szeged, Hungary

    Imma Pérez-Salamó, Bogáta Boros & László Szabados

Authors
  1. Imma Pérez-Salamó
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bogáta Boros
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. László Szabados
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to László Szabados .

Editor information

Editors and Affiliations

  1. Instituto Gulbenkian de Ciência, Oeiras, Portugal

    Paula Duque

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Pérez-Salamó, I., Boros, B., Szabados, L. (2016). Screening Stress Tolerance Traits in Arabidopsis Cell Cultures. In: Duque, P. (eds) Environmental Responses in Plants. Methods in Molecular Biology, vol 1398. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3356-3_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3356-3_19

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3354-9

  • Online ISBN: 978-1-4939-3356-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation