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Screening Stress Tolerance Traits in Arabidopsis Cell Cultures

  • Imma Pérez-Salamó
  • Bogáta Boros
  • László SzabadosEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)

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.

Key words

In vitro selection Agrobacterium Transformation cDNA library Gene isolation 

Notes

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.

References

  1. 1.
    Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55:307–319CrossRefPubMedGoogle Scholar
  2. 2.
    Chinnusamy V et al (2002) Screening for gene regulation mutants by bioluminescence imaging. Sci STKE 2002:1–10Google Scholar
  3. 3.
    Alonso JM, Ecker JR (2006) Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7:524–536CrossRefPubMedGoogle Scholar
  4. 4.
    Koiwa H et al (2006) Identification of plant stress-responsive determinants in Arabidopsis by large-scale forward genetic screens. J Exp Bot 57:1119–1128CrossRefPubMedGoogle Scholar
  5. 5.
    Papdi C et al (2010) Genetic screens to identify plant stress genes. Methods Mol Biol 639:121–139CrossRefPubMedGoogle Scholar
  6. 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 92:10580–10584PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Eswaran N et al (2010) Yeast functional screen to identify genetic determinants capable of conferring abiotic stress tolerance in Jatropha curcas. BMC Biotechnol 10:23PubMedCentralCrossRefPubMedGoogle Scholar
  8. 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 79:555–568CrossRefPubMedGoogle Scholar
  9. 9.
    Shi J et al (1995) Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean. Plant J 8:381–390CrossRefPubMedGoogle Scholar
  10. 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 23:115–122CrossRefPubMedGoogle Scholar
  11. 11.
    McCoy TJ (1987) Characterization of alfalfa (Medicago sativa L.) plants regenerated from selected NaCl tolerant cell lines. Plant Cell Rep 6:417–422CrossRefPubMedGoogle Scholar
  12. 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 59:77–92CrossRefPubMedGoogle Scholar
  13. 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 71:757–760CrossRefPubMedGoogle Scholar
  14. 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:e23519PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Matheka JM et al (2008) In vitro Selection and characterization of drought tolerant somaclones of tropical maize (Zea mays L). Biotechnol Adv 7:641–650Google Scholar
  16. 16.
    Pistelli L et al (2012) Novel Prunus rootstock somaclonal variants with divergent ability to tolerate waterlogging. Tree Physiol 32:355–368CrossRefPubMedGoogle Scholar
  17. 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 26:1413–1420CrossRefPubMedGoogle Scholar
  18. 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 80:359–365CrossRefPubMedGoogle Scholar
  19. 19.
    Guenzi AC et al (1992) Genetic analysis of a grass dwarf mutation induced by wheat callus culture. Theor Appl Genet 84:952–957PubMedGoogle Scholar
  20. 20.
    Kaeppler SM et al (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188CrossRefPubMedGoogle Scholar
  21. 21.
    Wang QM, Wang L (2012) An evolutionary view of plant tissue culture: somaclonal variation and selection. Plant Cell Rep 31:1535–1547CrossRefPubMedGoogle Scholar
  22. 22.
    Koncz C et al (1992) T-DNA insertional mutagenesis in Arabidopsis. Plant Mol Biol 20:963–976CrossRefPubMedGoogle Scholar
  23. 23.
    Azpiroz-Leehan R, Feldmann KA (1997) T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends Genet 13:152–156CrossRefPubMedGoogle Scholar
  24. 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 255–277Google Scholar
  25. 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 53:247–259CrossRefPubMedGoogle Scholar
  26. 26.
    Weigel D et al (2000) Activation tagging in Arabidopsis. Plant Physiol 122:1003–1013PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Jeong DH et al (2002) T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol 130:1636–1644PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Nakazawa M et al (2003) Activation tagging, a novel tool to dissect the functions of a gene family. Plant J 34:741–750CrossRefPubMedGoogle Scholar
  29. 29.
    Wan S et al (2009) Activation tagging, an efficient tool for functional analysis of the rice genome. Plant Mol Biol 69:69–80CrossRefPubMedGoogle Scholar
  30. 30.
    LeClere S, Bartel B (2001) A library of Arabidopsis 35S-cDNA lines for identifying novel mutants. Plant Mol Biol 46:695–703CrossRefPubMedGoogle Scholar
  31. 31.
    Ichikawa T et al (2006) The FOX hunting system: an alternative gain-of-function gene hunting technique. Plant J 48:974–985CrossRefPubMedGoogle Scholar
  32. 32.
    Nakamura H et al (2007) A genome-wide gain-of function analysis of rice genes using the FOX-hunting system. Plant Mol Biol 65:357–371CrossRefPubMedGoogle Scholar
  33. 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 364:250–257CrossRefPubMedGoogle Scholar
  34. 34.
    Papdi C et al (2008) Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system. Plant Physiol 147:528–542PubMedCentralCrossRefPubMedGoogle Scholar
  35. 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 165:319–334PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Rigo G et al (2012) Transformation using controlled cDNA overexpression system. Methods Mol Biol 913:277–290PubMedGoogle Scholar
  37. 37.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  38. 38.
    Mathur J et al (1995) A simple method for isolation, liquid culture, transformation and regeneration of Arabidopsis thaliana protoplasts. Plant Cell Rep 14:221–226PubMedGoogle Scholar
  39. 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. 53-74Google Scholar
  40. 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 77:205–211CrossRefPubMedGoogle Scholar
  41. 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 12:175–179CrossRefPubMedGoogle Scholar
  42. 42.
    Kieran PM et al (1997) Plant cell suspension cultures: some engineering considerations. J Biotechnol 59:39–52CrossRefPubMedGoogle Scholar
  43. 43.
    Zhong JJ (2001) Biochemical engineering of the production of plant-specific secondary metabolites by cell suspension cultures. Adv Biochem Eng Biotechnol 72:1–26CrossRefPubMedGoogle Scholar
  44. 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 547:107–115CrossRefGoogle Scholar
  45. 45.
    Mustafa NR (2011) Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protoc 6:715–742CrossRefPubMedGoogle Scholar
  46. 46.
    Moscatiello R et al (2013) Plant cell suspension cultures. Methods Mol Biol 953:77–93PubMedGoogle Scholar
  47. 47.
    Offringa R, van der Lee F (1995) Isolation and characterization of plant genomic DNA sequences via (inverse) PCR amplification. Methods Mol Biol 49:181–195PubMedGoogle Scholar
  48. 48.
    Springer NM (2010). Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB. Cold Spring Harb Protoc 2010: pdb prot5515Google Scholar
  49. 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 1:4PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Imma Pérez-Salamó
    • 1
  • Bogáta Boros
    • 1
  • László Szabados
    • 1
    Email author
  1. 1.Institute of Plant BiologyBiological Research CentreSzegedHungary

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