Skip to main content
SpringerLink
Log in
SpringerLink
Log in

Transformation Using Controlled cDNA Overexpression System

  • Protocol
  • First Online:
Plant Salt Tolerance

Part of the book series: Methods in Molecular Biology ((MIMB,volume 913))

Abstract

The controlled cDNA overexpression system (COS) was developed to identify novel regulatory genes in model plants as well as in other species that might have a particular valuable trait. The COS system (Papdi et al. Plant Physiol 147:528–542, 2008) is composed of a random cDNA library prepared in a T-DNA plant expression vector, under the control of the estradiol-inducible XVE promoter. Large-scale genetic transformation of Arabidopsis thaliana generates a transgenic plant population with randomly inserted cDNA clones. Overexpression of the inserted cDNA can create selectable phenotypes, allowing the facile identification and cloning of the responsible genes. Here we describe protocols to create and use the COS system for diverse purposes in plant biology.

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 84.99
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 109.99
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. Hansen G, Chilton MD (1999) Lessons in gene transfer to plants by a gifted microbe. Curr Top Microbiol Immunol 240:21–57

    PubMed  CAS  Google Scholar 

  2. Koncz C, Martini N, Mayerhofer R et al (1989) High-frequency T-DNA-mediated gene tagging in plants. Proc Natl Acad Sci USA 86:8467–8471

    Article  PubMed  CAS  Google Scholar 

  3. Bechtold N, Jaudeau B, Jolivet S et al (2000) The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana. Genetics 155:1875–1887

    PubMed  CAS  Google Scholar 

  4. Tague BW, Mantis J (2006) In planta Agrobacterium-mediated transformation by vacuum infiltration. Methods Mol Biol 323: 215–223

    PubMed  Google Scholar 

  5. Hsing YI, Chern CG, Fan MJ et al (2007) A rice gene activation/knockout mutant resource for high throughput functional genomics. Plant Mol Biol 63:351–364

    Article  PubMed  CAS  Google Scholar 

  6. Jeong DH, An S, Kang HG et al (2002) T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol 130:1636–1644

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  8. Bouche N, Bouchez D (2001) Arabidopsis gene knockout: phenotypes wanted. Curr Opin Plant Biol 4:111–117

    Article  PubMed  CAS  Google Scholar 

  9. Koncz C, Nemeth K, Redei GP et al (1992) T-DNA insertional mutagenesis in Arabidopsis. Plant Mol Biol 20:963–976

    Article  PubMed  CAS  Google Scholar 

  10. Weigel D, Ahn JH, Blazquez MA et al (2000) Activation tagging in Arabidopsis. Plant Physiol 122:1003–1013

    Article  PubMed  CAS  Google Scholar 

  11. Alvarado MC, Zsigmond LM, Kovacs I et al (2004) Gene trapping with firefly luciferase in Arabidopsis. Tagging of stress-responsive genes. Plant Physiol 134:18–27

    Article  PubMed  CAS  Google Scholar 

  12. Koo J, Kim Y, Kim J et al (2007) A GUS/luciferase fusion reporter for plant gene trapping and for assay of promoter activity with luciferin-dependent control of the reporter protein stability. Plant Cell Physiol 48:1121–1131

    Article  PubMed  CAS  Google Scholar 

  13. Meissner R, Chague V, Zhu Q et al (2000) Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato. Plant J 22:265–274

    Article  PubMed  CAS  Google Scholar 

  14. Yamamoto YY, Tsuhara Y, Gohda K et al (2003) Gene trapping of the Arabidopsis genome with a firefly luciferase reporter. Plant J 35:273–283

    Article  PubMed  CAS  Google Scholar 

  15. Alonso JM, Stepanova AN, Leisse TJ et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657

    Article  PubMed  Google Scholar 

  16. Rios G, Lossow A, Hertel B et al (2002) Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes. Plant J 32:243–253

    Article  PubMed  CAS  Google Scholar 

  17. Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshaar B (2003). An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53:247–259

    Article  PubMed  CAS  Google Scholar 

  18. Samson F, Brunaud V, Balzergue S et al (2002) FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants. Nucleic Acids Res 30:94–97

    Article  PubMed  CAS  Google Scholar 

  19. Szabados L, Kovacs I, Oberschall A et al (2002) Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. Plant J 32:233–242

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  21. Kondou Y, Higuchi M, Takahashi S et al (2009) Systematic approaches to using the FOX hunting system to identify useful rice genes. Plant J 57:883–894

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  24. Papdi C, Joseph MP, Pérez-Salamó I, et al (2009). Genetic technologies for the identification of Arabidopsis genes controlling environmental stress responses. Funct Plant Biol 36:696–720

    Google Scholar 

  25. Kuhn JM, Boisson-Dernier A, Dizon MB et al (2006) The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA. Plant Physiol 140:127–139

    Article  PubMed  CAS  Google Scholar 

  26. Banno H, Ikeda Y, Niu QW, Chua NH (2001). Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell 13: 2609–2618

    PubMed  CAS  Google Scholar 

  27. Yokotani N, Ichikawa T, Kondou Y et al (2009) Tolerance to various environmental stresses conferred by the salt-responsive rice gene ONAC063 in transgenic Arabidopsis. Planta 229:1065–1075

    Article  PubMed  CAS  Google Scholar 

  28. Du J, Huang YP, Xi J et al (2008) Functional gene-mining for salt-tolerance genes with the power of Arabidopsis. Plant J 56:653–664

    Article  PubMed  CAS  Google Scholar 

  29. Szabados L, Salamó IP, Papdi C et al (2009) Functional gene mining in Arabidopsis and related species. Presented at PlantGem Congress, Lisbon, Portugal

    Google Scholar 

  30. Salamó I, Szabados L (2011) Identification of novel regulatory factors of plant stress responses using new genetic approaches. Presented at international conference on plant gene discovery technologies, Vienna

    Google Scholar 

  31. Sambrook J, MacCallum P, Russel D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor’s Laboratory Press, New York

    Google Scholar 

  32. Koncz C, Martini N, Szabados L, et al (1994) Specialized vectors for gene tagging and expression studies. In: Gelvin SB (ed) Plant molecular biology manual. pp 1–22

    Google Scholar 

  33. Wise AA, Liu Z, Binns AN (2006) Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol Biol 343:43–53

    PubMed  CAS  Google Scholar 

  34. 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 

  35. Wang K (2006) Agrobacterium protocols. Humana, Totowa

    Google Scholar 

  36. Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–266

    PubMed  CAS  Google Scholar 

  37. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  38. Katavic V, Haughn GW, Reed D et al (1994) In planta transformation of Arabidopsis thaliana. Mol Gen Genet 245:363–370

    Article  PubMed  CAS  Google Scholar 

  39. 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  PubMed  CAS  Google Scholar 

  40. Koiwa H, Bressan RA, Hasegawa PM (2006) Identification of plant stress-responsive determinants in Arabidopsis by large-scale forward genetic screens. J Exp Bot 57:1119–1128

    Article  PubMed  CAS  Google Scholar 

  41. Papdi C, Leung J, Joseph MP et al (2010) Genetic screens to identify plant stress genes. Methods Mol Biol 639:121–139

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Authors are indebted for Mary Prathiba Joseph for critical reading and correcting the manuscript. Research was supported by OTKA Grants no. K-68226, K-81765, HuRo Cross border Cooperation Programme HURO/0801/167 and COST Action FA0605.

Author information

Authors and Affiliations

  1. Institute of Plant Biology, Biological Research Center, Szeged, Hungary

    Gábor Rigó, Csaba Papdi & László Szabados

Authors
  1. Gábor Rigó
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Csaba Papdi
    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. School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, 7001, Tasmania, Australia

    Sergey Shabala

  2. INRA-SupAgro, BPMP-IBIPGC, place Viala 1, Montpellier, 34060, France

    Tracey Ann Cuin

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Rigó, G., Papdi, C., Szabados, L. (2012). Transformation Using Controlled cDNA Overexpression System. In: Shabala, S., Cuin, T. (eds) Plant Salt Tolerance. Methods in Molecular Biology, vol 913. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-986-0_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-986-0_19

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-985-3

  • Online ISBN: 978-1-61779-986-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation