Skip to main content
SpringerLink
Log in

Virus-Induced Gene Silencing of the Alkaloid-Producing Basal Eudicot Model Plant Eschscholzia californica (California Poppy)

  • Protocol
  • First Online:
Virus-Induced Gene Silencing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 975))

Abstract

Eschscholzia californica (California poppy), a member of the basal eudicot family of the Papaveraceae, is an important species to study alkaloid biosynthesis and the effect of alkaloids on plant metabolism. More recently, it has also been developed as a model system to study the evolution of plant morphogenesis. While progress has been made towards establishing methods for generating genetically modified cell culture lines, transcriptome data and gene expression analysis, the stable transformation and subsequent regeneration of transgenic plants has proven extremely time consuming and difficult. Here, we describe in detail a method to transiently down regulate expression of a target gene by virus-induced gene silencing (VIGS) and the subsequent analysis of the VIGS treated plants. VIGS in E. californica allows for the study of gene function within 2 to 3 weeks after inoculation, and the method proves very efficient, enabling the rapid analysis of gene functions.

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 139.00
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. Wang W, Lu A-M, Ren Y et al (2009) Phylogeny and classification of Ranunculales: evidence from four molecular loci and morphological data. Perspect Plant Ecol Evol Syst 11(2):81–110

    Article  Google Scholar 

  2. Kadereit JW, Blattner FR, Jork KB et al (1995) The phylogeny of the Papaveraceae s.l.: morphological, geographical and ecological implications. Plant Syst Evol 9:133–145

    Google Scholar 

  3. Becker A, Gleissberg S, Smyth DR (2005) Floral and vegetative morphogenesis in California poppy (Eschscholzia californica Cham). Int J Plant Sci 166(4):537–555

    Article  CAS  Google Scholar 

  4. Bennett MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Philos Trans R Soc Lond B Biol Sci 274(933):227–274

    Article  PubMed  CAS  Google Scholar 

  5. Barakat A, Wall K, Leebens-Mack J et al (2007) Large-scale identification of microRNAs from a basal eudicot (Eschscholzia californica) and conservation in flowering plants. Plant J 51(6):991–1003

    Article  PubMed  CAS  Google Scholar 

  6. Carlson J, Leebens-Mack JH, Wall PK et al (2006) EST database for early flower development in California poppy (Eschscholzia californica Cham., Papaveraceae) tags over 6000 genes from a basal eudicot. Plant Mol Biol 62(3):351–369

    Article  PubMed  Google Scholar 

  7. Wall PK, Leebens-Mack J, Chanderbali AS et al (2009) Comparison of next generation sequencing technologies for transcriptome characterization. BMC Genomics 10:347

    Article  PubMed  Google Scholar 

  8. Zahn LM, Ma X, Altman NS et al (2010) Comparative transcriptomics among floral organs of the basal eudicot Eschscholzia californica as reference for floral evolutionary developmental studies. Genome Biol 11(10):R101

    Article  PubMed  CAS  Google Scholar 

  9. Park SU, Facchini PJ (2000) Agrobacterium-mediated genetic transformation of California poppy, Eschscholzia californica Cham., via somatic embryogenesis. Plant Cell Rep 19(10):1006–1012

    Article  CAS  Google Scholar 

  10. Orashakova S, Lange M, Lange S et al (2009) The CRABS CLAW ortholog from California poppy (Eschscholzia californica, Papaveraceae), EcCRC, is involved in floral meristem termination, gynoecium differentiation and ovule initiation. Plant J 58(4):682–693

    Article  PubMed  CAS  Google Scholar 

  11. Angelova S, Buchheim M, Frowitter D et al (2010) Overproduction of alkaloid phytoalexins in California poppy cells is associated with the co-expression of biosynthetic and stress-protective enzymes. Mol Plant 3(5):927–939

    Article  PubMed  CAS  Google Scholar 

  12. Liscombe DK, Ziegler J, Schmidt J et al (2009) Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species. Plant J 4:729–743

    Article  Google Scholar 

  13. Vogel M, Lawson M, Sippl W et al (2010) Structure and mechanism of sanguinarine reductase, an enzyme of alkaloid detoxification. J Biol Chem 285(24):18397–18406

    Article  PubMed  CAS  Google Scholar 

  14. Weiss D, Baumert A, Vogel M et al (2006) Sanguinarine reductase, a key enzyme of benzophenanthridine detoxification. Plant Cell Environ 29(2):291–302

    Article  PubMed  CAS  Google Scholar 

  15. Donaire L, Barajas D, Martínez-García B et al (2008) Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs. J Virol 82(11):5167–5177

    Article  PubMed  CAS  Google Scholar 

  16. Waterhouse PM, Fusaro AF (2006) Plant science. Viruses face a double defense by plant small RNAs. Science 313(5783):54–55

    Article  PubMed  CAS  Google Scholar 

  17. Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130(3):413–426

    Article  PubMed  CAS  Google Scholar 

  18. Kalantidis K, Schumacher HT, Alexiadis T et al (2008) RNA silencing movement in plants. Biol Cell 100(1):13–26

    Article  PubMed  CAS  Google Scholar 

  19. Liu Y, Schiff M, Marathe R et al (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30(4):415–429

    Article  PubMed  CAS  Google Scholar 

  20. Yellina A, Orashakova S, Lange S et al (2010) Floral homeotic C function genes repress specific B function genes in the carpel whorl of the basal eudicot California poppy (Eschscholzia californica). EvoDevo 1(1):1–13

    Article  Google Scholar 

  21. Koncz C, Schell J (1986) The promoter of TLDNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204(3):383–396

    Article  CAS  Google Scholar 

  22. Mersereau M, Pazour GJ, Das A (1990) Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90(1):149–151

    Article  PubMed  CAS  Google Scholar 

  23. Shen WJ, Forde BG (1989) Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 17(20):8385

    Article  PubMed  CAS  Google Scholar 

  24. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622

    Article  PubMed  CAS  Google Scholar 

  25. Dorak MT (2006) Real-time PCR. In: Owen E (ed) BIOS Advanced Methods. Taylor & Francis Group, New York, p 127

    Google Scholar 

Download references

Acknowledgments

The E. californica work in A.B.’s lab is largely funded by the German Research Foundation (DFG grants BE 2547/6-1, 6-2, 7-2, 8-1).

Author information

Authors and Affiliations

  1. Plant Evodevo Group, Justus-Liebig-Universität Gießen, Gießen, Germany

    Dawit G. Tekleyohans & Annette Becker

  2. School of Geography, Environment and Earth Sciences, University of Wellington, Wellington, New Zealand

    Sabrina Lange

Authors
  1. Dawit G. Tekleyohans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sabrina Lange
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Annette Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Annette Becker .

Editor information

Editors and Affiliations

  1. Institute of Botany, Justus-Liebig-University Gießen, Heinrich-Buff-Ring 38, Gießen, 35392, Germany

    Annette Becker

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Tekleyohans, D.G., Lange, S., Becker, A. (2013). Virus-Induced Gene Silencing of the Alkaloid-Producing Basal Eudicot Model Plant Eschscholzia californica (California Poppy). In: Becker, A. (eds) Virus-Induced Gene Silencing. Methods in Molecular Biology, vol 975. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-278-0_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-278-0_7

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-277-3

  • Online ISBN: 978-1-62703-278-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation