Confocal Imaging of Seeds

  • Protocol
  • First Online:
Seed Dormancy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2830))

  • 46 Accesses

Abstract

In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Understanding such a complex biological process requires microscopy techniques able to unveil the seed internal morphological structure. Seed thickness and relatively low permeability make conventional tissue staining techniques impractical unless combined with time-consuming dissecting methods. Here, we describe two techniques to imaging the three-dimensional structure of Arabidopsis seeds by confocal laser scanning microscopy. Both procedures, while differing in their time of execution and resolution, are based on cell wall staining of seed tissues with fluorescent dyes.

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

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. O’Brien IEW, Reutelingsperger CPM, Holdaway KM (1997) Annexin-V and TUNEL use in monitoring the progression of apoptosis in plants. Cytometry 29:28–33

    Article  PubMed  Google Scholar 

  2. O’Brien IEW, Murray BG, Baguley BC et al (1998) Major changes in chromatin condensation suggest the presence of an apoptotic pathway in plant cells. Exp Cell Res 241:46–54

    Article  PubMed  Google Scholar 

  3. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461

    Article  CAS  PubMed  Google Scholar 

  4. Zhu H (2012) Propidium iodide staining of cells for FACS analysis. Bio Protocol 2. https://doi.org/10.21769/BioProtoc.195

  5. Darzynkiewicz Z, Huang X, Zhao H (2017) Analysis of cellular DNA content by flow cytometry. Curr Protoc Immunol 2017:5.7.1–5.7.20

    Google Scholar 

  6. Jones K, Kim DW, Park JS et al (2016) Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae. BMC Plant Biol 16:1–8

    Google Scholar 

  7. Truernit E, Bauby H, Dubreucq B et al (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20:1494–1503

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rae AE, Wei X, Flores-Rodriguez N et al (2021) Super-resolution fluorescence imaging of arabidopsis thaliana transfer cell wall ingrowths using pseudo-schiff labelling adapted for the use of different dyes. Plant Cell Physiol 61:1775–1787

    Article  Google Scholar 

  9. Flütsch S, Distefano L, Santelia D (2018) Quantification of starch in guard cells of Arabidopsis thaliana. Bio Protoc 8:e2920

    Google Scholar 

  10. Lu J, Le HR, Gómez-Páez DM et al (2021) The nucellus: between cell elimination and sugar transport. Plant Physiol 185:478–490

    CAS  PubMed  Google Scholar 

  11. Xu W, Fiume E, Coen O et al (2016) Endosperm and nucellus develop antagonistically in Arabidopsis seeds. Plant Cell 28:1343–1360

    Google Scholar 

  12. Coen O, Lu J, Xu W et al (2020) A TRANSPARENT TESTA transcriptional module regulates endothelium polarity. Front Plant Sci 10:1–14

    Article  Google Scholar 

  13. Haseloff J (2003) BioImaging old botanical techniques for new microscopes. Biotechniques 34:1174–1182

    Article  CAS  PubMed  Google Scholar 

  14. Moreno N, Bougourd S, Haseloff J et al (2006) Imaging plant cells. Handbook of biological confocal microscopy, vol 166, 3rd edn, pp 769–787

    Book  Google Scholar 

  15. Truernit E, Siemering KR, Hodge S et al (2006) A map of KNAT gene expression in the Arabidopsis root. Plant Mol Biol 60:1–20

    Article  CAS  PubMed  Google Scholar 

  16. Musielak TJ, Schenkel L, Kolb M et al (2015) A simple and versatile cell wall staining protocol to study plant reproduction. Plant Reprod 28:161–169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Buda GJ, Isaacson T, Matas AJ et al (2009) Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy. Plant J 60:378–385

    Article  CAS  PubMed  Google Scholar 

  18. Tofanelli R, Vijayan A, Scholz S et al (2019) Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy. Plant Methods 15:1–13

    Article  CAS  Google Scholar 

  19. Benerini Gatta L, Cadei M, Balzarini P et al (2012) Application of alternative fixatives to formalin in diagnostic pathology. Eur J Histochem 56(2):e12

    Article  CAS  PubMed  Google Scholar 

  20. Schneitz K, Hülskamp M, Pruitt RE (1995) Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. Plant J 7:731–749

    Google Scholar 

  21. Provost A, Rousset C, Bourdon L et al (2019) Innovative particle standards and long-lived imaging for 2D and 3D dSTORM. Sci Rep 9:1–13

    Article  CAS  Google Scholar 

  22. Ursache R, Andersen TG, Marhavý P et al (2018) A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. Plant J 93:399–412

    Article  CAS  PubMed  Google Scholar 

  23. Coen O, Lu J, Xu W et al (2019) Deposition of a cutin apoplastic barrier separating seed maternal and zygotic tissues. BMC Plant Biol 19:1–20

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work has benefited from the support of IJPB’s Plant Observatory technological platforms and was founded by the PERISEED ANR (ANR-18-CE20-0009) and Labex Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS) grants.

The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Enrico Magnani .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Gómez-Páez, DM., Magnani, E. (2024). Confocal Imaging of Seeds. In: Kawakami, N., Sato, K. (eds) Seed Dormancy. Methods in Molecular Biology, vol 2830. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3965-8_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3965-8_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3964-1

  • Online ISBN: 978-1-0716-3965-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Navigation