Skip to main content
SpringerLink
Log in

Protoplast Isolation and Staining

  • Chapter
  • First Online:
Plant Microtechniques and Protocols

Abstract

The successful isolation of mesophyll protoplasts from plant species has become a versatile tool for in vivo imaging of subcellular structures. Taking advantage of the various cytochemical probes available, the subcellular localization of specific organelles can be visualized in live protoplasts. In an isolated system, monitoring of the dynamics of organelle movement in response to external stimuli, stresses or an exogenous substance can be substantially facilitated. The isolation of a pure population of non-stressed, healthy protoplasts critically affects the reliability and reproducibility of these studies. In this chapter, we detail a standard protocol for the isolation of live mesophyll protoplasts from leaves of the model plant, Arabidopsis thaliana. We also consider the critical factors for empirical optimization of protoplast isolation procedures for succulent species such as Kalanchoe daigremontiana, Bienertia sinuspersici and Lampranthus spectabilis.

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

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 299.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 379.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 379.99
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. Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187:962–963

    Article  Google Scholar 

  2. Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Miao Y, Jiang L (2007) Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc 2:2348–2353

    Article  CAS  PubMed  Google Scholar 

  4. Lung SC, Yanagisawa M, Chuong SDX (2011) Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici. Plant Cell Rep 30:473–484

    Article  CAS  PubMed  Google Scholar 

  5. Gallie DR, Lucas WJ, Walbot V (1989) Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell 1:301–311

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Cormeau J, Barthou H, Jauneau A, Petitprez M et al (2002) Cellular import of synthetic peptide using a cell-permeable sequence in plant protoplasts. Plant Physiol Biochem 40:1081–1086

    Article  CAS  Google Scholar 

  7. Hong SY, Seo PJ, Cho SH, Park CM (2012) Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon. J Plant Biol 55:390–397

    Article  CAS  Google Scholar 

  8. Bergman P, Glimelius K (1993) Electroporation of rapeseed protoplasts: transient and stable transformation. Physiol Plant 88:604–611

    Article  CAS  Google Scholar 

  9. Garcia R, Pimentel E, Somonte D, Mena J et al (1998) Isolation and transient transformation of stem and leaf protoplasts from sweet potato (Ipomoea batatas L). Asia Pac J Mol Biol 6:145–151

    Google Scholar 

  10. Mazarei M, Al-Ahmad H, Rudis MR, Stewart CN (2008) Protoplast isolation and transient gene expression in switchgrass, Panicum virgatum L. Biotech J 3:354–359

    Article  CAS  Google Scholar 

  11. Guo J, Morrell-Falvey JL, Labbe JL, Muchero W et al (2012) Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PloS ONE 7:e44908

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Pitzschke A, Persak H (2012) Poinsettia protoplasts: a simple, robust and efficient system for transient gene expression studies. Plant Methods 8:14

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Chen S, Tao L, Zeng L, Vega-Sanchez M et al (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7:417–427

    Article  CAS  PubMed  Google Scholar 

  14. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

    Article  CAS  PubMed  Google Scholar 

  15. Eeckhaut T, Lakshmanan PS, Deryckere D, Bockstaele EV, Huylenbroeck JV (2013) Progress in plant protoplast research. Planta 238:991–1003

    Article  CAS  Google Scholar 

  16. Faraco M, Di Sansebastiano GP, Spelt K, Koes RE, Quattrochio FM (2011) One protoplast is not the other. Plant Physiol 156 474–478

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Galston AW, Altman A, Kaur-Sawhney R (1978) Polyamines, ribonuclease and the improvement of oat leaf protoplasts. Plant Sci Lett 11:69–79

    Article  CAS  Google Scholar 

  18. Walter M, Chaban C, Schutze K, Batistic O et al. (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438

    Article  CAS  PubMed  Google Scholar 

  19. Durieu P, Ochatt SJ (2000) Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplast. J Exp Bot 5:1237–1242

    Article  Google Scholar 

  20. Henn HJ, Wingender R, Schnabl H (1998) Regeneration of fertile interspecific hybrids from protoplast fusions between Helianthus annuus L and Helianthus species. Plant Cell Rep 18:220–234

    Article  CAS  Google Scholar 

  21. Hu Q, Andersen SB, Dixelius C, Hansen LN (2002) Production of fertile intergeneric somatic hybrids between Brassica napus and Sinapis arvensis for the enrichment of the rapeseed gene pool. Plant Cell Rep 21:147–152

    Article  CAS  Google Scholar 

  22. Kisaka H, Kisaka M, Kanno A, Kameya T (1998) Intergeneric somatic hybridization of rice (Oryza sativa L) and barley (Hordeum vulgare L) by protoplast fusion. Plant Cell Rep 17:362–367

    Article  CAS  Google Scholar 

  23. Orczyk W, Przetakiewicz J, Nadolska-Orczyk A (2003) Somatic hybrids of Solanum tuberosum: application to genetics and breeding. Plant Cell Tissue Org Cult 74:1–13

    Article  CAS  Google Scholar 

  24. Sutiojono E, Nonhebel HM, Kantharajah AS (1998) Factors affecting protoplast culture of Cucumis melo “Green Delica”. Ann Bot 81:775–777

    Article  CAS  Google Scholar 

  25. Crowder AJ, Landgren CR, Rockwood LL (1979) Cultivar differences in starch content and protoplast yields from root cortical explants of Pisum sativum. Physiol Plant 46:85–88

    Article  Google Scholar 

  26. Chang MM, Loescher WH (1991) Effects of preconditioning and isolation conditions on potato (Solanum tuberosum L. cv. Russet Burbank) protoplast yield for shoot regeneration and electroporation. Plant Sci 73:103–109

    Article  Google Scholar 

  27. Morris P, Thain JF (1980) Comparative studies of leaf tissue and isolated mesophyll protoplasts. J Exp Bot 31:83–95

    Article  CAS  Google Scholar 

  28. Rose RJ (1980) Factors that influence the yield, stability in culture and cell wall regeneration of spinach mesophyll protoplasts. Aust J Plant Physiol 7:713–725

    CAS  Google Scholar 

  29. Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23:131–171

    Article  CAS  PubMed  Google Scholar 

  30. Hasegawa H, Sato M, Suzuki M (2002) Efficient plant regeneration from protoplasts isolated from long-term, shoot primordial-derived calluses of garlic (Allium sativum). J Plant Physiol 159:445–449

    Article  Google Scholar 

  31. Dovzhenko A, Dal Bosco C, Meurer J, Koop HU (2003) Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana. Protoplasma 222:107–111

    Article  CAS  PubMed  Google Scholar 

  32. Pan ZG, Liu CZ, Murch SJ, El-Demerdash M, Saxena PK (2003) Plant regeneration from mesophyll protoplasts of the Egyptian medicinal plants Artemisia judaica L. and Echinops spinosissimus Turra. Plant Sci 165:681–687

    Article  CAS  Google Scholar 

  33. Hooley R (1982) Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid. Planta 154:29–40

    Article  CAS  PubMed  Google Scholar 

  34. Dovzhenko A, Koop HU (2003) Sugarbeet (Beta vulgaris L.): shoot regeneration from callus and callus protoplasts. Planta 217:374–381

    Article  CAS  PubMed  Google Scholar 

  35. Schmidt, R., Poole, R.J. (1980) Isolation of protoplasts and vacuoles from storage tissue of red beet. Plant Physiol 66:25–28

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Barsby TL, Yarrow SA, Shepard JF (1988) A rapid and efficient alternative procedure for regeneration of plants from hypocotyl protoplasts of Brassica napus. Plant Cell Rep 5:101–103

    Article  Google Scholar 

  37. Rybczyfiski JJ (1989) Plant regeneration and protoplast culture of Browollia speciosa. Plant Cell Rep 8:383–386

    Article  Google Scholar 

  38. Coutts RHA, Wood KR. (1975) The isolation and culture of cucumber mesophyll protoplasts. Plant Sci Lett 4:189–193

    Article  Google Scholar 

  39. Pan ZG, Liu CZ, Sobayed SMA, Saxena PK (2004) Plant regeneration from mesophyll protoplasts of Echinacea purpurea. Plant Cell Tissue Org Cult 77:251–255.

    Article  CAS  Google Scholar 

  40. Firoozabady E, DeBoer DL (1986) Isolation, culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense). Plant Cell Rep 5:127–131

    Article  CAS  PubMed  Google Scholar 

  41. Bohorova NE, Cocking EC, Power JB (1986) Isolation, culture and callus regeneration of protoplasts of wild and cultivated Helianthus species. Plant Cell Rep 5:256–258

    Article  CAS  PubMed  Google Scholar 

  42. Sarhan F, Cesar D (1988) High yield isolation of mesophyll protoplasts from wheat, barley and rye. Physiol Plant 72:337–342

    Article  CAS  Google Scholar 

  43. Tudses N, Premjet S, Premjet D (2014) Optimal conditions for high-yield protoplast isolations of Jatropha curcas L and Ricinus communis L. Am-Eurasian J Agric Environ Sci 14:221–230

    Google Scholar 

  44. Engler DE, Grogan RG (1982) Isolation, culture and regeneration of lettuce leaf mesophyll protoplasts. Plant Sci Lett 2B:228–229

    Google Scholar 

  45. Sinha A, Wetten AC, Caligari PDS (2003) Optimization of protoplast production in white lupin. Biol Plant 47:21–25

    Article  Google Scholar 

  46. Anthony P, Davey MR, Power JB, Lowe KC (1995) An improved protocol for the culture of cassava leaf protoplasts. Plant Cell Tissue Org Cult 42:299–302

    Article  CAS  Google Scholar 

  47. Nagata T, Takebe I (1971) Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta 99:12–20

    Article  CAS  PubMed  Google Scholar 

  48. Edwards E, Lilley RM, Craig S, Hatch MD (1979) Isolation of intact and functional chloroplasts from mesophyll and bundle sheath protoplasts of the C4 plant Panicum miliaceum Plant Physiol 63:821–827

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Crepy L, Barros LM, Valente VRN (1986) Callus production from leaf protoplast of various cultivars of bean (Phaseolus vulgaris L.). Plant Cell Rep 5:124–126

    Article  CAS  PubMed  Google Scholar 

  50. Russell JA, Mccown BH (1986) Techniques for enhanced release of leaf protoplasts in populus. Plant Cell Rep 5:284–287

    Article  CAS  PubMed  Google Scholar 

  51. Wu SC, Kuniyuki AH (1985) Isolation and culture of almond protoplasts. Plant Sci 41:55–60

    Article  CAS  Google Scholar 

  52. Gregory DW, Cocking EC (1965) The large-scale isolation of protoplasts from immature tomato fruit. J Cell Biol 24:143–146

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Shepard JF, Totten RE (1977) Mesophyll cell protoplasts of potato. Plant Physiol 60:313–316

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  54. Binding H, Nehls R, Schieder O, Sopory SK, Wenzel G (1978) Regeneration of mesophyll protoplasts isolated from dihaploid clones of Solanum tuberosum. Physiol Plant 43:52–54

    Article  Google Scholar 

  55. Sairam RV, Seetharama N, Devi PS, Verma Al et al (1999). Culture and regeneration of mesophyll-derived protoplasts of sorghum (Sorghum bicolour L. Moench). Plant Cell Rep 18:972–977

    Article  CAS  Google Scholar 

  56. Nishimuram, Hara-Nishimura I, Robinson SP (1984) Isolation of metabolically competent protoplasts from grapevine leave. Plant Sci Lett 37:171–175

    Article  Google Scholar 

Download references

Acknowledgments

This research was supported by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Waterloo Start-Up Fund to SDXC.

Author information

Authors and Affiliations

  1. Department of Biology, University of Waterloo, Waterloo, Ontario, Canada

    Shiu-Cheung Lung, Sarah Schoor, Dustin Sigurdson, Makoto Yanagisawa, Kelly Yeung & Mi Qi Liu

  2. School of Biological Sciences, University of Hong Kong, Hong Kong SAR, China

    Shiu-Cheung Lung

  3. Department of Biology, University of Waterloo, 200 University Avenue West, N2L 3G1, Waterloo, Ontario, Canada

    Simon D. X. Chuong

Authors
  1. Shiu-Cheung Lung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah Schoor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dustin Sigurdson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Makoto Yanagisawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kelly Yeung
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mi Qi Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Simon D. X. Chuong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Simon D. X. Chuong .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, University of Calgary, CALGARY, Alberta, Canada

    Edward Chee Tak Yeung

  2. Department of Plant Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

    Claudio Stasolla

  3. Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

    Michael John Sumner

  4. Center for Basic Research in Digestive Disease, Mayo Clinic , Rochester, Minnesota, USA

    Bing Quan Huang

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Lung, SC. et al. (2015). Protoplast Isolation and Staining. In: Yeung, E., Stasolla, C., Sumner, M., Huang, B. (eds) Plant Microtechniques and Protocols. Springer, Cham. https://doi.org/10.1007/978-3-319-19944-3_12

Download citation

Publish with us

Policies and ethics

Search

Navigation