Skip to main content
Log in

Efficient protoplast isolation and transient gene expression system for Phalaenopsis hybrid cultivar ‘Ruili Beauty’

  • Plant Tissue Culture
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

With the release of the Phalaenopsis equestris (Schauer) Rchb.f. genome database, more in-depth studies of Phalaenopsis spp. will be carried out in the future. Transient gene expression in protoplasts is a useful system for gene function analysis, which is especially true for Phalaenopsis, whose stable genetic transformation is difficult and extremely time-consuming. In this study, juvenile leaves from aseptic Phalaenopsis seedlings were used as the starting material for protoplast isolation. After protocol refinement, the highest yield of viable protoplasts [5.94 × 106 protoplasts g−1 fresh weight (FW)] was achieved with 1.0% (w/v) Cellulase Onozuka R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M D-mannitol, with an enzymolysis duration of 6 h. As indicated by transient expression of green fluorescent protein (GFP), a transformation efficiency of 41.7% was achieved with 20% (w/v) polyethylene glycol (PEG-4000), 20 μg plasmid DNA, 2 × 105 mL−1 protoplasts, and a transfection duration of 30 min. The protocol established here will be valuable for functional studies of Phalaenopsis genes.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.

Similar content being viewed by others

References

  • Cai J, Liu X, Vanneste K, Proost S, Tsai W-C, Liu K-W, Chen L-J, He Y, Xu Q, Bian C, Zheng Z, Sun F, Liu W, Hsiao Y-Y, Pan Z-J, Hsu C-C, Yang Y-P, Hsu Y-C, Chuang Y-C, Dievart A, Dufayard J-F, Xu X, Wang J-Y, Wang J, Xiao X-J, Zhao X-M, Du R, Zhang G-Q, Wang M, Y-Y S, Xie G-C, Liu G-H, Li L-Q, Huang L-Q, Luo Y-B, Chen H-H, Van de Peer Y, Liu Z-J (2015) The genome sequence of the orchid Phalaenopsis equestris. Nat Genet 47:65–72

    Article  CAS  PubMed  Google Scholar 

  • Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang G-L (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 

  • Duquenne B, Eeckhaut T, Werbrouck S, Van Huylenbroeck J (2007) Effect of enzyme concentrations on protoplast isolation and protoplast culture of Spathiphyllum and Anthurium. Plant Cell Tissue Organ Cult 91:165–173

    Article  CAS  Google Scholar 

  • Hsieh M-H, Pan Z-J, Lai P-H, Lu H-C, Yeh H-H, Hsu C-C, Wu W-L, Chung M-C, Wang S-S, Chen W-H, Chen H-H (2013) Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids. J Exp Bot 64:3869–3884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang H, Wang Z, Cheng J, Zhao W, Li X, Wang H, Zhang Z, Sui X (2013) An efficient cucumber (Cucumis sativus L.) protoplast isolation and transient expression system. Sci Hortic 150:206–212

    Article  CAS  Google Scholar 

  • Julkifle AL, Rathinam X, Sinniah UR, Subramaniam S (2010) Optimisation of transient green fluorescent protein (GFP) gene expression in Phalaenopsis violacea orchid mediated by Agrobacterium tumefaciens-mediated transformation system. Aust J Basic Appl Sci 4:3424–3432

    CAS  Google Scholar 

  • Kao KN, Michayluk MR (1974) A method for high-frequency intergeneric fusion of plant protoplasts. Planta 115:355–367

    Article  CAS  PubMed  Google Scholar 

  • Khentry Y, Paradornuvat A, Tantiwiwat S, Phansiri S, Thaveechai N (2006) Protoplast isolation and culture of Dendrobium Sonia ‘Bom 17’. Kasetsart J –Nat Sci 40:361–369

    Google Scholar 

  • Kobayashi T, Kameya T, Ichihashi S (1993) Plant regeneration from protoplasts derived from callus of Phalaenopsis. Plant Tissue Cult Lett 10:267–270

    Article  CAS  Google Scholar 

  • Locatelli F, Vannini C, Magnani E, Coraggio I, Bracale M (2003) Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount. Plant Cell Rep 21:865–871

    CAS  PubMed  Google Scholar 

  • Luehrsen KR, de Wet JR, Walbot V (1992) Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol 216:397–414

    Article  CAS  PubMed  Google Scholar 

  • Maas C, Werr W (1989) Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts. Plant Cell Rep 8:148–151

    Article  CAS  PubMed  Google Scholar 

  • Maddumage R, Fung RMW, Weir I, Ding H, Simons JL, Allan AC (2002) Efficient transient transformation of suspension culture-derived apple protoplasts. Plant Cell Tissue Organ Cult 70:77–82

    Article  CAS  Google Scholar 

  • Mishiba K-I, Chin DP, Mii M (2005) Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination. Plant Cell Rep 24:297–303

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Pindel A (2007) Optimization of isolation conditions of Cymbidium protoplasts. Folia Hort 19:79–88

    Google Scholar 

  • Qiao Y-X, Zhang Y-P, Wang G-L, Chen C, Wang Y (2008) Optimization of the method for isolating protoplast of Phalaenopsis amabilisBl. Plant Physiol Comm 44:1177–1180 (in Chinese)

    Google Scholar 

  • Ratanasanobon K, Seaton KA (2013) Protoplast isolation for species in the Chamelaucium group and the effect of antioxidant enzymes (superoxide dismutase and catalase) on protoplast viability. In Vitro Cell Dev Biol – Plant 49:593–598

    Article  CAS  Google Scholar 

  • Rezazadeh R, Niedz RP (2015) Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design. Plant Cell Tissue Organ Cult 122:585–604

    Article  CAS  Google Scholar 

  • 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 

  • Shrestha BR, Tokuhara K, Mii M (2007) Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis. Plant Cell Rep 26:719–725

    Article  CAS  PubMed  Google Scholar 

  • Su V, Hsu BD (2003) Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers. Biotechnol Lett 25:1933–1939

    Article  CAS  PubMed  Google Scholar 

  • Sun S, Furtula V, Nothnagel EA (1992) Mechanical release and lectin labeling of maize root protoplasts. Protoplasma 169:49–56

    Article  Google Scholar 

  • Xu J, Li J, Cui L, Zhang T, Wu Z, Zhu P-Y, Meng Y-J, Zhang K-J, Yu X-Q, Lou Q-F, Chen J-F (2017) New insights into the roles of cucumber TIR1 homologs and miR393 in regulating fruit/seed set development and leaf morphogenesis. BMC Plant Biol 17:130

    Article  PubMed  PubMed Central  Google Scholar 

  • Yao L, Liao X, Gan Z, Peng X, Wang P, Li S, Li T (2016) Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.) Sci Hortic 209:14–21

    Article  CAS  Google Scholar 

  • Yoo S-D, Cho Y-H, 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 

  • Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7:30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao W, Yang W, Wei C, Sun G (2011) A simple and efficient method for isolation of pineapple protoplasts. Biotechnol Biotechnol Equip 25:2464–2467

    Article  CAS  Google Scholar 

  • Zhou B, Nie Y-Z, Zhang X-L, Li Y-H (2008) Protoplast isolation of Brassica rapa ‘Tsuda’ turnip and transient expression of GFP. Lett Biotechnol 19:542–544 (in Chinese)

    Google Scholar 

Download references

Acknowledgments

The authors would like to thank Drs. Ji Li and Jian Xu of Nanjing Agricultural University for providing vectors and help with transient expression experiments.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 31372101).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Guangdong Wang.

Additional information

Editor: Mark Jordan

Electronic supplementary material

ESM 1

(DOCX 11.7 kb).

ESM 2

(DOCX 11.7 kb).

ESM 3

(DOCX 11.9 kb).

ESM 4

(DOCX 11.5 kb).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, J., Liao, X., Zhou, S. et al. Efficient protoplast isolation and transient gene expression system for Phalaenopsis hybrid cultivar ‘Ruili Beauty’. In Vitro Cell.Dev.Biol.-Plant 54, 87–93 (2018). https://doi.org/10.1007/s11627-017-9872-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-017-9872-z

Keywords

Navigation