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

  • Plant Tissue Culture
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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.

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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).

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Authors and Affiliations

  1. Department of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China

    Jinlan Li, Xuezhu Liao, Shushan Zhou, Song Liu, Li Jiang & Guangdong Wang

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  1. Jinlan Li
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  2. Xuezhu Liao
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  3. Shushan Zhou
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  4. Song Liu
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  5. Li Jiang
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Correspondence to Guangdong Wang.

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Editor: Mark Jordan

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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

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