Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca)

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Polyethylene glycol (PEG)-mediated transient expression system in plant protoplasts has been widely used in a variety of plants for gene function characterization. However, such a system has not been developed for strawberry. In this study, we report a method for obtaining high quality and high yield protoplasts from strawberry leaves. In addition, we developed an efficient transient gene expression system based on the PEG-mediated method, and the system has been successfully applied to studies of protein expression, protein subcellular localization and protein–protein interaction in strawberry. Furthermore, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable targeted mutagenesis of the FvTCP17 gene in strawberry protoplasts. Taken together, our results demonstrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9-mediated genome editing in strawberry.

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We report a highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9‐mediated genome editing in strawberry.

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The present study was financially supported by Grants from the Opening Foundation of Key Laboratory of Fruit Breeding Technology, the Ministry of Agriculture and Rural Affairs of China (NYB201705-3), the Natural Science Foundation of Henan (162300410329), and the project for Agriculture Sci-Tech of Xi’an (20193020YF008NS008). We also thank Bruce Levine of University of Maryland for careful editing of this manuscript.

Author information

JYF conceived the research. YJG performed all treatments with assistance of YLL, PPB, DJW, YYM, and Y.H. YJG and YYM analyzed the data. YH provided assistance in constructing vectors for subcellular localization. YJG, JYF, HCZ and YQW contributed to the writing of the manuscript. All authors read and approved the final manuscript.

Correspondence to Jia-Yue Feng.

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Communicated by Wenwu Guo.

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Supplementary material 1 (TIF 65687.9 kb). Fig. S1 Transient gene expression in strawberry protoplasts. The effect of incubation time on transfection efficiency. Fluorescent (left panel) and bright field (right panel) microscopic images were taken using a fluorescence microscope with a × 20 objective lens. The scale bar represents 50 μm

Supplementary material 2 (TIF 765.3 kb). Fig. S2 The T-DNA region of the pKSE401 vector. LB, Left border of T-DNA; RB, Right border of T-DNA; U6-26p, Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator; 2 × 35Sp, 2 × 35S promoter; 3 × FLAG, 3 × FLAG tag; NLS, Nuclear localization signal; zCas9, Zea mays codon-optimized Cas9; Nost, nos gene terminator; KanR, Kanamycin resistance gene; 35St, 35S terminator

Supplementary material 3 (TIF 40282.5 kb). Fig. S3 Vitality identification of strawberry protoplasts a The integrity of freshly isolated strawberry protoplasts was observed using bright-field conditions using a fluorescence microscope and a × 40 objective. b Freshly isolated protoplasts were stained for viability with FDA and visualized as above with a × 40 objective lens. The scale bar represents 50 μm


Supplementary material 4 (DOCX 16.6 kb). Table S1. Primers used in this study. The gene ID comes from the NCBI database ( and the strawberry genome database (

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Gou, Y., Li, Y., Bi, P. et al. Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca). Plant Cell Tiss Organ Cult (2020).

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  • Strawberry
  • Protoplast isolation
  • Transient gene expression
  • CRISPR/Cas9
  • FvTCP17