Nondestructive and Quantitative Analysis of Cell Wall Regeneration in the Medicinal Macrofungus Ganoderma lingzhi by a Membrane-Fusing Fluorescent Probe

Ganoderma is a prize medicinal macrofunguswitha broad range of pharmaceutical values. To date, various attemptshave been made to cultivate Ganoderma to improvethe production of secondary metabolites with pharmacological activity.Among the adopted techniques, protoplast preparation and regenerationare indispensable. However, the evaluation of protoplasts and regeneratedcell walls usually relies on electron microscopy assays, which requiretime-consuming and destructive sample preparation and merely providelocalized information in the selected area. In contrast, fluorescenceassays enable sensitive real-time detection and imaging invivo. They can also be applied to flow cytometry, providinga collective overview of every cell in a sample. However, for macrofungisuch as Ganoderma, the fluorescence analysis of protoplastsand regenerated cell walls is difficult owing to the hindrance ofthe homologous fluorescent protein expression and the lack of an appropriatefluorescence marker. Herein, a specific plasma membrane probe, TAMRAperfluorocarbon nucleic acid probe (TPFN), is proposed for the nondestructiveand quantitative fluorescence analysis of cell wall regeneration.Exploiting the perfluorocarbon membrane-anchoring chains, hydrophilicnucleic acid linker, and fluorescent dye TAMRA, the probe is provento be selective, soluble, and stable, enabling rapid fluorescencedetection of a protoplast sample free of transgenic expression orimmune staining. Based on the TPFN and flow cytometry techniques,a quantitative approach is constructed to monitor the process of cellwall growth in a fast, quantitative, and high-throughout manner, andthe obtained results are consistent with those of conventional electronmicroscopy. In principle, with slight modifications or integration,the proposed probe and approach can be adapted to the preparationof cell protoplasts, inspection of cell wall integrity under environmentalstress, and programmable membrane engineering for cytobiology andphysiology research.

Automated summary

This study proposes a specific plasma membrane probe and combines it with flow cytometry to establish a rapid, quantitative, and high-throughput method for monitoring cell wall growth. The obtained results are consistent with those of conventional electron microscopy. The proposed probe and approach can be adapted to various applications including the preparation of cell protoplasts, inspection of cell wall integrity under environmental stress, and programmable membrane engineering for cytobiology and physiology research.