Fluorescent Protoplast Imaging of Apple Bitter Pit

The fruits were consistently light, and the fruit size was uniform. The apples were cut into four parts on average, and each portion of pulp was taken from near the peduncle to near the calyx. Seven healthy apples and six apples showing bitter pit symptoms were used to isolate the protoplasts from the pulp cells, which were then fluorescently stained with fluo-4/AM (Qiu et al., 2020 (link)). The final concentration of fluo-4/AM (Dojindo Laboratories, Kumamoto, Japan) was 5 μmol/L. Since the excitation wavelength of fluo-4/AM is 490 nm, GFP was selected as the light cube. The viability of cells was then determined under a fluorescent microscope (EVOS Auto 2, Thermo Fisher Scientific, United States). The fluorescence results were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., MD, United States), according to our published methods (Qu et al., 2016 (link)).