Polyethylene glycol (PEG)-mediated protoplast fusion is widely used to generate somatic hybrids; however, fusion efficiency largely depends on PEG concentration, which influences both membrane destabilization and cell viability. In this study, we systematically evaluated PEG-mediated fusion across five Brassicaceae species: Brassica rapa var. perviridis, Brassica rapa var. pekinensis, Brassica oleracea var. capitata, Eruca vesicaria subsp. sativa, and Arabidopsis thaliana. Protoplast viability was maintained up to 10% (w/v) PEG but declined markedly above 15%, whereas fusion frequency increased with PEG concentration and reached 70–90% at ≥10% PEG. To integrate these effects, we defined protoplast fusion efficiency (PFE) as the product of viability and fusion frequency. All species exhibited maximal PFE within a PEG range of 7.5–15%, indicating a shared optimal window that balances fusogenic activity and cytotoxicity. Interspecific fusion assays performed at 10% PEG successfully generated hybrid cells across all pairwise combinations, with efficiency varying by species pair. The fusion rates were highest between closely related B. rapa subspecies and lowest in combinations involving Arabidopsis thaliana. Notably, interspecific fusion efficiency correlated with phylogenetic proximity. Collectively, these findings provide quantitative guidelines for optimizing PEG-mediated fusion in Brassicaceae species and demonstrate that genetic relatedness influences interspecific fusion success.

Autofluorescence derived from plant tissues, originating from cell walls, chloroplasts, and various endogenous pigments, often interferes with fluorescence imaging and hampers quantitative analysis. Although numerous fluorescence-based techniques have been developed for plant cell studies, autofluorescence remains a persistent technical obstacle. An LED-based autofluorescence removal system (TiYO), originally developed for animal neural tissues, has recently been shown to selectively reduce tissue autofluorescence without compromising specific fluorescence signals. In this study, we examined whether this system can be applied to plant materials.

The effectiveness of TiYO-mediated LED irradiation was evaluated using tissue sections of plantlets of Torenia fournieri, gametophytes of Equisetum arvense, and callus tissues of Marchantia polymorpha. The degree of autofluorescence quenching varied with plant material and fluorescence wavelength. Blue autofluorescence was only weakly reduced in Torenia tissues and M. polymorpha callus, whereas other autofluorescence components were efficiently suppressed. In DAPI-stained E. arvense gametophytes and M. polymorpha callus cells, strong autofluorescence severely impaired nuclear visualization; however, 120 min of TiYO treatment markedly reduced autofluorescence, enabling clear observation and quantification of individual nuclei.

These results demonstrate that TiYO-mediated LED irradiation, originally designed for animal tissues, is also applicable to plant materials and can improve fluorescence imaging by reducing autofluorescence while preserving specific fluorescence signals. This approach provides a useful option for enhancing fluorescence-based analyses in plant cell and developmental biology.