Optimization of PEG-Mediated protoplast fusion and its phylogenetic determinants in Brassicaceae species

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.