FLASH: A high-efficiency soybean-native protoplast platform for real-time protein localization and interaction studies

Soybean ( Glycine max (L.) Merr.), is one of the world’s most important oilseed and economic crops, yet its cell-biology-oriented gene-function studies remain hampered by low transformation efficiency and poor detection of fluorescent tags such as GFP. Researchers therefore routinely resort to heterologous systems like Arabidopsis or Nicotiana benthamiana , risking mis-localization and artifactual activity of soybean proteins and compromising physiological relevance. A robust, soybean-based platform is urgently needed. In this study, we introduce FLASH ( F luorescent L ocalization A ssay for S ubcellular in H airy-root-derived callus protoplasts). Transgenic hairy roots expressing fluorescent proteins are bulked into callus, from which protoplasts are isolated in a single step, > 80% of cells display bright, stable fluorescence within 24 h. Using FLASH we monitored, in real time, light-induced assembly of GmCRY photobodies in both the nucleus and cytoplasm, and confirmed nuclear co-localization of GmCRY1b with STF1. FLASH offers a high-fidelity, high-throughput solution for subcellular localization, protein–protein interaction and phase-separation studies of soybean proteins.