An Efficient Fluorescence‐Activated Protoplast Sorting (FAPS) and Regeneration Protocol for Canola (<i>Brassica napus)</i>

Abstract Protoplast sorting and purification methods are powerful tools enabling the enrichment of cellular subpopulations for basic and applied studies in plant sciences. Fluorescence‐activated protoplast sorting (FAPS) is an efficient method to isolate specific protoplast populations based on innate features (size and autofluorescence) or expression of fluorescent proteins. FAPS‐based methods have recently been deployed in single‐cell purification for single‐cell RNA sequencing–based transcriptional profiling studies. Protoplast sorting methods integrated with the ability to culture and recover whole plants add value to functional genomics and gene editing applications. Enriching cells expressing nucleases linked to fluorescent proteins can maximize knockout or knockin editing efficiencies and minimize toxic and off‐target effects. Here, we report the protocol for protoplast preparation, sterile cell sorting, culture, and downstream regeneration of plants from canola protoplasts. This protocol can be successfully applied to all totipotent protoplast methods that can regenerate into whole plants. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Preparation of transfected canola protoplasts for sorting Basic Protocol 2 : Fluorescence‐activated protoplast sorting Basic Protocol 3 : Bead culture of sorted protoplasts and recovery of plantlets