A protoplast-based method to visualize early cell biological events in plant cellular reprogramming and regeneration.

Plant developmental biology lacks cell-based experimental systems comparable to the organoids and live-imaging platforms that have transformed mechanistic discovery in animal research. To address this gap, we present a robust, trackable protoplast regeneration platform in Arabidopsis thaliana that enables high-resolution, time-resolved analysis from the single-cell stage through microcolony formation and early regenerative development. Protoplasts are embedded in thin alginate matrices containing fluorescent fiducial beads, maintaining physical separation and allowing repeated return to the same cells over days to weeks. This design supports long-term imaging using epifluorescence, confocal and lattice light-sheet microscopy, enabling visualization of cell-cycle re-entry, asymmetric division, organelle dynamics, dedifferentiation, redifferentiation and regenerative competence. Fluorescent reporters for nuclei, membranes, microtubules, Golgi and hormone signaling further permit observation of subcellular organization and signaling heterogeneity during early reprogramming. Together, this platform provides an accessible, scalable system for studying plant cellular plasticity at single-cell resolution and offers a foundation for plant organoid-like models. By enabling high-resolution study of regeneration from single cells, this method expands the experimental toolkit and supports efforts to overcome species- and genotype-dependent barriers to transformation and plant biotechnology.