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

Bibliographic Details
Title:	A protoplast-based method to visualize early cell biological events in plant cellular reprogramming and regeneration.
Authors:	Reed KM; School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA., Masri AA; School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA., Hanrahan AJ; School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA., Ng EL; Facility for Advanced Imaging and Microscopy, Fralin Life Sciences Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA., Li M; Donald Danforth Plant Sciences Center, St. Louis, MO 63132, USA., Bargmann BOR; School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Source:	Development (Cambridge, England) [Development] 2026 Sep 15; Vol. 153 (18). Date of Electronic Publication: 2026 Sep 15.
Publication Type:	Journal Article
Language:	English
Journal Info:	Publisher: Company Of Biologists Limited Country of Publication: England NLM ID: 8701744 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1477-9129 (Electronic) Linking ISSN: 09501991 NLM ISO Abbreviation: Development Subsets: MEDLINE
Imprint Name(s):	Publication: Cambridge Eng : Company Of Biologists Limited Original Publication: [Cambridge] : Company of Biologists, [c1987-
MeSH Terms:	Protoplasts/metabolism , Protoplasts/cytology , Protoplasts/physiology , Arabidopsis/cytology , Arabidopsis/physiology , Regeneration/physiology , Cellular Reprogramming/physiology , Plant Cells
Abstract:	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. (© 2026. Published by The Company of Biologists.)
Competing Interests:	Competing interests The authors declare no competing or financial interests.
Grant Information:	IOS 2314549 Division of Integrative Organismal Systems; IOS 2424273 Division of Integrative Organismal Systems; VA-160262 National Institute of Food and Agriculture; VA-136423 National Institute of Food and Agriculture; National Science Foundation
Contributed Indexing:	Keywords: Live imaging; Protoplast; Regeneration
Entry Date(s):	Date Created: 20260909 Date Completed: 20260915 Latest Revision: 20260915
Update Code:	20260915
DOI:	10.1242/dev.205715
PMID:	42714221
Database:	MEDLINE

Description
ISSN:	1477-9129
DOI:	10.1242/dev.205715