Protocol for real-time imaging, polar protein quantification, and targeted laser ablation of regenerating shoot progenitors in Arabidopsis

Summary: Here, we provide a protocol for real-time tracking of regenerating shoot progenitors, combined with polar protein quantification and targeted laser ablation of callus cells in Arabidopsis. Using Arabidopsis strains expressing GFP-labeled polar auxin efflux carrier, PINFORMED 1 (PIN1) protein, we detail steps to prepare the callus for time-lapse confocal imaging and track the progenitors expressing PIN1-GFP, followed by mapping and quantifying PIN1 polarity using Fiji/ImageJ. We then describe targeted laser ablation of cells and subsequent time-lapse imaging to study regeneration.For complete details on the use and execution of this protocol, please refer to Varapparambath et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.