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

Abstract

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 .

Code and data availability

This is a methods/protocol paper. It explicitly states 'This study did not generate/analyze any datasets or code.' The only software mentioned (Fiji/ImageJ, ZEN) are generic third-party tools, not authors' analysis code or paper-specific assets. No public phenotype datasets, images, models, or code deposits are present

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