Protoplast Preparation and Fluorescence-Activated Cell Sorting for the Evaluation of Targeted Mutagenesis in Plant Cells

Ward Decaestecker; Norbert Bollier; Rafael Andrade Buono; Moritz K Nowack; Thomas B Jacobs

doi:10.1007/978-1-0716-2164-6_15

Protoplast Preparation and Fluorescence-Activated Cell Sorting for the Evaluation of Targeted Mutagenesis in Plant Cells

Methods Mol Biol. 2022:2464:205-221. doi: 10.1007/978-1-0716-2164-6_15.

Authors

Ward Decaestecker^{1

2}, Norbert Bollier^{1

2}, Rafael Andrade Buono^{1

2}, Moritz K Nowack^{1

2}, Thomas B Jacobs^{3

4}

Affiliations

¹ Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
² VIB Center for Plant Systems Biology, Ghent, Belgium.
³ Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium. [email protected].
⁴ VIB Center for Plant Systems Biology, Ghent, Belgium. [email protected].

PMID: 35258835
DOI: 10.1007/978-1-0716-2164-6_15

Abstract

Fluorescence-activated cell sorting (FACS) allows for the enrichment of specific plant cell populations after protoplasting. In this book chapter, we describe the transformation and protoplasting of an Arabidopsis thaliana cell suspension culture (PSB-D, derived from MM2d) that can be used for the evaluation of CRISPR vectors in a subpopulation of cells. We also describe the protoplasting of Arabidopsis thaliana cells from the roots and stomatal lineage for the evaluation of tissue-specific gene editing. These protocols allow us to rapidly and accurately quantify various CRISPR systems in plant cells.

Keywords: CRISPR; Cell suspension cultures; FACS; Lateral root cap; Protoplasts; Stomata.

MeSH terms

Arabidopsis* / genetics
CRISPR-Cas Systems
Flow Cytometry / methods
Mutagenesis
Plant Cells
Protoplasts*