Abstract
Mesophyll protoplasts freshly isolated from leaves are a useful research system in plants. However, cell walls in woody plants contain more pectin, making mesophyll protoplasts isolation difficult in Populus. This has limited their application in biochemical, molecular, cellular, genetic, genomic, transcriptomic, and proteomic assays. In this protocol, a simple and efficient method to prepare and transfect mesophyll protoplasts of Populus tomentosa is presented in detail. Leaves of P. tomentosa plants grown in tissue culture media were pre-treated in D-mannitol solution and then digested with an enzyme solution. After washing with W5 and MMg buffers, the protoplasts were incubated in PEG/Ca2+ solution with plasmid for transfection. The mesophyll protoplasts isolated were used to express the histone variant H2B fused with green fluorescent protein (GFP) for confocal microscopy imaging. This "P. tomentosa mesophyll protoplasts preparation and transfection" system provides a useful tool for studying woody plants using a variety of applications, including gene expression, subcellular localization, protein-protein interaction, chromatin immunoprecipitation, western blot, single-cell sequencing, and genome editing.