Protoplast isolation and transfection in a 96-well plate

The method for protoplast isolation is based on the "Tape-Arabidopsis Sandwich” protocol reported by Wu et al. 2009 Plant Methods (https://doi.org/10.1186/1746-4811-5-16 ). The Tape Sandwich method is recommended for preparing large numbers of protoplasts due to the ease with which it releases protoplast cells from a high number of leaves. The transfection method is based on Yoo et al., (2007) "Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis" Nature Protocols (https://doi.org/10.1038/nprot.2007.199 ) This protocol provides a detailed description of the isolation, handling and pipetting steps necessary to obtain healthy protoplasts and also consistent transfection results in 96 well plates.