Isolation and Transfection of Nicotiana benthamiana Mesophyll Protoplasts

Protoplasts, being plant cells without cell walls, can efficiently uptake large macromolecules such as DNA, RNA, and proteins. This feature makes them exceptionally effective for transient gene expression experiments. This chapter provides a detailed protocol for the efficient isolation of mesophyll protoplasts from the model plant Nicotiana benthamiana. It also covers the subsequent transfection of these isolated protoplasts using PEG-CaCl₂ method with plasmid DNA, offering a comprehensive guide to ensure high-quality protoplast preparation and transfection for a successful genetic manipulation.