Protoplast Isolation and Transfection in Maize

Protoplast-based transient gene expression platforms can be used to study a range of questions concerning gene regulation. Crucial to the success of these studies is the isolation of large quantities of healthy protoplasts from the tissue of interest. Herein, we describe protocols for isolating and transfecting maize mesophyll protoplasts for gene expression studies. The isolation protocol yields approximately 1.8-1.9 × 10⁷ protoplasts with 80-90% viability from 6 g of etiolated leaf tissue, and the polyethylene glycol-mediated transfection protocol results in 55-58% transfection efficiency. The transfection protocol describes the use of a dual-expression vector that carries the coding sequence for two fluorescent proteins (FPs), one driven by a constitutive promoter for normalization for transfection efficiency and the other driven by the construct of interest. The use of a dual-FP expression vector eliminates the need for co-transfection and separate steps for enzymatic/substrate processing as required for luciferase-based assays. These protocols have been tested on leaf tissue from the maize genotypes B73 and PHR03 and, as written, can be completed in 24 h.