Optimized protocol for isolation and transient expression of placenta-originated protoplast in pepper (Capsicum annuum L.)

Abstract

Pepper (Capsicum annuum L.) is one of the most widely cultivated species and is highly valued for its pungency. The pungency of pepper is primarily attributed to a group of chemical compounds known as capsaicinoids. These compounds are synthesized in the placental tissue of pepper fruits through the activation of specific genes and enzymes, thereby contributing to their pungency. Protoplast-based gene expression systems have been considered an efficient method for gene function studies, protein-protein interactions, promoter analysis, and subcellular localization. Here, we optimized an efficient protocol for isolating protoplasts from pepper placental tissues and the polyethylene glycol (PEG)-mediated transient expression of green fluorescent proteins (GFP). Several factors affecting GFP expression in intact protoplasts were evaluated and optimized in this study. Protoplast isolation was carried out using 2.0% cellulase “onozuka” R-10 and 0.3% macerozyme R-10 solution. Different amounts of plasmid DNA and various incubation times for transfection with 40% PEG resulted in different transfection efficiencies (78–85%). The highest GFP transformation efficiency was observed when 120 µL protoplast suspension (3 × 10⁷ to 5 × 10⁷/mL) was mixed with 15 µg plasmid DNA and incubated for 25 min with an equal volume of 40% PEG. The improved protocol described in this study can be helpful for the isolation and transfection of pepper placenta-originated protoplasts and for the rapid investigation of pepper gene functions.