Enhancing genetic modification in recalcitrant plants: An investigation in chili (<i>Capsicum annuum</i>) through the optimized tape sandwich protoplast isolation and polyethylene glycol-mediated transfection

Chili presents challenges for <i>Agrobacterium</i>-mediated transfection due to its highly recalcitrant nature. One way to overcome this challenge is by using PEG-mediated transfection of protoplasts, which enhances the likelihood of successfully introducing transgenes into the cells. The tape sandwich method for isolating chili leaf protoplasts was optimized by adjusting enzyme concentrations and incubation duration, resulting in a high yield of 1.3×10⁶ cells ml^-1 per 0.1 g of leaves. The efficiency of transfecting GFP-encoding plasmids and Cas9 protein using PEG molecules of different sizes was also examined. The highest plasmid transfection efficiency was achieved with 5 µg of plasmid in 50 µl^-1, with an average efficiency of 48.71%. For Cas9 protein transfection, the most effective treatment involved using 1000 µg of protein in 100 µl^-1, mediated by 40% PEG 4000, resulting in an average efficiency of 2.94% due to protein aggregation. Nevertheless, this optimized protocol reduces the time required for chili protoplast isolation and enhances plasmid transfection efficiency by nearly 50%.