Establishment of a PEG-Assisted Protoplast Transfection System in Musa acuminata cv. Berangan (AAA) Using a CRISPR/Cas9 Ribonucleoprotein Complex

Banana is an important food crop worldwide. However, as banana production is largely affected by pests, diseases, and environmental stresses, there is a need to develop stress-resistant banana varieties. Although modern breeding and conventional genetic engineering techniques exist, they may not provide sufficient precision or speed in introducing desired traits and enhancing banana resilience. Hence, this study focuses on the development of an efficient protoplast isolation and DNA-free CRISPR/Cas9 ribonucleoprotein-mediated protoplast transformation protocols for Berangan cultivar. A total of 1.54 × 10 7 protoplasts/g FW were isolated from immature male flower buds using an enzymatic mixture of 1% cellulase RS, 1% macerozyme R-10 and 0.15% pectolyase Y-23. Applying 10 min-vacuum infiltrations twice and 0.5 M mannitol significantly increased the number of protoplasts. The isolated protoplasts were transfected with pC-AMBIA1304-GFP and CRISPR/Cas9 ribonucleoprotein complex targeting the stress-related banana Sugar Transport Protein 13 ( STP13 ) using a polyethylene glycol solution. Following 15 minutes of transfection, 76.89% of the treated protoplasts were GFP-positive. DNA sequence analysis confirmed the presence of small deletions (1–3 bp) at the target sites of STP13 with a mutation rate of 4.40–4.90%, indicating that the protocols are suitable for use to modify the genomes of protoplasts of bananas.