We developed an optimized CRISPR/Cas9 gene editing system using maize mesophyll protoplasts to enable rapid evaluation of guide RNA (gRNA) activity. Using the tropical inbred line Tzi8, we improved protoplast isolation and transfection protocols, achieving high yields of 17.88 × 10⁶ viable protoplasts per gram fresh weight while extending post-transfection viability. Etiolated seedlings and vertical leaf cutting significantly enhanced protoplast recovery and viability. A transfection efficiency of ~ 50% was achieved using 10 µg of plasmid DNA; higher DNA inputs did not result in significant gains, resulting in a more resource-efficient approach. Protoplast viability was maintained for up to seven days post-transfection, allowing for downstream applications that require extended incubation. This optimized system was used to assess the editing efficiency of nine gRNAs targeting three key floral repressors (ZmCCT9, ZmCCT10, and ZmRap2.7) across four maize genotypes (Tzi8, CML277, B73, and B104). These floral repressor genes are involved in the photoperiod sensitivity of tropical maize, a major challenge in the effort to introduce tropical maize germplasm into temperate breeding programs. Editing efficiencies ranged from 0.4% to 23.7%, with some variation observed between gRNAs and genotypes. Although protoplast-based assays do not currently enable plant regeneration, this platform offers a rapid method for in vivo gRNA validation, reducing assay time from months to days. This work expands the gene editing toolkit for tropical maize, supporting efforts to overcome breeding barriers through gene editing.
