CRISPR/Cas9-mediated knockout of NYC1 gene for the development of stay-green variety in Zoysia matrella (L.) Merrill

Abstract Zoysia matrella is a widely used C4 warm-season turfgrass for landscaping, golf courses, and sports fields. Here, we used the CRISPR/Cas9 system to targetthe Non-Yellow Coloring1 (ZmNYC1) gene in the highly heterozygous allotetraploid Z. matrella 'Wakaba', aiming to generate a novel stay-green variety. Of 441 Agrobacterium-infected calli, 22 (5.0%) were transformed, and 14 of these (63.6%) showed targeted mutations through cleaved amplified polymorphic sequences analysis. Sequencing analysis revealed mutations mostly consisting of 1 or 2 bp indels, occurring 2 to 4 bp upstream of the PAM sequence. Regenerated plants exhibited five ZmNYC1 target locus genotypes, including homozygous mutants with a complete knockout of all four alleles in the T0 generation. Under dark treatment, ZmNYC1-mutated plants displayed suppressed chlorophyll b (Chl b) degradation, leading to higher chlorophyll content and Chl b, with a lower chlorophyll a/chlorophyll b ratio compared to the wild type (WT). Additionally, during winter simulation, mutant with a complete knockout retained greenness longer than the WT. This is the first successful use of CRISPR/Cas9 genome editing in zoysiagrass. The knockout mutants of the ZmNYC1 gene would serve as valuable breeding material for developing improved zoysiagrass varieties that can maintain their green color for longer periods, even during winter dormancy.