Improving perennial ryegrass transformation protocols with developmental regulators

Abstract The development of genetically modified (GM) or gene edited (GE) turfgrass requires transformation systems that are both efficient and broadly applicable across genotypes. However, traditional Agrobacterium -mediated callus culture methods remain limited by low transformation efficiency, extended culture durations, and strong genotype dependence. In this study, we compare a modified classical callus culture protocol with an approach that incorporates the developmental regulator genes WUSCHEL2 ( wus2 ) and BABY BOOM ( bbm ), along with an inducible Cre recombinase and a dual luciferase assay to test variable promoter strengths in perennial ryegrass ( Lolium perenne L.). We show that traditional protocols failed to regenerate plants, despite successful callus formation and transgene expression. In contrast, the developmental regulator system enabled efficient callus induction and plant regeneration independent of genotype. This optimized protocol significantly reduces the time and genotype constraints of perennial ryegrass transformation, offering a practical platform for advanced genetic engineering applications of an important turf and forage grass.