An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1

Background: Perennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system. Results: In this report, a highly efficient protoplast isolation (5.6 x 10(7) protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein-protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time. Conclusion: This efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein-protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.