An Efficient System for Mesophyll Protoplast Isolation, Purification, and Transformation in Loquat: Studies on Fluorescent Marker Analysis and Subcellular Localization

Loquat (Eriobotrya japonica Lindl.) is one of the most important subtropical evergreen fruit trees. However, due to the lack of widely applicable genetic transformation platforms, the research about gene functional characterization and molecular mechanisms is largely confined. In this study, the efficient protocol of protoplast isolation (the enzyme solution composed of 2.4% macerozyme R-10, 4.8% cellulase RS, dissolved in a 0.6 M mannitol solution) and the method of protoplast purification (CPW solution containing 5% sucrose and 11% mannitol) have been achieved with protoplast yields of 12.6 × 106/g·FW, reaching a viability rate of up to 91%. A protoplast transient gene expression system has been established with an efficiency of approximately 40% using GFP reporter gene. Using this reliable and efficient system, the protein localization characteristics of transcription factor EjDELLA, EjbHLH79, and marker gene OsPHT4 were also utilized for further analysis. To our knowledge, this is the first report on establishing an efficient system for protoplast isolation, purification, and transformation of loquat mesophyll. The system reported here will definitely promote rapid progress in breeding, genetic transformation, and molecular research.