Highly efficient endosperm and pericarp protoplast preparation system for transient transformation of endosperm-related genes in wheat

Plant protoplasts constitute a versatile system for transient gene expression and have frequently been used in high-throughput to screen and identify functional characterization of plant genes. Wheat ( Triticum aestivum L.) is one of the most important crops for our daily life. Endosperm-trait related genes are associated with grain yield or quality in wheat. However, very few studies have explored on the use of protoplasts isolated from endosperm and pericarp tissue of developing grain. In this study, endosperm tissues of developing wheat grains at 8 DPA (days post-anthesis) were collected. It was shown that, after being digested with the enzymolysis solution containing 13% mannitol for 2 h, total 1.1×10 5 of intact protoplasts containing 80% vital individuals were isolated from 0.6 g samples. Pericarp protoplasts were successfully purified from wheat grains at 4 DPA using the optimized method. To identify the activity of the protoplasts, transcription factor TaABI5 and amyloplast protein TaSSIIIa were transfected to the protoplasts, and they were successfully localized in the nucleus and the surface of starch granule, respectively. It is an effective and reproductive method for endosperm and pericarp protoplast isolation and of great importance to further investigate gene’s functions and regulations related to endosperm development and differentiation in plants.