Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in Catalpa bungei

Catalpa bungei is an important ornamental plant and timber species that originated in China. With the forthcoming C. bungei genome sequence, an efficient and convenient transformation system should be developed. The transient gene expression system using protoplasts is fast and efficient and can be applied to study the functions of genes in C. bungei. In this study, the main factors influencing the isolation of protoplasts from C. bungei were optimized by orthogonal design. The results showed that when the enzymolysis solution contained 3% Cellulase RS (w/v), 2% Macerozyme R-10 (w/v), and 0.4 M mannitol and the enzymolysis time was 8 h, the protoplast yield was 1 × 106 protoplasts/g fresh weight [FW]. The viability of protoplasts was approximately 90 % under optimal enzymolysis conditions. Furthermore, using GFP as the reporter gene, we verified the feasibility of PEG-mediated protoplast transformation of C. bungei. We established an efficient protoplast isolation procedure and feasible transient gene expression system. This methodology, when combined with genetics and omics techniques, will allow further study of the functions of the genes in C. bungei.