Mesophyll protoplast isolation technique and flow cytometry analysis of ancient Platycladus orientalis (Cupressaceae)

Platycladus orientalis (Cupressaceae) is a native tree species used widely for landscaping and afforestation in arid and semiarid areas in Asia. Ancient P. orientalis trees not only have an important humanistic and historical value but also have a significant scientific value in aging mechanism research. Mesophyll protoplasts are an important material for studies of plant regeneration, transient gene expression, and senescence. Although an easy and effective technique of mesophyll protoplast isolation from mature trees is urgently needed, the isolation parameters have great material specificity. The young tissue of plants is an ideal material for protoplast preparation. In this study, we employed an orthogonal experiment and several single-variable experiments to determine the main factors influencing the successful isolation of mesophyll protoplasts and established an efficient technique for isolating mesophyll protoplasts from the young scale leaves of ancient P. orientalis. The optimal parameters for mesophyll protoplast isolation are as follows: mechanical homogenization of the leaf tissue, 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, 1.0% ligninase, 0.7 M mannitol (pH 5.8), and 16 h of incubation. Two centrifugations (100 x g for 3 min and 500 x g for 5 min) were repeated 2 times to obtain purified protoplast suspension. The yield and viability of protoplasts under optimal conditions were 4.8 x 106 g FW-1 (per gram fresh weight) and 82.5%, respectively. The results of flow cytometry analysis showed that the isolated protoplasts had ideal viability to meet the demands of further protoplast-based research.