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

Abstract

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 × g for 3 min and 500 × 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 × 10 6 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.

Genetic Diversity and Population Genetic Structure of Ancient Platycladus orientalis L. (Cupressaceae) in the Middle Reaches of the Yellow River by Chloroplast Microsatellite Markers

“…Platycladus orientalis (L.) Franco, belonging to the genus Platycladus (Cupressaceae) of evergreen coniferous tree species, is the main afforestation tree species in the arid regions of the Loess Plateau of Northwest China [4][5][6]. Due to its long lifespan, beautiful trunk, sacred status and important economic value, P. orientalis has been planted in gardens, temples and the vicinity of mausoleums since ancient times [7]. It is the most widely planted cemetery tree species in China [8].…”

Effect of Enzyme Treatments on Protoplast Isolation from Leaves of Vetiver (Vetiveria spp.)

“…Ratanasanobon and Seaton (2013) studied on protoplast isolation in Chamelaucium group plants with longer than 6 h of incubation time, protoplasts could not be released when treated with 2% cellulase and 1% macerozyme. Zhou et al (2019) reported that period of incubation significantly affects the yield and viability of protoplast in Platycladus orientalis, yield of viable protoplasts decreased after 16 h of incubation time. Different species have different requirements which influence the success of protoplast isolation.…”

Efficient protoplast isolation from ovule-derived embryogenic callus inCitrusvolkameriana

“…The most favorable protoplast isolation efficiency was determined in the combination of 2% cellulase and 0.2% pectinase formulated protoplast digestion solution.The cellulase concentrations appeared to be crucial for callus protoplast isolation. Zhou et al (2019) reported that different cellulase and pectinase concentrations in the protoplast digestion solution remarkably affected protoplast yield. However, using cellulase at a concentration of 4% reduced the number of protoplasts isolated from the ovule-derived embryogenic callus of C. volkameriana in the present study.…”