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

Qianyi ZHOU,Fei ZHAO,Tian ZHANG,Yiming LI,Zhaohong JIANG,Zhong ZHAO,Hailan ZHU

doi:10.3906/tar-1805-62

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 × 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.

Highlights

The protoplast is a naked bioactive cell from which the cell wall has been removed, usually by enzyme digestion
The effects of different enzyme combinations and enzyme concentrations on protoplast isolation We chose five enzymes according to the published literature (Table 1) and the results of preliminary experiments
For the protoplast isolation results, experiments E2, E3, and E5 indicated that Cellulase R-10, Pectolyase Y-23, and ligninase are indispensable for ensuring that protoplasts can be isolated

Summary

Introduction

The protoplast is a naked bioactive cell from which the cell wall has been removed, usually by enzyme digestion. Protoplasts can take up the source material of protoplasm such as DNA, plasmids, viruses, bacteria, and organelles in a variety of ways, including heat shock, electroporation, bacteria incubation, and high pH. These features make protoplasts a useful tool for genetic manipulation and gene transfer in plant cell engineering (Puite, 1992). Since the first isolation by Cocking in tomato root tips in 1960 (Cocking, 1960), additional progress in isolating the protoplasts of other horticultural plants, including wheat, barley, corn, soybean, rice, cotton, and potato, has been reported.

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