An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts.

Sun Sun,Li Li,Wang Wang,Chen Chen,Jiang Jiang,Zheng Zheng,Tang Tang,Liang Liang,Yuan Yuan,Luo Luo,Gong Gong,Zhang Zhang

doi:10.3390/plants8100385

Sun Sun, Li Li + Show 10 more

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https://doi.org/10.3390/plants8100385

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Abstract

In this study, we report the isolation and purification of protoplasts from Chinese kale (Brassica oleracea var. alboglabra) hypocotyls, and their transient gene expression transformation and subcellular localization of BaMYB75 (Bol042409). The upshot is that the vintage protocol included 5-d hypocotyls that were enzymatically hydrolyzed for 8 h in enzyme solution (3.0% cellulase, 0.5% pectolase, and 0.5 M mannitol), and the protoplasts were purified by precipitation. The total yield of protoplasts was 8 × 105 protoplast g−1 fresh weight, and the protoplasts’ viability was 90%. The maximum transformation efficiency obtained by using green fluorescent protein (GFP) as a detection gene was approximately 45% when the polyethylene glycol (PEG)4000 concentration was 40% and transformation time was 20 min. In addition, BaMYB75 was ultimately localized in the nucleus of Chinese kale hypocotyl protoplasts, verifying the validity and reliability of this transient transformation system. An effective and economical hypocotyl protoplast isolation, purification, and transformation system was established for Chinese kale in this study. This effectively avoided interference of chloroplast autofluorescence compared to using mesophyll cells, laying the foundation for future research in the molecular biology of Brassica vegetables.

Highlights

The plant protoplast refers to the entire cell without the cell wall, which is capable of absorbing foreign substances such as DNA plasmids and viruses [1,2,3,4]
The combination of 3.0% cellulase and 0.5% pectolase was determined to be the optimal combination of enzymes to isolate protoplasts from Chinese kale hypocotyls
The results showed that the BaMYB75 gene was detected in the nucleus of hypocotyl protoplasts

Summary

Introduction

The plant protoplast refers to the entire cell without the cell wall, which is capable of absorbing foreign substances such as DNA plasmids and viruses [1,2,3,4]. Mechanical and enzymatic methods have been used to separate protoplasts. The process of mechanical protoplast separation is very complicated and the yield is low, while the enzymatic method separates a large number of protoplasts in a short time. The optimal combination of enzyme solution and digestion time differs between species [5,6], making it necessary to develop an effective and suitable separation scheme for each species. Compared with stable gene expression in transgenic plants, transient gene expression is faster and more convenient [7,8]—especially using CRISPR/Cas technology—and the advantage of transient

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