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Updated: Jun 25, 2025
A Robotic Platform for High-throughput Protoplast Isolation and Transformation
Published on: September 27, 2016
Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver
Bin Hu1,2, Mingyang Dong1,2, Ruonan Liu2
1Key Laboratory of Plant Resources Conservation and Utilization, College of Biological Resources and Environmental Sciences, Jishou University, 416000 Jishou, Hunan, China.
Background:
Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides.
Methods:
In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method.
Results:
Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%.
Conclusions:
This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.
