IMR Press / FBL / Volume 29 / Issue 5 / DOI: 10.31083/j.fbl2905187
Open Access Original Research
Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver
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1 Key Laboratory of Plant Resources Conservation and Utilization, College of Biological Resources and Environmental Sciences, Jishou University, 416000 Jishou, Hunan, China
2 College of Biology and Environmental Sciences, Jishou University, 416000 Jishou, Hunan, China
3 National and Local United Engineering Laboratory of Integrative Utilization of Eucommia ulmoides, 416000 Jishou, Hunan, China
*Correspondence: zhouqiang@jsu.edu.cn (Qiang Zhou)
Front. Biosci. (Landmark Ed) 2024, 29(5), 187; https://doi.org/10.31083/j.fbl2905187
Submitted: 6 January 2024 | Revised: 17 February 2024 | Accepted: 24 April 2024 | Published: 14 May 2024
Copyright: © 2024 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
Abstract

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.

Keywords
E. ulmoides
stem
protoplast isolation
transient transformation
Funding
32160388/National Natural Science Foundation of China
Figures
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