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Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important
Rui Ren1,2, Jie Gao1, Dongmei Yin2
1Guangdong Key Laboratory of Ornamental Plant Germplasm Innovation and Utilization, Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China.
Frontiers in Plant Science
|March 4, 2021
Summary
Researchers developed an efficient orchid protoplast isolation protocol using leaf base tissues, achieving high yields and over 80% transfection efficiency for molecular studies in orchids and other monocots.
Area of Science:
- Plant Science
- Molecular Biology
- Biotechnology
Background:
- Efficient protoplast isolation systems are crucial for plant research but remain challenging for many horticultural plants, including orchids.
- Orchids, widely cultivated for ornamental and medicinal purposes, lack well-established protoplast isolation and transient expression protocols.
Purpose of the Study:
- To establish a highly efficient protoplast isolation protocol for orchids and other monocot crops.
- To develop a reliable transient expression system for functional gene analysis in orchids.
Main Methods:
- Optimized enzymatic digestion conditions using specific concentrations of D-mannitol, cellulose, macerozyme, and 2-mercaptoethanol for 6-hour digestion.
- Isolated protoplasts from various orchid tissues (young leaves, flower pedicels, root tips) and monocot crops (maize, rice), focusing on leaf base tissues.
- Utilized polyethylene glycol (PEG)-mediated transfection for transient expression studies in isolated Cymbidium leaf base protoplasts.
Main Results:
- Developed a protocol yielding high numbers of viable protoplasts from leaf bases of various orchids (e.g., Cymbidium: ~2.50 × 107/g FW) and monocot crops (maize: 3.25 × 107/g FW, rice: 4.31 × 107/g FW).
- Achieved over 80% transfection efficiency using PEG-mediated delivery in Cymbidium leaf base protoplasts.
- Successfully applied the system to analyze CsDELLA-mediated gibberellin signaling, including subcellular localization and gene regulation studies via transient overexpression and silencing.
Conclusions:
- The established leaf base protoplast isolation and transient expression system is highly efficient and versatile for orchids and other monocot crops.
- This system offers a valuable tool for cellular and molecular research, particularly for species lacking efficient in vivo genetic transformation methods.
