Related Experiment Video
Updated: Aug 15, 2025
10:12
A Robotic Platform for High-throughput Protoplast Isolation and Transformation
Published on: September 27, 2016
14.4K
A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts
Dongxu Yang1, Yongyu Zhao1, Yumei Liu1
1Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China.
Frontiers in Plant Science
|December 29, 2022
Summary
An efficient broccoli protoplast isolation and transient transformation system was developed. This method yields high-viability protoplasts for gene function studies and subcellular localization, advancing plant science research.
Area of Science:
- Plant Biotechnology
- Molecular Biology
- Genetics
Background:
- Transient transformation of plant protoplasts is crucial for gene function studies and understanding plant development.
- Optimizing key factors is essential for efficient protoplast transformation.
Purpose of the Study:
- To establish an efficient and universal broccoli protoplast isolation and transient transformation system.
- To optimize plasmid concentration, PEG4000 concentration, and genotype selection for enhanced transformation efficiency.
Main Methods:
- Optimized enzyme concentrations (cellulase R-10, pectolyase Y-23) and hydrolysis time for broccoli cotyledon protoplast isolation.
- Evaluated transformation efficiency using two plasmid vectors (PHG-eGFP, CP507-YFP) in broccoli B1 protoplasts.
- Performed subcellular localization of three target genes (CRa, Bol029100, Bol031350) using the developed system.
Main Results:
- Achieved high protoplast yield (>5×106/mL/g) and viability (>95%).
- Demonstrated high average transformation efficiencies of 61.4% for PHG-eGFP and 41.7% for CP507-YFP.
- Successfully localized the products of all three target genes to the nucleus.
Conclusions:
- The developed system provides high-yield, high-viability broccoli protoplasts for reliable transient transformation.
- This research offers significant technical support for advanced plant science research, including single-cell sequencing and gene function analysis.
