Related Experiment Video
Updated: Dec 8, 2025
A Robotic Platform for High-throughput Protoplast Isolation and Transformation
Published on: September 27, 2016
Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein
Shaoping Wu1,2, Haocheng Zhu3,4, Jinxing Liu3
1College of Horticulture, Hunan Agricultural University, Changsha, China.
Background:
To date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system.
Results:
Protoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems.
Conclusions:
The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.
More Related Videos
12:59High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
Published on: April 30, 2016
09:22A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses
Published on: January 25, 2018
Related Concept Videos
01:28CRISPR/Cas9 Genome Editing
02:50Transgenic Plants
The first-ever transgenic plant was a tobacco plant developed in 1983 that showed resistance against the tobacco mosaic virus. Since then, many transgenic plants have been developed and commercialized for improving the agricultural, ornamental, and horticultural value of a crop plant. Transgenic...
01:59CRISPR