Protoplasts Isolation and Transient Transformation System Optimization for Poplar 84K (Populus alba × Populus...

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Forest Genetics

Background:

• Stable genetic transformation in poplar is time-consuming, hindering rapid gene functional analysis.
• Efficient gene function studies are crucial for molecular breeding in poplar.

Purpose of the Study:

• To optimize a high-yield protoplast isolation and transient transformation protocol for poplar.
• To establish a rapid and reliable platform for gene functional studies in poplar.

Main Methods:

• Systematic optimization of enzymatic digestion for poplar leaf protoplast isolation.
• Refinement of polyethylene glycol (PEG)-mediated transformation parameters for protoplasts.
• Evaluation of protoplast yield, viability, and transfection efficiency.

Main Results:

• An optimal enzymatic solution (3% cellulase R-10, 0.3% macerozyme R-10, 0.8% pectolyase R-10, 0.4 M mannitol) yielded 12.9 × 10^6 protoplasts/g FW with 93.45% viability after 3h digestion.
• Optimized PEG-mediated transformation (60 µg DNA, 40% PEG 4000, 20 min incubation) achieved 68.67% transfection efficiency.
• The protocol provides high-yield protoplast isolation and efficient transient transformation.

Conclusions:

• The developed transient expression system is reliable, rapid, and effective for poplar gene functional studies.
• This platform supports analyses like subcellular localization, protein-protein interactions, and gene expression.
• The optimized protocol facilitates molecular breeding applications in poplar.