Development of a protoplast isolation system for functional gene expression and characterization using petals of...

Protoplasts preparation and purification have been frequently used in plant genetics and breeding studies, whereas application of protoplasts in woody plants is still in its infancy. Although transient gene expression using purified protoplasts is well-documented and widely used in model plants and agriculture crops, no instance of either stable transformation or transient gene expression in the woody plant Camellia Oleifera has as of yet been reported. Here, we developed a protoplast preparation and purification method using C. oleifera petals by optimizing osmotic condition with D-mannitol and polysaccharide-degrading enzyme concentrations for petal cell wall digestion, to reach a high efficiency of protoplast productivity and viability. The achieved protoplasts yield was approximately 1.42 × 10⁷ cells per gram of petal material and the viability of protoplasts was up to 89%. In addition, we explored influencing factors of protoplast transformation, including concentrations of PEG4000 and plasmid DNA. The transformation efficiency of 81% could be reached under the optimized condition. This protoplast isolation and transient expression system were deployed to further identify the functional regulation of C. oleifera related genes and the subcellular distribution of their encoded products. In summary, the protoplast isolation and transient expression system we established using oil-tea tree petals is an efficient, versatile and time-saving system, being suitable for gene function characterization and molecular mechanism analysis.