DNA Delivery to Intact Plant Cells by Casein Nanoparticles with Confirmed Gene Expression

This research presents gene expression after DNA delivery into intact plant cells by protein nanoparticles. The DNA delivery is carried out by casein nanoparticles (CNPs). A plasmid harboring the red fluorescent protein DsRed sequence is absorbed to the CNPs surface by electrostatic interaction and served as a model DNA in this study, and its expression is monitored by the fluorescence of the DsRed protein. The zeta potential of the CNPs is tuned by altering the pH to obtain sufficient electrostatic interaction between the CNPs and the DsRed plasmid for successful DNA delivery into the cells of the model plant Nicotiana benthamiana. The CNPs are covalently modified with the green fluorescent dye 6-Aminofluorescein (6-AF) to determine their location in the plant. To assess the ability of the CNPs to deliver DNA into the cells, CNP/6-AF/DsRed plasmid electrostatic conjugates are infiltrated into N. benthamiana leaves. Confocal fluorescence microscopy results showed successful intracellular and nucleus uptake of the conjugates at pH 4.5 and a concentration of 2 mg mL⁻¹ at CNPs: DsRed plasmid ratio of 1:0.01. The successful gene expression is confirmed by RT-PCR and qRT-PCR. The first appearance of the emitted red signal of the DsRed protein is observed 24 h post-infiltration.