PEGylated Positively Charged Fluorescent Silica Nanoparticles for Enhanced Gene Delivery in Canola ( Brassica napus )

Genetic transformation of Brassica napus (canola) using conventional methods remains limited by low efficiency and tissue-specific barriers, but nanoparticle (NP)-mediated delivery offers a promising complementary approach to overcome these limitations. In this study, a PEGylated, positively charged fluorescent silica nanoparticle (SiO2NP–TMAC/FITC/PEG) was developed and evaluated for DNA delivery and real-time tracking in canola protoplasts and microspores. This multifunctional platform integrates quaternary ammonium groups for efficient DNA binding and enhanced cellular interaction, FITC for fluorescence-based monitoring, and PEG for improved stability, reduced cytotoxicity, and enhanced delivery performance. Together, these features enable a robust and versatile nanocarrier system capable of efficiently delivering plasmid DNA while allowing simultaneous tracking of cellular internalization and transformation efficacy. Protoplasts were isolated from both leaf and hypocotyl tissues, and microspores were obtained from unopened floral buds. Fluorescent reporter plasmids were complexed with functional SiO2NPs, and uptake efficiency and cell viability were assessed using fluorescein diacetate and trypan blue staining. Among the four nanocarrier designs, PEG surface modification improved DNA uptake and cell viability compared with non-PEGylated SiO2 nanoparticles. The multifunctional SiO2NP–TMAC/FITC/PEG platform showed the best delivery and tracking performance, demonstrating a strong synergistic effect and highlighting its promise for efficient gene delivery. Among protoplast sources, hypocotyl-derived protoplasts exhibited superior transformation efficiency and more sustained expression than leaf-derived protoplasts, while in microspores, developmental stages strongly influenced uptake, and larger buds (3–4 mm) were more amenable to NP transformation. Transient transformation results demonstrate the feasibility of NP-mediated gene delivery in canola and provide practical insights for optimizing dose, exposure time, and cell type. These data will allow for the integration of NP delivery with established transformation methods and could further enhance efficiency and facilitate future efforts toward stable integration in B. napus.