Developing sustainable and eco-friendly approaches to plant propagation, development, and protection is a common goal for the scientific community. Plant cell culturing enables us to obtain plant clones and produce biomolecules under controlled conditions. The same principle can be applied to the harvesting of extracellular vesicles (EVs). These nanosized structures are key players in cell communication and stress response by carrying, protecting and delivering important biomolecules. Raising interest in the scientific community, EVs have been tested successfully as nanocarriers in therapeutics and biotechnology. However, despite their potential, there remains a gap in research regarding scalable and reliable sources for EV production. Our goals were to optimize EV production and isolation from induced poplar callus cell lines (Populus tremula x P. alba) and load these with RNA to validate their functionality as nanocarriers. We were able to isolate 2.5 x 1010 EVs/g, highlighting the potential of these lines to mass produce them. Furthermore, RNA loaded into EVs through electroporation was internalized into Botrytis cinerea hyphae, reassuring their potential in protecting and delivering cargo. Our findings contribute to EV characterization and demonstrate that RNA-delivery through EV transport could be a safe and effective method for future EV-based technologies in plant protection.