Single-walled carbon nanotube-mediated plasmid DNA delivery in the model monocot Brachypodium distachyon

Plant genetic engineering is a valuable approach for improving stress tolerance, crop productivity, and quality traits. However, its progress is limited by the drawbacks of traditional transformation methods. Commonly used techniques, such as Agrobacterium-mediated transformation and biolistics, are restricted by genotype dependency, lengthy tissue culture steps, and low efficiency, particularly in monocot species. Therefore, the development of alternative gene delivery strategies remains an ongoing research focus in plant biotechnology. Nanoparticle-based delivery systems have recently gained attention due to their physiochemical properties, biocompatibility, and potential for alternative gene delivery approaches in plant systems.

We investigated the use of single-walled carbon nanotubes (SWCNTs) as nanocarriers for plasmid DNA delivery in the model monocot plant Brachypodium distachyon. SWCNTs were functionalized with polyethyleneimine (PEI) and plasmid DNA (pCAMBIA1301) was loaded via electrografting. Successful complex formation was confirmed with transmission electron microscopy (TEM), atomic force microscopy (AFM), and zeta potential measurements.

The TEM and AFM analyses revealed that the SWCNTs retained a thin, elongated needle-like structure, consistent with their ability to pass through the plant cell wall. Zeta potential measurements showed a negative surface charge of -43 mV for COOH-SWCNTs that shifted to +56 mV after PEI functionalization, enabling electrostatic binding of plasmid DNA. pDNA-SWCNT complexes at different mass ratios were applied to both callus and seed explants to evaluate their delivery potential. Successful delivery was confirmed by b-glucuronidase (GUS) reporter gene expression in both callus tissues and seeds, with ImageJ-based quantification showing that the 3:1 SWCNT:pDNA ratio yielded the highest mean signal intensity.

Our findings show that the SWCNT-based system enabled the delivery of a large plasmid (~12 kb), supporting transient reporter gene expression in monocot tissues. Overall, the data suggest that SWCNT-mediated delivery represents a promising technique for DNA delivery in monocots, and further optimization is expected to improve its efficiency.