(135b) 1-Dimensional Carbon Nanoparticles for Functional Biomolecule Delivery to Mature Plants

Plants are at the core of environmental sustainability efforts, natural product synthesis of pharmaceuticals, and food security for a growing population under a changing global climate. Traditional plant breeding strategies are slow, laborious, and lack precise control over plant genotype and phenotype. Over the past several decades, remarkable progress has been made in biotechnology with the improvement of genome editing and sequencing tools. Owing to these recent advancements, plant bioengineering can be leveraged to create crops that are resistant to disease and drought. In pharmaceuticals and therapeutics, genetically engineered plants can be used to synthesize valuable small-molecule drugs and recombinant proteins. Furthermore, bioengineered plants may provide cleaner and more efficient biofuels.

Despite the promise of plant biotechnology, the presence of the plant cell wall has posed a transport limitation to delivery of exogenous biomolecules to plant cells, such that conventional methods of biomolecule delivery (Agrobacterium and gene gun) are inefficacious for plant biotechnology¹. Thus, the plant cell wall is the primary bottleneck of plant genetic engineering. To date, plant biotechnology lacks a method that allows passive delivery of diverse biomolecules into a broad range of plant species without the aid of external force and without causing tissue damage. We posit carbon nanotechnology as a key driver in the creation of a transformational tool to address delivery challenges in plants, and to enhance the throughput of plant genetic engineering.

Under certain surface chemistries, high aspect ratio nanomaterials such as carbon nanotubes (CNTs) have shown to traverse extracted chloroplast and plant membranes with several figures of merit: high aspect ratio, exceptional tensile strength, high surface area-to-volume ratio, and biocompatibility. When bound to CNTs, biomolecules are protected from cellular degradation, exhibiting superior biostability compared to free biomolecules. Herein, we present a CNT-based platform that permits diverse conjugation chemistries to deliver functional biomolecules into both model and consumer food crop plants with high efficiency and no toxicity². Surface-modified CNTs are covalently-functionalized with cationic polymers (such as polyethylenimine - PEI) to load DNA via electrostatic attraction, for DNA delivery into mature arugula (dicot) and wheat (monocot) leaves. Through this platform, we obtain strong transient protein expression, with efficiencies comparable to Agrobacterium-mediated and higher than gene gun gene delivery. We also show nanotube-based transient protein expression in arugula protoplasts with 85% transformation efficiency. Lastly, we achieve 95% transient gene silencing in Nicotiana benthamiana leaves through the delivery of small interfering RNA molecules with pristine SWCNTs. This study establishes efficient transient gene expression and silencing in mature plants through passive carbon nanotube-mediated delivery of functional biomolecules, and can enable high-throughput genetic plant transformations for a variety of plant biotechnology applications.

1. Demirer, G.S., Landry, M.P. Delivering Genes to Plants. AIChE SBE (2017)
2. Demirer, G.S. et al. (2018) High Aspect Ratio Nanomaterials Enable Biomolecule Delivery and Transgene Expression or Silencing in Mature Plants. bioRxiv DOI: 10.1101/179549