Nanoparticle-Guided Biomolecule Delivery for Transgene Expression and Gene Silencing in Mature Plants

Abstract

Genetic engineering of plants is at the core of environmental sustainability efforts, natural product synthesis of pharmaceuticals, and agricultural crop engineering to meet the needs of a growing population and changing global climate. The physical barrier presented by the cell wall has limited the ease and throughput with which exogenous biomolecules can be delivered to plants. Current techniques suffer from host range limitations, low transformation efficiencies, toxicity, and unavoidable DNA integration into the host genome. The central aim of our research is to bring plant science up to speed with modern genome editing biology. Herein, we demonstrate efficient diffusion-based plasmid DNA and small interfering RNA (siRNA) delivery into two species of mature plants with a suite of pristine and chemically-functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong transient protein expression is accomplished in mature Eruca sativa (arugula) leaves with covalently-functionalized or pristine single-walled and multi-walled carbon nanotubes, with efficiencies comparable to agrobacterium. We also demonstrate nanotube-based transient protein expression in cell wall-free arugula protoplasts with 85% transformation efficiency. Lastly, we demonstrate a second nanoparticle-based strategy in which siRNA is delivered and activated in the Nicotiana benthamiana plant cell cytosol, effectively silencing a gene with 95% efficiency. Our work provides a promising tool for species-independent, targeted, and passive delivery of genetic material, without transgene integration, into plant cells for rapid and parallelizable testing of plant genotype-phenotype relationships.