UC Berkeley

Losses in crop yield from climate variations and proliferation of insects and pathogens threaten to leave a large proportion of the world’s population vulnerable, predominantly those from low-income regions whose livelihoods are dependent on agribusiness. Moreover, rising populations and increasing reliance on plants for energy, food, medicine, and chemicals provide additional stress on our conventional agricultural system. To ensure agricultural sustainability and food security, there is a cogent need to engineer crop varieties with traits such as abiotic and biotic stress resistance and increased production of useful plant products. Traditional plant breeding to produce cultivars with desired phenotypes is too time and resource consuming to support future sustainability, and breeding lacks controls over acquired traits other than the traits of interest. Plant genetic engineering is a promising alternative to traditional plant breeding, though is primarily limited by the efficient delivery of genetic engineering biomolecules such as RNA, DNA, and proteins across plant biological barriers. This includes the cellulosic and multi-layered plant cell wall and double-layered membrane of the cell, nucleus, chloroplast, and mitochondrion. Nanoparticles have emerged as promising materials for use as biomolecule carriers into plant systems. Owing to their highly tunable chemical and physical properties, nanoparticles can be synthesized and functionalized to achieve targeted localization and cargo release. The full potential of nanoparticles in agriculture remains underexplored; nanomaterials and conjugation approaches have yet to be tested, and there remains a lack of design heuristics towards engineering nanoparticles in agriculture. Functional nanoparticle design is a complex, multivariable optimization process that necessitates a fundamental understanding of nanoparticle interactions with plants across various length scales, as well as probing structure-function relationships.

This dissertation presents a holistic overview of RNA, DNA, and protein delivery to plants. I develop a microscopy and molecular biology-based workflow for probing nanoparticle-plant interactions, specifically, assessing how nanoparticle morphology impacts transport within a plant leaf and their cargo delivery capabilities. This study focused on using gold nanoparticles for foliar delivery of small-interfering RNA, and revealed that contrary to expectations, nanoparticle entry into cells is not necessary for efficient siRNA cargo delivery. Subsequently, I contribute a novel system to the nanoparticle delivery toolbox by designing and validating a single-walled carbon nanotube (SWNT)-based system for plasmid DNA delivery to plants. Notably, while traditional DNA delivery methods result in undesired transgene integration into plants, this SWNT-based platform achieves DNA delivery and gene expression without transgene integration. In addition to gold nanoparticles and SWNTs, in recent years, multiple nanoparticle-mediated systems have been shown to deliver RNA and DNA. However, there remains a dearth of literature demonstrating protein delivery into walled plant cells. I outline the unique challenges and potential strategies that can be used to advance nanoparticle-mediated protein delivery. All the studies outlined in this dissertation have been conducted on the bench-scale, though an eventual goal will involve translating this platform to widescale field utilization. I summarize the potential obstacles to field translation and detail approaches to probing nanoparticle behavior and function in a complex biological environment.

The findings presented in this dissertation lay the foundation for uncovering structure-function relationships for nanoparticles as biomolecule delivery vehicles. Furthermore, they represent unique formulations to add to the toolbox of RNA and DNA delivery. In sum, this work strives to advance RNA, DNA, and protein delivery to plants and provides a roadmap towards achieving translation of these technologies from the lab to the field.