Enabling DNA Complexation and Plant Transformation with Cell-Penetrating Peptides

Conventional techniques for plant genetic engineering have challenges including genotype and species dependence, tissue damage, and complicated progeny regeneration. Several types of nanoparticles have been shown to be able to transport biomacromolecules across the cell membrane efficiently in mammalian cells and there is promise in adapting these technologies for gene delivery in plants. Cationic peptide sequences have been shown to form complexes with DNA for transport in plants, but efficiency is still limited. Three cationic peptide sequences were synthesized on a solid-state peptide synthesizer, purified with HPLC, and verified with LCMS. These sequences were then complexed with plasmid DNA in various N/P ratios (Nitrogen/Phosphate) to determine the optimal complexation ratio. The size and zeta potential of the formulations were measured with DLS. Complexes were formed at an N/P ratio of 2 with a 35S-GFP-NosT plasmid and incubated with N. benthamiana callus cells. Fluorescent microscopy was used to check for GFP expression in the plant cells, but no fluorescence was observed. Complexes were formed again at an N/P ratio of 2 with a Dual-luciferase plasmid. These were infiltrated into N. Benthamiana leaves and incubated with N. Benthamiana and tomato callus cells. Luminescence was quantified after the addition of D-luciferin to each sample. No luminescence was observed with the dual-luciferase peptide complexes. Agrobacterium tumefaciens was used as a positive control and displayed expression of each reporter gene. Nanoparticle-mediated gene delivery in plants has shown promise, but barriers to entry still require investigation to achieve efficient in planta transformation.

Being able to learn and work in Dr. Christopher Alabi’s and Dr. Joyce Van Eck’s lab was full of so much learning in ways I could not expect. It was enriching at Cornell and the Boyce Thompson Institute to see how motivated and helpful people in the research community here are. I worked with my mentor, Nathan Piligian, with the help of Ritesh Kumar on a project to use nanoparticles to improve the process of genetically engineering plants. I was glad to both create and understand nanoparticles in the chemistry lab and to test their effects in plants and cell cultures. The techniques and approaches I experienced were eye-opening and left me feeling much more capable as a researcher. On top of invigorating research, Ithaca is such a great place to explore, and it was so fun being able to do it with so many awesome people in the program.