Journal
NATURE PROTOCOLS
Volume 14, Issue 10, Pages 2954-2971Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-019-0208-9
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Funding
- Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI)
- Stanley Fahn PDF Junior Faculty Grant [PF-JFA-1760]
- Beckman Foundation Young Investigator Award
- USDA AFRI award
- Gordon and Betty Moore Foundation
- USDA NIFA award
- USDA-BBT EAGER award
- Chan-Zuckerberg Foundation
- FFAR New Innovator Award
- Schlumberger Foundation
- National Natural Science Foundation of China [21605153]
- UC Berkeley Molecular Imaging Center (Gordon and Betty Moore Foundation)
- QB3 Shared Stem Cell Facility
- Innovative Genomics Institute (IGI)
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Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. We have demonstrated efficient plasmid DNA delivery into intact plants of several species with functionalized high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs), enabling efficient DNA delivery into a variety of non-model plant species (arugula, wheat, and cotton) and resulting in high protein expression levels without transgene integration. Herein, we provide a protocol that can be implemented by plant biologists and adapted to produce functionalized single-walled CNTs (SWNTs) with surface chemistries optimized for delivery of plasmid DNA in a plant species-independent manner. This protocol describes how to prepare, construct, and optimize polyethylenimine (PEI)-functionalized SWNTs and perform plasmid DNA loading. The authors also provide guidance on material characterization, gene expression evaluation, and storage conditions. The entire protocol, from the covalent functionalization of SWNTs to expression quantification, can be completed in 5 d.
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