Successful delivery of nucleic acids in plant cells forms the basis of transformation and gene editing protocols. There is an urgent need for new targeted delivery methods for these bio-macromolecules. In this proposal we aim to develop a method, using nanoparticles (NPs) as vehicle, to transport and release nucleic acids into intact plant cells that have regenerative capacity. This would enable gene expression and gene editing, without the need for (trans)gene integration.

Successful delivery of (foreign) DNA, RNA, and/or proteins in target cells forms the basis of plant transformation and genome editing protocols. Transformation and regeneration of plants are major bottlenecks in these protocols 1. The goal of this project is to solve these bottlenecks by creating a well-defined and controlled method to deliver nucleic acids into intact plant cells, that have regenerative capacity, by means of nanoparticles (NPs) which we will develop. Delivering bio-macromolecules like DNA and RNA will enable (transient) gene expression to test any gene of interest, and to express editing tools like CRISPR/Cas to introduce stable gene edits (Fig. 1A) 2,3. Additionally, delivering DNA or RNA would make it possible to, for instance, induce flowering and silence or activate a gene.
We propose to investigate the application of NPs to create a method to transfer plasmid DNA directly into intact plant cells. Delivering nucleic acids in animal cells using NPs has been well studied, but for plants the research is still in its infancy 4. Intact plant cells contain a cell wall, which poses a major barrier 3. NP-based bio-macromolecule delivery into plant cells is a challenging procedure, and requires a multidisciplinary approach 5, which will be achieved in this project by the unique collaboration between the laboratories of Plant Breeding (PBR) and BioNanoTechnology (BNT) at WUR. In a systematic way, and based on promising preliminary results, we aim to test dendrimer-based nanomaterials to find an optimal system to internalize nucleic acids into intact plant cells and organelles therein, like the nucleus.

This project fits well within the Key Technologies: ST2 Biotechnology and Breeding. As a true key technology, it can, in essence, contribute to many future innovations, such as MMIP A2 and A4, since we aim to develop a novel bio-macromolecule delivery technique to complement classical breeding with new plant breeding techniques. By providing a technique to control binding, transport, release, and activation of bio-macromolecules such as plasmid DNA, in plant cells with regeneration capacity, this will form a basis for gene function analysis, regeneration, and genome editing of recalcitrant crops and ornamental plants, like pepper and alstroemeria. This project will thus provide a toolbox of enabling technologies that contribute to a sustainable future. It would, for instance, contribute to the reduction of the amount of chemicals used in crop and ornamental plant protection by enabling breeders to test potential resistance or susceptibility genes in their preferred plant of interest, to produce a resistant variety and consequently reduce the need for chemicals to be used by growers to combat diseases in the field.

Within the project that is proposed here, we will initially design nucleic acid delivery methods using model plants like Nicotiana tabacum, N. benthamiana, and crops like tomato and potato. Moreover, with the broad consortium on board of this project, we will be able to test methodologies on a wide range of dicot and monocot crop and ornamental plants, with eminent economic and societal importance. Furthermore, our embedding in the laboratory of Plant Breeding (PBR) at WUR, gives us ample possibilities to disseminate knowledge and methodologies to complement research at various areas of Plant Sciences. Our colleagues at PBR work on, for instance, biotic and abiotic stress resistance (including a wide range of pathogens and pests), bio-based economy, growth and development, and quality. The research is conducted on in a wide range of plant species.