Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana.

There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium-based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC.

Fast-TrACC: A Rapid Method for Delivering and Testing Gene Editing Reagents in Somatic Plant Cells.

The production of transgenic or gene edited plants requires considerable time and effort. It is of value to know at the onset of a project whether the transgenes or gene editing reagents are functioning as predicted. To test molecular reagents transiently, we implemented an improved, Agrobacterium tumefaciens-based co-culture method called Fast-TrACC (Fast Treated Agrobacterium Co-Culture). Fast-TrACC delivers reagents to seedlings, allowing high throughput, and uses a luciferase reporter to monitor and calibrate the efficiency of reagent delivery. We demonstrate the use of Fast-TrACC in multiple solanaceous species and apply the method to test promoter activity and the effectiveness of gene editing reagents.

Plant gene editing through de novo induction of meristems.

Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.