Search

Application of new genome editing technologies for plant improvement depends on efficient delivery of editing components into plant cells. For editing of canola, which is an important oil crop, CRISPR/Cas9 and Agrobacterium-mediated transformation of hypocotyl or stem segments have been the primary methods used. Here, we present the development of an efficient and high-throughput system for protoplast isolation, DNA-free editing, and production of edited canola plants. Protoplast polyethylene glycol (PEG)–mediated transfection was conducted with ribonucleoproteins (RNPs) comprising LbCas12a and a single guide RNA. Edits were found at a frequency of approximately 40% in protoplasts cultured for 2 d, in callus colonies derived from these protoplasts, and in regenerated plants. Out of 30 plants that were regenerated from the protoplasts, 12 were edited. Six of them were with biallelic mutations and another six had monoallelic mutations. Targeted deletions from 2 to 9 nucleotides were identified in the T0 edited plants. Analysis of T1 progeny confirmed that the RNP-based, DNA-free edits using this canola protoplast system are heritable.

Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers’ expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids.