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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.

Glyphosate is a broad spectrum herbicide that inhibits the activity of endogenous plant 5-enolpyruvylshlkimate-3-phosiahate synthase (EPSPS). Glyphosate resistance has been engineered in multiple crops by constitutive expression of an EPSPS variant from Agrobacterium sp. CP4 (CP4 EPSPS) that binds glyphosate less efficiently. The first glyphosate-resistant cotton (Gossypium hirsutum L.) event, MON 1445, was developed utilizing this CP4 EPSPS and commercially introduced in 1997. Due to insufficient expression of CP4 EPSPS in male reproductive tissues, over-the-top glyphosate applications must be limited to the four-leaf stage of growth. In an attempt to generate events with complete constitutive expression, multiple constructs consisting of different promoters driving cp4 epsps were transformed into cotton. The MON 88913 event was identified as providing exceptional vegetative and reproductive resistance to glyphosate based on more than 8 yr of field testing. Cotton containing MON 88913 has a comparable agronomic profile to the respective negative isoline, the event is stably inherited over multiple generations, and the insertion site is simple with minimal disruption of the cotton genome. As a result, the MON 88913 event was commercially introduced in 2006 as Roundup Ready Flex Cotton (Monsanto Co., St. Louis, MO).

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.