Pea (Pisum sativum L.) is an annual cool-season legume crop. Owing to its role in sustainable agriculture as both a rotation and a cash crop, its global market is expanding and increased production is urgently needed. For both technical and regulatory reasons, neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production. In answer to this challenge, CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years. However, there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea. We developed a transient transformation system of hairy roots, mediated by Agrobacterium rhizogenes strain K599, to validate the efficiency of a CRISPR/Cas9 system. Further optimization resulted in an efficient vector, PsU6.3-tRNA-PsPDS3-en35S-PsCas9. We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase (PsPDS) gene, causing albinism, by Agrobacterium-mediated genetic transformation. This is the first report of successful generation of gene-edited pea plants by this route.

The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3 (BE3) and targeted activation-induced cytidine deaminase (CDA) (target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase (UDG) inhibitor (UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a high-fidelity and high-efficiency solution for rice genomic base editing.