Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.

Ascorbic acid (AsA) is a key antioxidant and nutrient in plants, regulating reactive oxygen species (ROS) levels and maintaining cellular redox homeostasis. The AsA recycling pathway sustains AsA pools by restoring its oxidized forms, ensuring intracellular balance. Among the enzymes involved, monodehydroascorbate reductase (MDHAR) is important for the regeneration of AsA from monodehydroascorbate. In this study, we analyzed the four MDHAR paralogs in Lactuca sativa using CRISPR/Cas9 to determine whether disruption of individual MDHAR genes could alter AsA levels in lettuce leaves. Unexpectedly, none of the knockouts caused long-term changes in leaf AsA content. Transcriptomic analyses at 14 and 28 days showed minimal effects on AsA recycling or biosynthesis genes, except MDHAR genes. However, several other genes indirectly implicated in AsA regulation displayed differential expression in all mutants compared to the wild type, suggesting the presence of a complex regulatory network. In particular, genes encoding transcription factors (TFs), such as mTERF15, COL9, UPBEAT1, NAC28, and NAC42, were differentially regulated in all MDHAR mutants compared to the wild type at 28 days. These findings indicate that, although AsA content remains unchanged, MDHAR single knockouts alter expression of other genes through which the plants may indirectly compensate to maintain redox homeostasis.