In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

Rice (Oryza sativa) varieties are generated through breeding programs focused on local requirements. In Chile, the southernmost rice producer, rice productivity relies on the use and generation of temperate japonica germplasms, which need to be adapted to the intensifying effects of climate change. Advanced biotechnological tools can contribute to these breeding programs; new technologies associated with precision breeding, including gene editing, rely on procedures such as regeneration and gene transfer. In this study, the local rice varieties Platino, Cuarzo, Esmeralda, and Zafiro were evaluated for somatic embryogenesis potential using a process that involved the combined use of auxins and cytokinins. An auxin-based (2,4-D) general medium (2N6) allowed for the induction of embryogenic masses in all the genotypes. After induction, masses required culturing either in N6R (kinetin; Platino) or N6RN (BAP, kinetin, IBA, and 2,4-D; Cuarzo, Esmeralda, and Zafiro) to yield whole plants using regeneration medium (N6F, no hormone). The sprouting rates indicated Platino as the most responsive genotype; for this reason, this variety was evaluated for gene transfer. Fifteen-day-old embryo masses were assayed for Agrobacterium-mediated transformation using the bacterial strain EHA105 harboring pFLC-Myb/HPT/GFP, a modified T-DNA vector harboring a geminivirus-derived replicon. The vector included the green fluorescent protein reporter gene, allowing for continuous traceability. Reporter mRNA was produced as early as 3 d after agroinfiltration, and stable expression of the protein was observed along the complete process. These achievements enable further biotechnological steps in these and other genotypes from our breeding program.

Enzymatic browning, driven by polyphenol oxidase (PPO), remains a major postharvest challenge for potato (Solanum tuberosum L.), reducing product quality, shelf life, and consumer acceptance. To mitigate this trait in the elite tetraploid cultivar ‘Yagana-INIA’, we applied a geminivirus-derived CRISPR–Cas9 system to edit the StPPO genes most highly expressed in tubers, StPPO1 and particularly StPPO2. A paired-gRNA strategy generated a double-cut deletion in StPPO1, while StPPO2 editing required a complementary single-gRNA screening workflow. High-resolution fragment analysis and sequencing identified three StPPO2-edited lines, including one that lacked GFP, Cas9, and Rep/RepA sequences, confirming a transgene-free editing outcome. Edited tubers exhibited visibly reduced browning relative to wild type, and biochemical assays showed decreased PPO activity consistent with targeted disruption of StPPO2. Amplicon sequencing verified monoallelic editing at the gRNA2 site in the non-transgenic line. These results demonstrate the utility of a replicon-based CRISPR system for achieving targeted, transgene-free edits in tetraploid potato and identify a non-GM StPPO2-edited line with improved postharvest quality under Chile’s regulatory framework.

Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of ‘Valencia’ and hybrid genotypes (CsH1–CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L⁻¹ GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in ‘Valencia’ and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.