Felipe Olivares

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.

Genetic transformation in Prunus persica genotypes could add single horticultural traits in existing cultivars without modifying their commercial characteristics. Despite this opportunity, the final setting of transformation protocols in the species endures two major limiting factors preventing the development of new varieties: (a) explants are recalcitrant in regenerating adventitious transformed shoots and (b) have a limited regeneration capability, usually extended to just a few genotypes (i.e., cultivar dependence). This protocol illustrates a procedure that has allowed the establishment of transformation methodologies in several varieties of the species and is presented here for Red Top and Elegant Lady; this methodology could help extend technical baselines for successful transformation procedures in other, potentially more recalcitrant, and newly developed cultivars or genus members.

The flowering and fruiting of sweet cherry (Prunus avium L.) depend on precise synchronization with seasonal events. During harsh autumn and winter conditions, floral buds enter dormancy to protect and prepare for the productive season. Dormancy release occurs after exposure to genotype-specific chilling temperatures, an event in which epigenetic reprogramming triggers further metabolic and gene expression activation. Similarly, several Arabidopsis ecotypes require chilling (vernalization) to transition from vegetative to floral states. At vernalization’s end, the decrease in the repressor complex formed by SHORT VEGETATIVE PHASE (SVP) and FLOWERING LOCUS C (FLC) allows FLOWERING LOCUS T (FT) to induce flowering. However, this alone does not fully explain the process. MicroRNAs (miRNAs) play a crucial role in gene regulation during plant development and environmental interactions, and miR396’s role during flower development and vernalization has been described in some plant species, although not for sweet cherry dormancy. We used ‘Regina’, a high-chill sweet cherry variety, to identify candidate small RNA molecules throughout dormancy, resulting in the detection of miR396. The transcript expression levels of the putative miRNA target genes were evaluated through quantitative PCR analyses of dormant buds. Additionally, an artificial sweet cherry miR396 was used to transform Arabidopsis Edi-0, a vernalization-requiring ecotype. Ectopic expression of this artificial molecule partially mirrored the effect on target genes observed in dormant buds and, more importantly, led to vernalization-independent flowering. Artificial miR396 expression also resulted in decreased FLC and increased SVP and FT transcript levels. These results could pave the way for future studies on the involvement of miR396 in the regulation of dormancy and flowering, with potential applications in improving crop resilience and productivity.

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.