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| Indexado |
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| DOI | 10.3390/PLANTS10091882 | ||||
| Año | 2021 | ||||
| Tipo | artículo de investigación |
Citas Totales
Autores Afiliación Chile
Instituciones Chile
% Participación
Internacional
Autores
Afiliación Extranjera
Instituciones
Extranjeras
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 com-plexities 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.
| Ord. | Autor | Género | Institución - País |
|---|---|---|---|
| 1 | Acha, Giovana | - |
Universidad de Santiago de Chile - Chile
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| 2 | VERGARA-GONZALEZ, RICARDO ALEJANDRO | Hombre |
Instituto de Investigaciones Agropecuarias - Chile
Inst Invest Agr Platina - Chile |
| 3 | MUNOZ-VILLAGRA, MARISOL DE LAS MERCEDES | Mujer |
Instituto de Investigaciones Agropecuarias - Chile
Inst Invest Agr Platina - Chile |
| 4 | Mora, Roxana | Mujer |
Instituto de Investigaciones Agropecuarias - Chile
Inst Invest Agr Platina - Chile |
| 5 | AGUIRRE-DUMENEZ, CARLOS | Hombre |
Instituto de Investigaciones Agropecuarias - Chile
Inst Invest Agr Platina - Chile |
| 6 | MUNOZ-DAVID, MANUEL ANDRES | Hombre |
Instituto de Investigaciones Agropecuarias - Chile
Inst Invest Agr Remehue - Chile |
| 7 | KALAZICH-BARASSI, JULIO CESAR | Hombre |
Universidad de Los Lagos - Chile
Univ Lagos - Chile |
| 8 | PRIETO-ENCALADA, HUMBERTO GODOFREDO | Hombre |
Instituto de Investigaciones Agropecuarias - Chile
Inst Invest Agr Platina - Chile |
| Fuente |
|---|
| Comisión Nacional de Investigación Científica y Tecnológica |
| FONDEF-Chile |
| MINAGRI |
| ANID |
| ANID/CONICYT |
| INIA-Chile (MINAGRI) |
| ANID-Fondecyt-Chile Postdoctoral |
| Agradecimiento |
|---|
| Funded by grants FONDEF-Chile #IT18I10102, INIA-Chile (MINAGRI) (no. 502955-70), and ANID-Fondecyt-Chile Postdoctoral (grant no. 3180079; R.V.). G.A. is an ANID/CONICYT Doctoral Fellow (no. 21170768). |
| Funded by grants FONDEF-Chile #IT18I10102, INIA-Chile (MINAGRI) (no. 502955-70), and ANID-Fondecyt-Chile Postdoctoral (grant no. 3180079; R.V.). G.A. is an ANID/CONICYT Doctoral Fellow (no. 21170768). |
| Funded by grants FONDEF-Chile #IT18I10102, INIA-Chile (MINAGRI) (no. 502955-70), and ANID-Fondecyt-Chile Postdoctoral (grant no. 3180079; R.V.). G.A. is an ANID/CONICYT Doctoral Fellow (no. 21170768). |