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Departamento Gestión de Conocimiento, Monitoreo y Prospección
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A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example
Indexado
WoS WOS:000701106500001
Scopus SCOPUS_ID:85114634493
DOI 10.3390/PLANTS10091882
Año 2021
Tipo artículo de investigación

Citas Totales

Autores Afiliación Chile

Instituciones Chile

% Participación
Internacional

Autores
Afiliación Extranjera

Instituciones
Extranjeras


Abstract



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.

Revista



Revista ISSN
Plants Basel 2223-7747

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Disciplinas de Investigación



WOS
Plant Sciences
Scopus
Sin Disciplinas
SciELO
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Publicaciones WoS (Ediciones: ISSHP, ISTP, AHCI, SSCI, SCI), Scopus, SciELO Chile.

Colaboración Institucional



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Autores - Afiliación



Ord. Autor Género Institución - País
1 Acha, Giovana - Universidad de Santiago de Chile - Chile
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

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Financiamiento



Fuente
Comisión Nacional de Investigación Científica y Tecnológica
FONDEF-Chile
MINAGRI
ANID
ANID/CONICYT
INIA-Chile (MINAGRI)
ANID-Fondecyt-Chile Postdoctoral

Muestra la fuente de financiamiento declarada en la publicación.

Agradecimientos



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).

Muestra la fuente de financiamiento declarada en la publicación.