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  1. Pubblicazioni

Knockout of phytoene desaturase gene using CRISPR/Cas9 in highbush blueberry

Articolo
Data di Pubblicazione:
2022
Abstract:
Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry (Vaccinium corymbosum L.), using Agrobacterium-mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase, whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro-cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry “Berkeley”, and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.
Tipologia CRIS:
03A-Articolo su Rivista
Keywords:
Vaccinium corymbosum L; de novo shoot organogenesis; gene editing; gene loss-of-function; phytoene desaturase
Elenco autori:
Vaia, Giuseppe; Pavese, Vera; Moglia, Andrea; Cristofori, Valerio; Silvestri, Cristian
Autori di Ateneo:
MOGLIA Andrea
Pavese Vera
Link alla scheda completa:
https://iris.unito.it/handle/2318/1885114
Link al Full Text:
https://iris.unito.it/retrieve/handle/2318/1885114/1068047/Vaia%20et%20al.pdf
Pubblicato in:
FRONTIERS IN PLANT SCIENCE
Journal
  • Aree Di Ricerca

Aree Di Ricerca

Settori (15)


LS9_8 - Applied plant sciences, plant breeding, agroecology and soil biology - (2022)

CIBO, AGRICOLTURA e ALLEVAMENTI - Agricoltura e Produzioni Vegetali

CIBO, AGRICOLTURA e ALLEVAMENTI - Allevamento e Produzioni Animali

CIBO, AGRICOLTURA e ALLEVAMENTI - Chimica e cibo

CIBO, AGRICOLTURA e ALLEVAMENTI - Farmacologia Veterinaria

CIBO, AGRICOLTURA e ALLEVAMENTI - Miglioramento e difesa delle colture

CIBO, AGRICOLTURA e ALLEVAMENTI - Tecnologie alimentari e microbiologia degli alimenti

MEDICINA, SALUTE e BENESSERE - Diagnostica e Imaging

MEDICINA, SALUTE e BENESSERE - Epidemiologia

MEDICINA, SALUTE e BENESSERE - Prevenzione e corretti stili di vita

MEDICINA, SALUTE e BENESSERE - Ricerca Traslazionale e Clinica

PIANETA TERRA, AMBIENTE, CLIMA, ENERGIA e SOSTENIBILITA' - Energia e Fonti Energetiche

PIANETA TERRA, AMBIENTE, CLIMA, ENERGIA e SOSTENIBILITA' - Foreste e Legno

PIANETA TERRA, AMBIENTE, CLIMA, ENERGIA e SOSTENIBILITA' - Risorsa suolo e ambiente

SCIENZE DELLA VITA e FARMACOLOGIA - Interazioni tra molecole, cellule, organismi e ambiente
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