Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.
Vitis vinifera L
genome editing
grapevine
lipofectamines
protoplast regeneration
recalcitrant genotypes
Journal
The Plant journal : for cell and molecular biology
ISSN: 1365-313X
Titre abrégé: Plant J
Pays: England
ID NLM: 9207397
Informations de publication
Date de publication:
22 Apr 2024
22 Apr 2024
Historique:
revised:
29
03
2024
received:
15
02
2024
accepted:
04
04
2024
medline:
22
4
2024
pubmed:
22
4
2024
entrez:
22
4
2024
Statut:
aheadofprint
Résumé
The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : European Union through the Next-Generation EU programme, Agritech National Research Center
Organisme : European Union through the Horizon Europe Research and Innovation Programme
ID : 101135088
Organisme : Italian Ministry of Agriculture Food and Forestry (MIPAAF) in the framework of the BIOTECH-VITECH project.
Informations de copyright
© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.
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