Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.Ĭastanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, helping to address the regulatory concerns related to genetically modified plants. The regeneration of GFP− protoplasts into whole plants was monitored throughout development, confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls. CRISPR/Cas9 activity, guided by two independent sgRNAs, was confirmed by the loss of GFP fluorescence. As proof of concept, a single-copy green fluorescent protein reporter gene (GFP) in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts. Here, we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. Although the direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has not been reported. The new method is therefore compatible with biotechnological applications such as gene transfer and genome editing.ĬRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding, offering enormous potential for crop improvement and food production. The protoplasts could also be transfected using the polyethylene glycol method, as confirmed using a plasmid carrying the yellow florescent protein marker gene. After ~ 6 months from the isolation of protoplasts, normal plants were regenerated, which were moved to the greenhouse. Then root elongation occurred after transferring on a medium with 0.5 µM 1-naphthaleneacetic. This step was necessary for the embryo to complete germination, allowing subsequent shoot elongation in response to light on a medium with 4 µM 6-benzylaminopurine. The somatic embryos were transferred to solid Nitsch’s medium supplemented with 30 g/L sucrose and 2 g/L gellan gum, and were maintained in the dark for 4 weeks. After 3–4 months, the protoplasts of both cultivars regenerated with similar efficiency into cotyledonal-stage somatic embryos. Isolated protoplasts were tested to confirm their viability and then cultivated using the disc-culture method, at a density of 1 × 10⁵ protoplasts/mL in solid Nitsch’s medium supplemented with 2 mg/L 1-naphthaleneacetic acid and 0.5 mg/L 6-benzylaminopurine. Embryogenic callus was induced successfully from stamens collected from immature flowers. Here we describe an efficient protocol for the induction of embryogenic callus, the isolation of protoplasts, and the regeneration of whole grapevine plants in two Italian grapevine cultivars. Protoplasts are useful research tools for basic and applied plant science, but the regeneration of whole plants from protoplasts is challenging in most of agronomically important crops, including grapevine (Vitis vinifera L.).
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