Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) 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.). 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. Embryogenic callus was induced successfully from stamens collected from immature flowers. 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. After 3–4 months, the protoplasts of both cultivars regenerated with similar efficiency into cotyledonal-stage somatic embryos. 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. 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. Then root elongation occurred after transferring on a medium with 0.5 µM 1-naphthaleneacetic. After ~ 6 months from the isolation of protoplasts, normal plants were regenerated, which were moved to the greenhouse. The protoplasts could also be transfected using the polyethylene glycol method, as confirmed using a plasmid carrying the yellow florescent protein marker gene. The new method is therefore compatible with biotechnological applications such as gene transfer and genome editing.