Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) cultivars

  • Edoardo Bertini
  • Giovanni Battista Tornielli
  • Mario Pezzotti
  • Sara ZenoniEmail author
Original Article


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.

Key message

This study reports an improved protocol for embryogenic callus induction, protoplast isolation and whole plant regeneration of two Vitis vinifera cultivars. Protoplasts showed high transfection efficiency.


Vitis vinifera Embryogenic callus Protoplast isolation Plant regeneration Protoplast transfection 



Poly-ethylene glycol


Yellow fluorescence protein


Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein


Fluorescein diacetate


1-Naphthaleneacetic acid






2-(N-morpholino)ethanesulfonic acid



We thank Mauro Commisso and Flavia Guzzo for the technical assistance and support. This study was supported by the POR FESR 2014-2020. DGR n. 1139 del 19.07.2017. Azione 1.1.4. Project VIT-VIVE and by the COST Action INTEGRAPE CA17111.

Author contributions

EB performed the protoplast isolation, the regeneration of whole plants from somatic embryos and the PEG-mediated transfection of protoplasts; MP conceived the study; SZ and GBT supervised the study and wrote the manuscript. All the authors contributed to the discussion of the results, reviewed the manuscript and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyUniversity of VeronaVeronaItaly

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