Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) cultivars
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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.
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.
KeywordsVitis vinifera Embryogenic callus Protoplast isolation Plant regeneration Protoplast transfection
Yellow fluorescence protein
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein
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.
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.
- Dal Bosco D, Sinski I, Ritschel PS, Camargo UA, Fajardo TVM, Harakava R, Quecini V (2018) Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation. Transgenic Res 27:379–396. https://doi.org/10.1007/s11248-018-0082-1 CrossRefGoogle Scholar
- Fasoli M, Dal Santo S, Zenoni S, Tornielli GB, Farina L, Zamboni A, Porceddu A, Venturini L, Bicego M, Murino V, Ferrarini A, Delledonne M, Pezzotti M (2012) The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program. Plant Cell 24:3489–3505. https://doi.org/10.1105/tpc.112.10023 CrossRefGoogle Scholar
- He R, Zhuang Y, Cai Y, Agüero CB, Liu S, Wu J, Deng S, Walker MA, Lu J, Zhang Y (2018) Overexpression of 9-cis-epoxycarotenoid dioxygenase cisgene in grapevine increases drought tolerance and results in pleiotropic effects. Front Plant Sci 9:970. https://doi.org/10.3389/fpls.2018.00970 CrossRefGoogle Scholar
- Rinaldo AR, Cavallini E, Jia Y, Moss SM, McDavid DA, Hooper LC, Robinson SP, Tornielli GB, Zenoni S, Ford CM, Boss PK, Walker AR (2015) A grapevine anthocyanin acyltransferase, transcriptionally regulated by vvmyba, can produce most acylated anthocyanins present in grape skins. Plant Physiol 169:1897–1916. https://doi.org/10.1104/pp.15.01255 Google Scholar
- Serrano A, Espinoza C, Armijo G, Inostroza-Blancheteau C, Poblete E, Meyer-Regueiro C, Arce A, Parada F, Santibáñez C, Arce-Johnson P (2017) Omics approaches for understanding grapevine berry development: regulatory networks associated with endogenous processes and environmental responses. Front Plant Sci 8:1486. https://doi.org/10.3389/fpls.2017.01486 CrossRefGoogle Scholar
- Subburaj S, Chung SJ, Lee C, Ryu SM, Kim DH, Kim JS, Bae S, Lee GJ (2016) Site-directed mutagenesis in Petunia hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep 35:1535–1544. https://doi.org/10.1007/s00299-016-1937-7 CrossRefGoogle Scholar
- Zamboni A, Di Carli M, Guzzo F, Stocchero M, Zenoni S, Ferrarini A, Tononi P, Toffali K, Desiderio A, Lilley KS, Pè ME, Benvenuto E, Delledonne M, Pezzotti M (2010) Identification of putative stage-specific grapevine berry biomarkers and omics data integration into networks. Plant Physiol 154:1439–1459. https://doi.org/10.1104/pp.110.160275 CrossRefGoogle Scholar
- Zhu YM, Hoshino Y, Nakano M, Takahashi E, Mii M (1997) Highly efficient system of plant regeneration from protoplasts of grapevine (Vitis vinifera L.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal. Plant Sci 123:151–157. https://doi.org/10.1016/S0168-9452(96)04557-8 CrossRefGoogle Scholar