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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
  • 106 Downloads

Abstract

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.

Keywords

Vitis vinifera Embryogenic callus Protoplast isolation Plant regeneration Protoplast transfection 

Abbreviations

PEG

Poly-ethylene glycol

YFP

Yellow fluorescence protein

CRISPR/Cas

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein

FDA

Fluorescein diacetate

NAA

1-Naphthaleneacetic acid

6-BAP

6-Benzylaminopurine

RNPs

Ribonucleoproteins

MES

2-(N-morpholino)ethanesulfonic acid

Notes

Acknowledgements

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.

References

  1. 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
  2. Davey MR, Anthony P, Power JB, Lowe C (2005) Plant protoplasts: status and biotechnological perspectives. Biotech Adv 23:131–171.  https://doi.org/10.1016/j.biotechadv.2004.09.008 CrossRefGoogle Scholar
  3. 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
  4. Fasoli M, Richter CL, Zenoni S, Bertini E, Vitulo N, Dal Santo S, Dokoozlian N, Pezzotti M, Tornielli GB (2018) The timing and order of the molecular events that mark the onset of berry ripening in grapevine. Plant Physiol 178:1187–1206.  https://doi.org/10.1104/pp.18.00559 CrossRefGoogle Scholar
  5. Fontes N, Silva R, Vignault C, Lecourieux F, Gerós H, Delrot S (2010) Purification and functional characterization of protoplasts and intact vacuoles from grape cells. BMC Res Notes 3:19.  https://doi.org/10.1186/1756-0500-3-19 CrossRefGoogle Scholar
  6. Franks T, He DG, Thomas M (1998) Regeneration of transgenic Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis. Mol Breed 4:321–333.  https://doi.org/10.1023/A:1009673619456 CrossRefGoogle Scholar
  7. 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
  8. Iocco P, Franks T, Thomas MR (2001) Genetic transformation of major wine grape cultivars of Vitis vinifera L. Transgenic Res 10:105–112.  https://doi.org/10.1023/A:100898961 CrossRefGoogle Scholar
  9. Jaillon O, Aury JM, No€el B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C et al (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463–467.  https://doi.org/10.1038/nature06148 CrossRefGoogle Scholar
  10. Kuhn N, Guan L, Dai ZD, Wu BH, Lauvergeat V, Gomès E, Li SH, Godoy F, Arce-Johnson P, Delrot S (2014) Berry ripening: recently heard through the grapevine. J Exp Bot 65:4543–4559.  https://doi.org/10.1093/jxb/ert395 CrossRefGoogle Scholar
  11. Li ZT, Kim KH, Dhekney SA, Jasinski JR, Creech MR, Gray DJ (2014) An optimized procedure for plant recovery from somatic embryos significantly facilitates the genetic improvement of Vitis. Hortic Res 1:14027.  https://doi.org/10.1038/hortres.2014.27 CrossRefGoogle Scholar
  12. Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, Velasco R, Kanchiswamy CN (2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7:1904.  https://doi.org/10.3389/fpls.2016.01904 CrossRefGoogle Scholar
  13. Osakabe Y, Liang Z, Ren C, Nishitani C, Osakabe K, Wada M, Komori S, Malnoy M, Velasco R, Poli M, Jung MH, Koo OJ, Viola R, Kanchiswamy CN (2018) CRISPR–Cas9-mediated genome editing in apple and grapevine. Nat Protoc 13:2844–2863.  https://doi.org/10.1038/s41596-018-0067-9 CrossRefGoogle Scholar
  14. Papadakis AK, Roubelakis-Angelakis KA (1999) The generation of active oxygen species differs in tobacco and grapevine mesophyll protoplasts. Plant Physiol 121:197–205.  https://doi.org/10.1104/pp.121.1.197 CrossRefGoogle Scholar
  15. Papadakis AK, Fontes N, Gerós H, Roubelakis-Angelakis KA (2009) Progress in grapevine protoplast technology. In: Roubelakis-Angelakis KA (ed) Grapevine molecular physiology & biotechnology, 2nd edn. Springer, New York, pp 429–460CrossRefGoogle Scholar
  16. Ren C, Liu X, Zhang Z, Wang Y, Duan W, Li S, Liang Z (2016) CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Sci Rep 6:32289.  https://doi.org/10.1038/srep32289 CrossRefGoogle Scholar
  17. Reustle G, Harst M, Alleweldt G (1995) Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenie tissue. Plant Cell Rep 15:238–241.  https://doi.org/10.1007/BF00193727 CrossRefGoogle Scholar
  18. 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
  19. Sarrion-Perdigones A, Vazquez-Vilar M, Palací J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D (2013) GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162:1618–1631.  https://doi.org/10.1104/pp.113.217661 CrossRefGoogle Scholar
  20. 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
  21. 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
  22. Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164.  https://doi.org/10.1038/nbt.3389 CrossRefGoogle Scholar
  23. Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR–Cas system. Mol Plant 6:1975–1983.  https://doi.org/10.1093/mp/sst119 CrossRefGoogle Scholar
  24. 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
  25. 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

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyUniversity of VeronaVeronaItaly

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