Skip to main content
SpringerLink
Log in

A simple and efficient protocol for transient transformation of sliced grape berries

  • Original Article
  • Published:
Protoplasma Aims and scope Submit manuscript

Abstract

Grape is an economically important crop but recalcitrant to Agrobacterium-mediated genetic transformation and in vitro regeneration. Here, we have developed a protocol for transient transformation of grapes by investigating the effects of explant pre-culture and duration of vacuum infiltration on transformation efficiency. Using sliced grape berries of “Shine-Muscat” (Vitis labrusca × Vitis vinifera) between the end of fruit expansion phase and the mature stage as explants, we firstly compared the effect of pre-culture explants into a susceptible state (incubation on Murashige and Skoog (MS) agar plate in the dark at 25 ± 1 °C for 48 h) with no pre-culture and then tested different vacuum infiltration times on transformation efficiency using β-glucuronidase (GUS) reporter system. Pre-culture increased the susceptibility of explants to the agrobacteria infection and increased transient transformation efficiency as assessed by histochemical GUS activity, with intense blue coloration compared with the faint staining observed in the non-susceptible explants. Using a Circulating Water Vacuum Pump system to facilitate agrobacteria entry into berry cells, we tested vacuum durations of 5, 10, and 15 min and observed that transformation efficiency increased with vacuum duration of infiltration. These results were confirmed by relative gene expression of GUS transgene as assessed by RT-qPCR and GUS activity assay. To further confirm the usefulness of our protocol, we transiently transformed grape berries with the hydrogen peroxide sensor gene VvHPCA3, and this was confirmed by gene expression analysis as well as increased sensitivity of the explants to hydrogen peroxide treatment. Overall, this study has resulted in a simple but efficient transient transformation protocol for grape berries and would be a valuable tool for the rapid testing of gene function and the study of key regulatory networks in this important crop.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

Source data for all the figures and tables are provided in the paper.

References

  • Aitken RJ (2018) Nitroblue tetrazolium (NBT) assay. Reprod Biomed Online 36:90–91

    Article  PubMed  Google Scholar 

  • An JP, Qu FJ, Yao JF, Wang XN, You CX, Wang XF, Hao J (2017) The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hortic Res 4:17023

    Article  PubMed  PubMed Central  Google Scholar 

  • Asande LK, Omwoyo RO, Oduor RO, Nyaboga EN (2020) A simple and fast Agrobacterium-mediated transformation system for passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa). Plant Methods 16:141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bakshi S, Sadhukhan A, Mishra S, Sahoo L (2011) Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration. Plant Cell Rep 30:2281–2292

    Article  CAS  PubMed  Google Scholar 

  • Campos G, Chialva C, Miras S, Lijavetzky D (2021) New technologies and strategies for grapevine breeding through genetic transformation. Front Plant Sci 12:767522

    Article  PubMed  PubMed Central  Google Scholar 

  • Chakrabarty R, Viswakarma N, Bhat SR, Kirti PB, Singh BD, Chopra VL (2002) Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower. J Biosci 27:495–502

    Article  CAS  PubMed  Google Scholar 

  • Du N, Pijut PM (2009) Agrobacterium-mediated transformation of Fraxinus Pennsylvanica hypocotyls and plant regeneration. Plant Cell Rep 28:915–923

    Article  CAS  PubMed  Google Scholar 

  • Gong P, Li Y, Tang Y, Wei R, Huijun Z, Wang Y, Zhang CH (2018) Vacuolar processing enzyme (VvβVPE) from Vitis vinifera, processes seed proteins during ovule development, and accelerates seed germination in VvβVPE heterologously over-expressed Arabidopsis. Plant Sci 274:420–431

    Article  CAS  PubMed  Google Scholar 

  • Gurusaravanan P, Vinoth S, Jayabalan N (2020) An improved Agrobacterium-mediated transformation method for cotton (Gossypium hirsutum L. ‘KC3’) assisted by microinjection and sonication. In Vitro Cell Dev Biol -Plant 56:111–121

    Article  CAS  Google Scholar 

  • Guzmán Y, Pugliese B, González CV, Travaglia C, Bottini R, Berli F (2021) Spray with plant growth regulators at full bloom may improve quality for storage of ‘superior seedless’ table grapes by modifying the vascular system of the bunch. Postharvest Biol Tec 176:111522

    Article  Google Scholar 

  • Haddadi F, Abd Aziz M, Abdullah SN, Tan SG, Kamaladini H (2015) An efficient Agrobacterium-mediated transformation of strawberry cv. Camarosa by a dual plasmid system. Molecules 20:3647–3666

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu D, Bent AF, Hou X, Li Y (2019) Agrobacterium-mediated vacuum infiltration and floral dip transformation of rapid-cycling Brassica rapa. BMC Plant Biol 19:1–9

    Article  Google Scholar 

  • Hwang HH, Yu M, Lai EM (2017) Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book 15:e0186

    Article  PubMed  PubMed Central  Google Scholar 

  • Jones HD, Doherty A, Sparks CA (2009) Transient transformation of plants. In: Gustafson JP (ed), Somers DJ (ed), Langridge P (ed) Plant Genomics. Methods in Molecular Biology™ (Methods and Protocols). Humana Press, New Jersey, pp 131–152.

  • Li J, Komori S, Sasaki K, Mimida N, Matsumoto S, Wada M, Soejima J, Ito Y, Masuda T, Tanaka N, Shigeta N (2011) Pre-culture before Agrobacterium infection to leaf segments and meropenem improves the transformation efficiency of apple (Malus × domestica Borkh.). J Jpn Soc Hortic Sci 80:244–254

    Article  CAS  Google Scholar 

  • Li J, Zhang D, Que Q, Chen X, Ouyang K (2019) Plant regeneration and Agrobacterium-mediated transformation of the miracle tree Neolamarckia cadamba. Ind Crop Prod 130:443–449

    Article  CAS  Google Scholar 

  • Li G, Hu S, Yang J, Zhao X, Kimura S, Schultz EA, Hou HW (2020) Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae). Plant Cell Rep 39:737–750

    Article  CAS  PubMed  Google Scholar 

  • Li S, Liu K, Yu S, Jia S, Chen S, Fu Y, Sun F, Luo QW, Wang YJ (2020) The process of embryo abortion of stenospermocarpic grape and it develops into plantlet in vitro using embryo rescue. Plant Cell Tiss Organ Cult 143:389–409

    Article  CAS  Google Scholar 

  • Li PY, Yu DD, Gu B, Zhang HJ, Liu QY, Zhang JX (2022) Overexpression of the VaERD15 gene increases cold tolerance in transgenic grapevine. Sci Hortic 293:110728

    Article  CAS  Google Scholar 

  • Li XF, Li SQ, Sun YD, Wang LL, Li M, Liu HN, Pei MS, Wei TL, Guo DL, Yu YH (2022) Isolation and functional validation of the VvZFP11 promoter associated with a signaling molecule and powdery mildew responses in grapevine. Sci Hortic 298:110980

    Article  CAS  Google Scholar 

  • Licciardello C, Perrone I, Gambino G, Talon M, Velasco R (2021) Functional genomics in fruit trees: from ‘Omics to sustainable biotechnologies. Front Plant Sci 12:729714

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu HN, Su J, Zhu YF, Yao GF, Allan AC, Ampomah-Dwamena C, Shu Q, Lin-Wang K, Zhang SL, Wu J (2019) The involvement of PybZIPa in light-induced anthocyanin accumulation via the activation of PyUFGT through binding to tandem G-boxes in its promoter. Hortic Res 6:134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu G, Massel K, Tabet B, Godwin ID (2020) Biolistic DNA delivery and its applications in sorghum bicolor. Methods Mol Biol 2124:197–215

    Article  CAS  PubMed  Google Scholar 

  • Liu HN, Shu Q, Lin-Wang K, Allan AC, Espley RV, Su J, Pei MS, Wu J (2021) The PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10 module regulates light-induced anthocyanin biosynthesis in red pear. Mol Horticulture 1:14

    Article  CAS  Google Scholar 

  • Liu HN, Pei MS, Wei TL, Yu YH, Guo DL (2021) Molecular cloning and expression analysis of hydrogen peroxide sensors under H2O2 and ROS inhibitor treatment in ‘Kyoho’ grape berry. Postharvest Biol Tec 180:111617

    Article  CAS  Google Scholar 

  • Liu S, Liu C, Wang X, Chen H (2021) Seed-specific activity of the Arabidopsis β-glucosidase 19 promoter in transgenic Arabidopsis and tobacco. Plant Cell Rep 40:213–221

    Article  CAS  PubMed  Google Scholar 

  • Liu HN, Pei MS, Wei TL, Yu YH, Guo DL (2022) ROS scavenger hypotaurine delays postharvest softening of ‘Kyoho’ grape by regulating pectin and cell metabolism pathway. Postharvest Biol Tec 186:111833

    Article  CAS  Google Scholar 

  • Moriyama A, Yamaguchi C, Enoki S, Aoki Y, Suzuki S (2020) Crosstalk pathway between trehalose metabolism and cytokinin degradation for the determination of the number of berries per bunch in grapes. Cells 9:2378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nikolić D, Banjanin T, Ranković-Vasić Z (2018) Variability and heredity of some qualitative and quantitative grapevine characteristics. Genetika 50:549–560

    Article  Google Scholar 

  • Ozyigit II, Yucebilgili Kurtoglu K (2020) Particle bombardment technology and its applications in plants. Mol Biol Rep 47:9831–9847

    Article  CAS  PubMed  Google Scholar 

  • Pawar BD, Jadhav AS, Kale AA, Chimote VP, Pawar SV (2013) Effect of explants, bacterial cell density and overgrowth-control antibiotics on transformation efficiency in tomato (Solanum lycopersicum L.). J Appl Hortic 15:95–99

    Article  Google Scholar 

  • Pei MS, Liu HN, Wei TL, Yu YH, Guo DL (2021) Detection and characterization of genome-wide genetic variation associated with the early-ripening phenotype of grape mutants. Sci Hortic 285:110195

    Article  CAS  Google Scholar 

  • Ren F, Ren C, Zhang Z, Duan W, Lecourieux D, Li S, Liang ZC (2019) Efficiency optimization of CRISPR/Cas9-mediated targeted mutagenesis in grape. Front Plant Sci 10:612

    Article  PubMed  PubMed Central  Google Scholar 

  • Ren Y, Jing Y, Kang X (2021) In vitro induction of tetraploid and resulting trait variation in Populus alba× Populus glandulosa clone 84 K. Plant Cell Tiss Organ Cult 146:285–296

    Article  CAS  Google Scholar 

  • Ricci A, Sabbadini S, Prieto H, Padilla IM, Dardick C, Li Z, Scorza R, Limera C, Mezzetti B, Perez-Jimenez M, Burgos L (2020) Genetic transformation in peach (Prunus persica L.): challenges and ways forward. Plants. 9:971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rodríguez-Pérez C, García-Villanova B, Guerra-Hernández E, Verardo V (2019) Grape seeds proanthocyanidins: an overview of in vivo bioactivity in animal models. Nutrients 11:2435

    Article  PubMed  PubMed Central  Google Scholar 

  • Safitri FA, Ubaidillah M, Kim KM (2016) Efficiency of transformation mediated by Agrobacterium tumefaciens using vacuum infiltration in rice (Oryza sativa L.). Plant Biotechnol J 43:66–75

    Article  Google Scholar 

  • Samuel T, Jesca LN, John A, Kenneth S, Andrew K (2019) Establishment of a transformation protocol for Uganda’s yellow passion fruit using the GUS gene. Afr J Biotechnol 18:416–425

    Article  Google Scholar 

  • Schröpfer S, Lempe J, Emeriewen OF, Flachowsky H (2022) Recent developments and strategies for the application of agrobacterium-mediated transformation of apple Malus × domestica Borkh. Front Plant Sci 13:928292

    Article  PubMed  PubMed Central  Google Scholar 

  • Sedaghati B, Haddad R, Bandehpour M (2019) Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): An important medicinal plant. Plant Cell Tiss Organ Cult 136:231–245

    Article  CAS  Google Scholar 

  • Sharma R, Modgil M, Sharma P, Saini U (2012) Agrobacterium-mediated transfer of chitinase gene in apple (Malus × domestica Borkh.) rootstock MM106. Indian J Hort 69:1–6

    Google Scholar 

  • Shin H, Oh SI, Son IC, Oh YJ, Park JY, Park JH, Kim D (2014) Berry cracking caused by powdery mildew (Uncinula necator) infection to ‘Fujiminori’ grapevines (Vitis sp.). Acta Hortic 1046:127–132

    Article  Google Scholar 

  • Shrawat AK, Becker D, Lörz H (2007) Agrobacterium tumefaciens-mediated genetic transformation of barley (Hordeum vulgare L.). Plant Sci 172:281–290

    Article  CAS  Google Scholar 

  • Simon-Gruita A, Pojoga MD, Constantin N, Duta-Cornescu G (2019) Genetic engineering in coffee. Caffeinated and Cocoa Based Beverages 8:447–488

    Article  Google Scholar 

  • Stavridou E, Τzioutziou NA, Madesis P, Labrou NE, Nianiou-Obeidat I (2019) Effect of different factors on regeneration and transformation efficiency of tomato (Lycopersicum esculentum) hybrids. Czech J Genet Plant Breed 55:120–127

    Article  CAS  Google Scholar 

  • Suma B, Keshavachandran R, Nybe E (2008) Agrobacterium tumefaciens mediated transformation and regeneration of ginger (Zingiber officinale Rosc.). J Trop Agric 46:38–44

    CAS  Google Scholar 

  • Sun S, Kang XP, Xing XJ, Xu XY, Cheng J, Zheng SW, Xing GM (2015) Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv. Hezuo 908) with improved efficiency. Biotechnol Biotec Eq 29:861–868

    Article  CAS  Google Scholar 

  • Sun YD, Luo WR, Li ZX, Li YH, Ni L (2017) Establishment of a high-efficiency genetic transformation system of cucumber (Cucumis sativus) using Csexpansin 10 (CsEXP10) gene. Int J Agric Biol 19:545–550

    Article  CAS  Google Scholar 

  • Tan J, Lin L, Luo H, Zhou S, Zhu Y, Wang X, Miao L, Wang H, Zhang P (2022) Recent progress in the regeneration and genetic transformation system of cucumber. Appl Sci 12:7180

    Article  CAS  Google Scholar 

  • Tzfira T, Li J, Lacroix B, Citovsky V (2004) Agrobacterium T-DNA integration: Molecules and models. Trends Genet 20:375–383

    Article  CAS  PubMed  Google Scholar 

  • Vasudevan V, Siva R, Krishnan V, Manickavasagam M (2020) Polyamines, sonication and vacuum infiltration enhances the Agrobacterium-mediated transformation in watermelon (Citrullus lanatus Thunb.). S Afr J Bot 128:333–338

    Article  CAS  Google Scholar 

  • Wang YR, Chen LM, Pan JS, He HL, Cai R (2006) Establishment of high effective regeneration system in cucumber (Cucumis Sativus L.) and agrobacterium tumefaciens mediated genetic transformation. J Shanghai Jiaotong Univ (agric Sci) 2:152–155

    Google Scholar 

  • Wang X, Tu M, Wang D, Liu J, Li Y, Li Z, Wang Y, Wang XP (2018) CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant Biotechnol J 16:844–855

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Liu H, Zhang M, Liu S, Hao Y, Zhang Y (2020) MdMYC2 and MdERF3 positively co-regulate α-farnesene biosynthesis in apple. Front Plant Sci 11:512844

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang Z, Li L, Ouyang L (2021) Efficient genetic transformation method for Eucalyptus genome editing. PLoS ONE 16:e0252011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xing Y, Yang Q, Ji Q, Luo Y, Zhang Y, Gu K, Wang D (2007) Optimization of Agrobacterium-mediated transformation parameters for sweet potato embryogenic callus using β-glucuronidase (GUS) as a reporter. Afr J Biotechnol 6:2578–2584

    Article  CAS  Google Scholar 

  • Yarra R, Jin L, Zhao Z, Cao H (2019) Progress in tissue culture and genetic transformation of oil palm: An overview. Int J Mol Sci 20:5353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yin XR, Xie XL, Xia XJ, Yu JQ, Ferguson IB, Giovannoni JJ, Chen KS (2016) Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening. Plant J 86:403–412

    Article  CAS  PubMed  Google Scholar 

  • Zhang C, Guan L, Fan X, Zheng T, Fang J (2019) Anatomical characteristics associated with different degrees of berry cracking in grapes. Sci Hortic 261:108992

    Article  Google Scholar 

  • Zhang XM, Wu YF, Li Z, Song CB, Wang XP (2021) Advancements in plant regeneration and genetic transformation of grapevine (Vitis spp.). J Integr Agr 20:1407–1434

    Article  Google Scholar 

  • Zhou YF, Massonnet M, Sanjak JS, Cantu D, Gaut BS (2017) Evolutionary genomics of grape (Vitis vinifera ssp. vinifera) domestication. Proc Natl Acad Sci USA 114:11715–11720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was financially supported by Natural Science Foundation of China (grant no. NSFC: U1904113 to D.L.G.), National Key Research and Development Program of China (grant no. 2018YFD1000105 to D.L.G.), and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (grant no. 21IRTSTHN021 to D.L.G.), Program for Science & Technology Innovation Talents in Universities of Henan Province (grant no. 21HASTIT035 to Y.H.Y.), Scientific and technological breakthroughs in Henan Province (grant no. 222102110083 to M.S.P.), PhD Research Startup Foundation of Henan University of Science and Technology (grant no. 13480068 to H.N.L.; grant no. 13480067 to M.S.P.).

Author information

Author notes

  1. Mao-Song Pei and Hai-Nan Liu contributed equally to this work.

Authors and Affiliations

  1. College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang, 471023, Henan Province, People’s Republic of China

    Mao-Song Pei, Hai-Nan Liu, Tong-Lu Wei, Yi-He Yu, Jia-Bing Jiao, Ying-Ying Lv & Da-Long Guo

  2. Henan Engineering Technology Research Center of Quality Regulation and Controlling of Horticultural Plants, Luoyang, 471023, People’s Republic of China

    Mao-Song Pei, Hai-Nan Liu, Tong-Lu Wei, Yi-He Yu, Jia-Bing Jiao, Ying-Ying Lv & Da-Long Guo

  3. The New Zealand Institute for Plant & Food Research Limited (PFR), Auckland, New Zealand

    Charles Ampomah-Dwamena

  4. Yanqing District Fruit Industry Service Station, Beijing, People’s Republic of China

    Feng Li

  5. Forestry Development Centre of Xiangfu District, Kaifeng, People’s Republic of China

    Hong-Chao Li

  6. Nong Fa Agricultural Science and Technology Company Limited, Luoyang, People’s Republic of China

    Xue-Jie Zhu

Authors
  1. Mao-Song Pei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hai-Nan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Charles Ampomah-Dwamena
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tong-Lu Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yi-He Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jia-Bing Jiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ying-Ying Lv
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Feng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hong-Chao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xue-Jie Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Da-Long Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.L.G. and M.S.P. conceived and designed the experiments. H.N.L. and M.S.P. provided experimental materials. Z.H.W., L.Y., Y.H.Y., and T.L.W. performed the preparatory work. M.S.P. and H.N.L. carried out the data analysis, conducted the molecular experiments, and wrote the paper. C.A.D. designed aspects of the study and revised the paper. All the authors have read and approved the final paper.

Corresponding author

Correspondence to Da-Long Guo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Communicated by Handling Editor: Peter Nick.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 4006 KB)

Supplementary file2 (PDF 293 KB)

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pei, MS., Liu, HN., Ampomah-Dwamena, C. et al. A simple and efficient protocol for transient transformation of sliced grape berries. Protoplasma 260, 757–766 (2023). https://doi.org/10.1007/s00709-022-01810-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-022-01810-w

Keywords

Search

Navigation