Skip to main content
SpringerLink
Log in

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

  • Original paper
  • Published:
Plant Cell, Tissue and Organ Culture Aims and scope Submit manuscript

Abstract

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.

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

Abbreviations

BA:

Benzyladenine

CaMV35S:

Cauliflower mosaic virus 35S

CTAB:

Cetyltrimethylammonium bromide

2,4-D:

2,4-Dichlorophenoxyacetic acid

EGFP:

Enhanced green fluorescence protein

GUS:

β-Glucuronidase

MU:

4-Methyl umbelliferone

MUG:

4-Methylumbelliferyl-b-D-glucuronide

NOA:

β-Naphthoxyacetic acid

NPTII:

Neomycin phosphotransferase II

PEM:

Pro-embryonic masses

SE:

Somatic embryos

References

  • Burrow MD, Chlan CA, Sen P, Murai N (1990) High frequency generation of transgenic tobacco plants after modified leaf disk cocultivation with Agrobacterium tumefaciens. Plant Mol Biol Rep 8:24–139

    Article  Google Scholar 

  • Das D, Reddy M, Upadhyaya S, Sopory S (2002) An efficient leaf disc culture method for the regeneration via somatic embryogenesis and transformation of grape (Vitis vinifera L.). Plant Cell Rep 20:999–1005

    Article  CAS  Google Scholar 

  • Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut rootstock. HortScience 19:507–509

    Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:12–14

    Google Scholar 

  • Garfinkel DJ, Nester EW (1980) Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol 144:732–743

    CAS  PubMed  Google Scholar 

  • Gray DJ (1995) Somatic embryogenesis in grape. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol. 2, Dordrecht, Kluwer Academic Publishers, Dordrecht, pp 191–217

    Google Scholar 

  • Gray DJ, Mortensen JA (1987) Initiation and maintenance of long term somatic embryogenesis from anthers and ovaries of Vitis longii 'Microsperma'. Plant Cell Tiss Org Cult 9:73–80

    Article  Google Scholar 

  • Gray DJ, Jayasankar S, Li Z (2005a) Vitis spp. Grape, Chap. 22. In: Litz RE (ed) Biotechnology of fruit and nut crops. CAB International Wallingford, Oxford, pp 672–706

    Google Scholar 

  • Gray DJ, Li ZT, Hopkins DL, Dutt M, Dhekney SA, Van Aman MM, Tattersall J, Kelley KT (2005b) Transgenic grapevines resistant to Pierce’s disease. 2005 Amer Soc Hort Sci Ann Meeting. HortScience 40:1104A

    Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    Article  CAS  Google Scholar 

  • Hoshino Y, Zhu YM, Nakano M, Takahashi E, Mii M (1998) Production of transgenic grapevine (Vitis vinifera L. cv. Koshusanjaku) plants by cocultivation of embryogenic calli with Agrobacterium tumefaciens and selecting secondary embryos. Plant Biotechnol 15:29–33

    CAS  Google Scholar 

  • Iocco P, Franks T, Thomas MR (2001) Genetic transformation of major wine grape varieties of Vitis vinifera L. Transgenic Res 10:105–112

    Article  CAS  PubMed  Google Scholar 

  • Jayasankar S, Bondada BR, Li Z, Gray DJ (2003) Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryonic masses. Am J Bot 90:973–979

    Article  Google Scholar 

  • Jayasankar S, Van Aman M, Cordts J, Dhekney S, Li Z, Gray DJ (2005) Long term storage of suspension culture-derived grapevine somatic embryos and regeneration of plants. In vitro Cell Dev Biol Plant 41:752–756

    Article  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  • Kaniewski WK; Thomas PE (1999) Field testing for virus resistance and agronomic performance in transgenic plants. Mol Biotechnol 12:101–115

    Article  Google Scholar 

  • Li Z, Jayasankar S, Gray DJ (2001) Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera). Plant Sci 160:877–887

    Article  CAS  PubMed  Google Scholar 

  • Li ZT, Jayasankar S, Gray DJ (2004) Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Trans Res 13:143–154

    Article  CAS  Google Scholar 

  • Li ZT, Dhekney S, Dutt M, Van Aman M, Tattersall J, Kelley KT, Gray DJ (2006) Optimizing Agrobacterium-mediated transformation of grapevine. In  vitro Cell Dev Biol Plant 42:220–227

    Article  Google Scholar 

  • Mauro MC, Toutain S, Walter B, Pinck L, Otten L, Coutos-Thevenot P, Deloire A, Barbier P (1995) High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene. Plant Sci 112:97–106

    Article  Google Scholar 

  • Mitsuhara I, Ugaki M, Hirochika M, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59

    CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nakano M, Hoshino Y, Mii M (1994) Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenes-mediated transformation of embryogenic calli. J Exp Bot 45:649–656

    Article  CAS  Google Scholar 

  • Perl A, Lotan O, Abu-Abied M, Holland D (1996) Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions. Nature Bio/Technol 14:624–628

    Article  CAS  Google Scholar 

  • Scorza R, Cordts JM, Ramming DW, Emershad RL (1995) Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants. Plant Cell Rep 14:589–592

    Article  Google Scholar 

  • Scorza R, Cordts JM, Gray DJ, Gonsalves D Emershad RL, Ramming DW (1996) Producing transgenic ‘Thompson Seedless’ grape (Vitis vinifera L.) plants. J Am Soc Hortic Sci 121:616–619

    Google Scholar 

  • Xue B, Ling KS, Reid CL, Krastanova S, Sekiya M, Momol EA, Sule S, Mozsar J, Gonsalves D, Burr TJ (1999) Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens. In vitro Cell Dev Biol Plant 35:226–231

    Article  CAS  Google Scholar 

  • Yamamoto T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hibi T, Hayashi T, Matsuta N (2000) Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Rep 19:639–646

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by the Florida Agricultural Experiment Station and the Florida Department of Agriculture and Consumer Services’ Viticulture Trust Fund.

Author information

Author notes

  1. M. Dutt

    Present address: Citrus Research and Education Center, University of Florida/IFAS, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

Authors and Affiliations

  1. Grape Biotechnology Core Laboratory, Mid-Florida Research and Education Center, University of Florida/IFAS, 2725 Binion Road, Apopka, FL, 32703-8504, USA

    Zhijian T. Li, S. A. Dhekney, M. Dutt & D. J. Gray

Authors
  1. Zhijian T. Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. A. Dhekney
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Dutt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D. J. Gray
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D. J. Gray.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Z.T., Dhekney, S.A., Dutt, M. et al. An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.). Plant Cell Tiss Organ Cult 93, 311–321 (2008). https://doi.org/10.1007/s11240-008-9378-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-008-9378-9

Keywords

Search

Navigation