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Plant Cell, Tissue and Organ Culture

, Volume 93, Issue 3, pp 311–321 | Cite as

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

  • Zhijian T. Li
  • S. A. Dhekney
  • M. Dutt
  • D. J. Gray
Original paper

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’.

Keywords

Agrobacterium tumefaciens Cocultivation EGFP GUS expression Transgenic plants Vitisvinifera 

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

Notes

Acknowledgements

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

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Zhijian T. Li
    • 1
  • S. A. Dhekney
    • 1
  • M. Dutt
    • 1
    • 2
  • D. J. Gray
    • 1
  1. 1.Grape Biotechnology Core Laboratory, Mid-Florida Research and Education CenterUniversity of Florida/IFASApopkaUSA
  2. 2.Citrus Research and Education CenterUniversity of Florida/IFASLake AlfredUSA

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