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High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) containing npt-II and antimicrobial peptide genes

  • Genetic Transformation and Hybridization
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Abstract

A reliable and efficient system for transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) plants via microprojectile bombardment was developed. Improvements over the previous biolistic transformation system included: (1) the use of gold particles for bombardment; (2) step-wise selection at 10 then 15 mg/l kanamycin; and (3) embryo induction at 27°C. Embryogenic cell cultures were either bombarded with pBI426, which contains the reporter gene gus (uidA) coding for β-glucuronidase (GUS), or were co-bombarded with pSAN237 carrying the npt-II (neomycin phosphotransferase II) selectable marker gene, and a second plasmid with an antimicrobial peptide gene. A large number of transient (7,883±1,928) and stable (46±32) blue spots per plate at 2 and 95 days after bombardment, respectively, were obtained according to GUS expression analyses. A total of 447 putative transgenic embryos was harvested from 84 bombarded plates. From these embryos, 242 (54%) were regenerated into plants within the first year of the experiment. Southern blot analyses confirmed integration of the transgenes into the grape genome. Co-transformation was tested with four separate antimicrobial constructs. The co-transformation frequency of unlinked genes was 48% as measured by polymerase chain reaction (PCR), and 56% as estimated by dot blot hybridization. Expression of the gus gene, and PCR and Southern blot analyses of npt-II and antimicrobial genes from regenerated plants document stable transformation of 'Chardonnay' and establish the parameters for highly-efficient biolistic transformation in V. vinifera.

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Abbreviations

AMP :

Antimicrobial peptide

DBH :

Dot blot hybridization

GM+NOA:

Glycerol and maltose liquid medium with β-naphthoxyacetic acid

gus :

β-Glucuronidase gene

GUS :

β-Glucuronidase

Km :

Kanamycin

Km R :

Kanamycin resistant

mag2 :

Magainin-2

MS :

Murashige and Skoog medium

MS/2 :

Half-strength Murashige and Skoog medium

nos :

Nopaline synthase

npt-II :

Neomycin phosphotransferase II gene

PCR :

Polymerase chain reaction

PGL :

Peptidyl-glycine-leucine

Pubq3 :

Arabidopsis ubiquitin-3 promoter

Pubq10-L :

Arabidopsis ubiquitin-10L promoter

Pubq11 :

Arabidopsis ubiquitin-11 promoter

SP :

Signal peptide

Tnos :

Nopaline synthase terminator

WPM :

Woody plant medium

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Acknowledgements

The authors are grateful to Dr. J. Sanford of FMS Foundation and Dr. F. Smith of Sanford Scientific Inc. for the magainin and PGL constructs. Drs. R. Seem and D. Gadoury suggested the use of these constructs. We thank Drs. S. Brown and G. Fermín (Cornell University) for critically reviewing this manuscript and for helpful discussions. This research was supported by Research Grant No. US-2759–96 from BARD, The United States-Israel Binational Agricultural Research and Development Fund, as well as grants from the Cornell Biotechnology Program, the USDA-Viticulture Consortium-East and the Kaplan Fund. J.R. Vidal was supported by a postdoctoral grant from the Spanish Ministry of Education and Science.

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Authors and Affiliations

  1. Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA

    J. R. Vidal, J. R. Kikkert, P. G. Wallace & B. I. Reisch

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  1. J. R. Vidal
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  2. J. R. Kikkert
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  3. P. G. Wallace
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  4. B. I. Reisch
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Correspondence to B. I. Reisch.

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Communicated by E.D. Earle

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Vidal, J.R., Kikkert, J.R., Wallace, P.G. et al. High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) containing npt-II and antimicrobial peptide genes. Plant Cell Rep 22, 252–260 (2003). https://doi.org/10.1007/s00299-003-0682-x

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  • DOI: https://doi.org/10.1007/s00299-003-0682-x

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