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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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