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Biolistic transformation of grapevine using minimal gene cassette technology

  • Genetic Transformation and Hybridization
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The use of minimal gene cassettes (MCs), which are linear DNA fragments (promoter+open reading frame+terminator) lacking the vector backbone sequence, was compared to the traditional use of whole circular plasmids (CPs) for transformation of grapevine. Embryogenic cell suspensions of ‘Chardonnay’ (Vitis vinifera L.) were transformed via particle co-bombardment using two nonlinked genes in either MCs or CPs. One construct contained the npt-II selectable marker and the second construct contained the MSI99 antimicrobial peptide gene. A total of five lines each from MC and CP treatments that showed positive signals by PCR for both the npt-II and MSI99 genes were selected. Southern blot analyses revealed up to five integration events in the DNA treatments. Transcription levels determined by semi-quantitative RT-PCR varied among transgenic lines. No significant differences were found in transgene transcription between lines from MC and CP transformation. The correlation between npt-II and MSI99 transcription levels was positive (P<0.05), however, no correlation between the transcription level and the number of integration events was observed. Transgenic lines presented a similar phenotype in leaf morphology and plant vigor compared to non-transgenic lines. Moreover, transgenic lines from both MC and CP DNA treatments produced fruit as did the non-transgenic lines in the third year of growth in the greenhouse. Our data confirm the effectiveness of the minimal cassette technology for genetic transformation of grapevine cultivars.

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circular plasmid(s)


minimal cassette(s)

MSI99 :

synthetic analog of magainin-2 gene


reverse transcription-polymerase chain reaction


  • Breitler JC, Labeyrie A, Meynard D, Legavre T, Guiderdoni E (2002) Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes. Theor Appl Genet 104:709–719

    Article  PubMed  CAS  Google Scholar 

  • Chen L, Marmey P, Taylor NJ, Brizard JP, Espinoza C, D’Cruz et al. (1998). Expression and inheritance of multiple transgenes in rice plants. Nat Biotechnol 16:1060–1064

    Article  PubMed  CAS  Google Scholar 

  • Christou P (1997) Rice transformation: bombardment. Plant Mol Biol 35:197–203

    Article  PubMed  CAS  Google Scholar 

  • Cluster PD, O’Dell M, Metzlaff M, Flavell RB (1994) Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. Plant Mol Biol 32:1197–1203

    Article  Google Scholar 

  • Colova-Tsolova V, Perl A, Krastanova S, Tsvetkov I, Atanassov A (2001) Genetically engineered grape for disease and stress tolerance. In: Roubelakis-Angelakis KA (ed) Molecular Biology and Biotechnology of the Grapevine. Kluwer Academic Publishers, The Netherlands, pp 441–432

    Google Scholar 

  • DeBuck S, DeWilde C, Van Montagu M, Depicker A (2000) T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol Breed 6:459–468

    Article  CAS  Google Scholar 

  • DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol 127:852–862

    Article  PubMed  CAS  Google Scholar 

  • Franks T, He DG, Thomas M (1998) Regeneration of transgenic Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis. Mol Breed 4:321–333

    Article  CAS  Google Scholar 

  • Fu X, Duc LT, Fontana S, Bong BB, Tinjuangjun P, Sudhakar D, Twyman RM, Christou P, Kohli A (2000) Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res 9:11–19

    Article  PubMed  CAS  Google Scholar 

  • Galet P.(1990) Cépages et Vignobles de France.Tome I-II. L'Ampélographie Francaise.Charles Dehan,Montpellier, France

    Google Scholar 

  • Gruber MY, Crosby WL (1993) Vectors for plant transformation. In: Glick BR, Thompson JE (eds) Methods in plant molecular biology and biotechnology. CRC Press, London, pp 89–119

    Google Scholar 

  • Hébert D, Kikkert JR, Smith FD, Reisch BI (1993) Optimization of biolistic transformation of embryogenic grape cell suspensions. Plant Cell Rep 12:585–589

    Article  Google Scholar 

  • Hunold R, Bronner R, Hahne G (1994) Early event in microprojectile bombardment, cell viability and particle location. Plant J 5:593–604

    Article  CAS  Google Scholar 

  • IPGRI (1997) Descriptors for grapevine (Vitis spp.). http://www.

  • Jakowitsch J, Papp I, Moscone E, Van Der Winden J, Matzke M, Matzke A (1999) Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans. Plant J 17:131–140

    Article  PubMed  CAS  Google Scholar 

  • Kikkert JR, Thomas MR, Reisch BI (2001) Grapevine genetic engineering. In: Roubelakis-Angelakis KA (ed) Molecular biology and biotechnology of the Grapevine. Kluwer Academic Publishers, The Netherlands, pp 393–410

    Google Scholar 

  • Kikkert JR, Vidal JR, Reisch BI (2004) Stable transformation of plant cell by particle bombardment/biolistics. In: Peña L (ed) Transgenic plants: methods and protocols. Humana Press, Totowa, NJ, USA, pp 61–78

    Chapter  Google Scholar 

  • Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70–73

    Article  CAS  Google Scholar 

  • Kohli A, Leech M, Vain P, Laurie D, Christou P (1998) Transgenic organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot-spots. Proc Natl Acad Sci USA 95:7203–7208

    Article  PubMed  CAS  Google Scholar 

  • Kohli A, Griffiths S, Palacios N, Twyman R, Vain P, Laurie D, Christou P (1999) Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J 17:591–601

    Article  PubMed  CAS  Google Scholar 

  • Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: Evidence for multiple complex patterns of integration. Plant J 11:945–957

    Article  PubMed  CAS  Google Scholar 

  • Kristyanne ES, Kim KS, Stewart JMcD (1997) Magainin 2 effects on the ultrastructure of five plant pathogens. Mycologia 89:353–360

    Article  CAS  Google Scholar 

  • Li ZT, Gray DJ (2003) Effect of five antimicrobial peptides on the growth of Agrobacterium tumefaciens, Escherichia coli and Xylella fastidiosa. Vitis 42:95–97

    CAS  Google Scholar 

  • Li Q, Lawrence CB, Xing HY, Babbitt RA, Bass WT, Maiti IB, Everett NP (2001) Enhanced disease resistance conferred by expression of an antimicrobial magainin analog in transgenic tobacco. Planta 212:635–639

    Article  PubMed  CAS  Google Scholar 

  • Loc NT, Tinjuangjun P, Gatehouse AMR, Christou P, Gatehouse JA (2002) Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance. Mol Breed 9:231–244

    Article  CAS  Google Scholar 

  • Lodhi MA, Ye GN, Weeden NF, Reisch BI (1994) A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12:6–13

    Article  CAS  Google Scholar 

  • Muller A, Kamisugi Y, Gruneberg R, Niedenhof I, Horold R, Meyer P (1999) Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum. J Mol Biol 291:29–46

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Renault AS, Deloire A, Letinois I, Kraeva E, Tesniere C, Ageorges A, Redon C, Bierne J (2000) β-1,3-Glucanase gene expression in grapevine leaves as a response to infection with Botrytis cinerea. Am J Enol Vitic 51:81–87

    CAS  Google Scholar 

  • Romano A, Raemakers K, Bernardi J, Visser R, Mooibroek H (2003) Transgene organization in potato after particle bombardment-mediated (co-)transformation using plasmids and gene cassettes. Transgenic Res 12:461–473

    Article  PubMed  CAS  Google Scholar 

  • Uzé M, Potrykus I, Sautter C (1999) Single-stranded DNA in the genetic transformation of wheat (Triticum aestivum L.): transformation frequency and integration pattern. Theor Appl Genet 99:487–495

    Article  Google Scholar 

  • Vidal JR, Kikkert JR, Wallace PG, Reisch BI (2003) 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

    Article  PubMed  CAS  Google Scholar 

  • Vidal JR, Kikkert JR, Malnoy MA, Wallace PG, Barnard J, Reisch BI (2006) Evaluation of transgenic ‘Chardonnay’ (Vitis vinifera) containing magainin genes for resistance to crown gall and powdery mildew. Transgenic Res 15:69–82

    Article  PubMed  CAS  Google Scholar 

  • Zasloff M (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci 84:5449–5453

    Article  PubMed  CAS  Google Scholar 

  • Zupan J, Muth TR, Draper O, Zambryski P (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23:11–28

    Article  PubMed  CAS  Google Scholar 

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The authors thank Dr. Herb Aldwinckle (Cornell University) for critically reviewing this manuscript and helpful discussions. We are grateful to Dr. John Sanford (FMS Foundation) and Dr. Franzine Smith (Sanford Scientific Inc.) for providing pSAN plasmids, Dr. John Barnard for supervising statistical analysis, and Steve Luce for excellent technical assistance. This research was supported by grants from the American Vineyard Foundation, the USDA Viticulture Consortium-East, and the New York Wine and Grape Foundation. J.R. Vidal was supported in part by Kaplan Funds and a postdoctoral grant from the Spanish Ministry of Education and Science.

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Correspondence to Jose R. Vidal.

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Communicated by L. Peña

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Vidal, J.R., Kikkert, J.R., Donzelli, B.D. et al. Biolistic transformation of grapevine using minimal gene cassette technology. Plant Cell Rep 25, 807–814 (2006).

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