Molecular Breeding

, Volume 4, Issue 4, pp 321–333 | Cite as

Regeneration of transgenic shape Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis

  • Tricia Franks
  • Ding Gang He
  • Mark Thomas


Different approaches to producing transgenic grapevines based on regeneration via embryogenesis were investigated. Embryogenic callus was initiated from anther tissue of Vitis vinifera cv. Sultana and three embryogenic culture types (embryogenic callus, tissue type I; proliferating embryos, tissue type II; and a suspension) were established. The three culture types were incolucaled with Agrobacterium tumefaciens harbouring a binary vector which contained a uidA reporter gene and either a hpt or nptII selectable marker gene or the cultures were bombarded with microprojectiles carrying a uidA/nptII binary vector. Transgenic plants were produced only from Agrobacterium transformation experiments. Transformed embryos were selected with kanamycin or hygromycin antibiotics and recovered with the highest efficiency from inoculated type I cultures. Southern analysis of genomic DNA extracted from ten transgenic plants showed that the number of T-DNA insertions in the genome ranged from 1 to at least 4. Evidence for methylation of the T-DNA at cytosine and adenine residues in transgenic plants was found by Southern analysis of DNA digested with two isoschizomer pairs of restriction endonucleases. No evidence for genotype alterations or somatic meiosis was found when DNA from 80 somatic embryos and seven plants regenerated from embryogenic culture were analysed at six sequence-tagged sites which are heterozygous in cv. Sultana. Expression of the uidA gene in in vitro grown leaves of transgenic plants was most often high and uniform but GUS staining was occasionally observed to be low and/or patchy. Transgenic plants and all plants regenerated from embryogenic culture produced red veined, lobed leaves which are uncharacteristic of the accepted ampelographic phenotype of Sultana. It is suggested that this phenotype may represent a juvenile growth stage.

Agrobacterium tumefaciens DNA methylation embryogenic culture grapevine sequence-tagged sites STSs somatic meiosis transgenic plants Vitis vinifera 


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  1. 1.
    Carrington JC, Freed DD: Cap-independent enhancement of translation by a plant potyvirus 5′ nontranslated region. J Virol 64: 1590–1597 (1990).Google Scholar
  2. 2.
    Chuch G, Lincoln C, Hake S: KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell 8: 1277–1289 (1996).Google Scholar
  3. 3.
    Deloire A, Charpentier M, Berlioz G, Colin A, Gimonnet G: Micropropagation of the grapevine: results of 10 years of experiments in the Champagne vineyard and results of the first vinifications. Am J Enol Viticult 46: 571–578 (1995).Google Scholar
  4. 4.
    Faure O, Aarrouf J, Nougarede A: Ontogenesis, differentiation and precocious germination in anther-derived somatic embryos of grapevine (Vitis vinifera L.): proembryogenesis. Ann Bot 78: 23–28 (1996).Google Scholar
  5. 5.
    Galet P, Morton LT: A Practical Ampelography: Grapevine Identification. Cornell University Press, (1979).Google Scholar
  6. 6.
    Gamborg OL, Miller RA, Ojima K: Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 1515–158 (1968).Google Scholar
  7. 7.
    Giorgetti L, Vergara MR, Evangelista M, Lo Schiavo F, Terzi M, Nuti Ronchi V: On the occurence of somatic meiosis in embryogenic carrot cell cultures. Mol Gen Genet 246: 657–662 (1995).Google Scholar
  8. 8.
    Gleave AP: A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20: 1203–1207 (1992).Google Scholar
  9. 9.
    Graham MW, Larkin PJ: Adenine methylation at dam sites increases transient gene expression in plant cells. Transgen Res 4: 324–331 (1995).Google Scholar
  10. 10.
    Grenan S: Micropropagation of Grapevine (Vitis vinifera L.). In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry. High-Tech and Micropropagation II, pp. 371–398. Springer-Verlag, Berlin/Heidelberg (1992).Google Scholar
  11. 11.
    Harding K, Benson EE, Roubelakisangelakis KA: Methylated DNA changes associated with the initiation and maintenance of Vitis vinifera in vitro shoot and callus cultures: a possible mechanism for age-related changes. Vitis 35: 79–85 (1996).Google Scholar
  12. 12.
    Hébert D, Kikkert JR, Smith FD, Reisch BI: Optimization of biolistic transformation of embryogenic grape cell suspensions. Plant Cell Rep 12: 585–589 (1993).Google Scholar
  13. 13.
    Kikkert JR, Hébert-Soule D, Wallace PG, Striem MJ, Reisch BI: Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions. Plant Cell Rep 15: 311–316 (1996).Google Scholar
  14. 14.
    Klein TM, Gradziel T, Fromm ME, Sanford JC: Factors influencing gene delivery into Zea mays cells by high-velocity microprojectiles. Bio/technology 6: 559–563 (1988).Google Scholar
  15. 15.
    Koruza B, Jelaska S: Influence of meristem culture and virus elimination on phenotypical modifications of grapevine (Vitis vinifera L. cv. Refosk). Vitis 32: 59–60 (1993).Google Scholar
  16. 16.
    Lebrun L, Branchard M: Embryogénèse ‘indéfinie’ chez Vitis sp. cultivé in vitro. 3ème Symp. Int. sur la Physiologie de la Vigne (Bordeaux): 38–41 (1987).Google Scholar
  17. 17.
    Lincoln C, Long J, Yamaguchi J, Serikawa K, Hake S: A knotted1-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants. Plant Cell 6: 1859–1876 (1994).Google Scholar
  18. 18.
    Mattanovich D, Rüker F, da Câmara Machado A, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H: Efficient transformation of Agrobacterium spp. by electroporation. Nucl Acids Res 17: 6747 (1989).Google Scholar
  19. 19.
    Mauro MC, Toutain S, Walter B, Pinck L, Otten L, Coutosthevenot P, Deloire A, Barbier P: High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene. Plant Sci 112: 97–106 (1995).Google Scholar
  20. 20.
    McClelland M, Nelson M, Raschke E: Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucl Acids Res 22: 3640–3659 (1994).Google Scholar
  21. 21.
    Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).Google Scholar
  22. 22.
    Nakano M, Hoshino Y, Mii M: Regeneration of transgenic plants of grapevine (Vitis viniferaL.) via Agrobacterium rhizogenes-mediated transformation of embryogenic calli. J Exp Bot 45: 649–656 (1994).Google Scholar
  23. 23.
    Nitsch JP, Nitsch C: Haploid plants from pollen grains. Science 163: 85–87 (1969).Google Scholar
  24. 24.
    Perl A, Lotan O, Abuabied M, Holland D: Establishment of an Agrobacterium-mediated transformation system for (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions. Nature Biotechnol 14: 624–628 (1996).Google Scholar
  25. 25.
    Perl A, Saad S, Sahar N, Holland D: Establishment of long-term embryogenic cultures of seedless Vitis vinifera cultivars: a synergistic effect of auxins and the role of abscisic acid. Plant Sci 104: 193–200 (1995).Google Scholar
  26. 26.
    Pintor-Toro JA: Adenine methylation in zein genes. Biochem Biophys Res Comm 147: 1082–1087 (1987).Google Scholar
  27. 27.
    Polito VS, McGranahan G, Pinney K, Leslie C: Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): implications for Agrobacteriummediated transformation. Plant Cell Rep 8: 219–221 (1989).Google Scholar
  28. 28.
    Rajasekaran K, Mullins MG: The origin of embryos and plantlets from cultured anthers of hybrid grapevines. J Enol Viticult 34: 108–113 (1983).Google Scholar
  29. 29.
    Rajasekaren K, Mullins MG: Embryos and plantlets from cultured anthers of hybrid grapevines. J Exp Bot 30: 399–407 (1979).Google Scholar
  30. 30.
    Rambaud C, Blervacq AS, Devaux P, Dubois T, Dubois J, Lammin F, Vasseur J: There is no somatic meiosis in embryogenic leaves of Cichorium. Ann Bot 78: 223–232 (1996).Google Scholar
  31. 31.
    Restrepo MA, Freed DD, Carrington JC: Nuclear transport of plant potyviral proteins. Plant Cell 2: 987–998 (1990).Google Scholar
  32. 32.
    Rogers JC, Rogers SW: Comparison of the effects of N 6-methyldeoxyadenosine and N 5-methyldeoxycytosine on transcription from nuclear gene promoters in barley. Plant J 7: 221–233 (1995).Google Scholar
  33. 33.
    Schneider S, Reustle G, Zyprian E: Detection of somaclonal variation in grapevine regenerants from protoplasts by RAPDPCR. Vitis 35: 99–100 (1996).Google Scholar
  34. 34.
    Scorza R, Cordts JM, Gray DJ, Gonsalves D, Emershad RL, Ramming DW: Producing transgenic ‘Thompson Seedless’ grape (Vitis vinifera L.) plants. J Am Soc Hort Sci 121: 616–619 (1996).Google Scholar
  35. 35.
    Scorza R, Cordts JM, Ramming DW, Emershad RL: Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants. Plant Cell Rep 14: 589–592 (1995).Google Scholar
  36. 36.
    Thomas M, Scott NN: Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theor and Appl Genet 86: 985–990 (1993).Google Scholar
  37. 37.
    Thomas MR, Cain P, Scott NS: DNA typing of grapevines: a universal methodology and database for describing cultivars and evaluating genetic relatedness. Plant Mol Biol 25: 939–949 (1994).Google Scholar
  38. 38.
    Thomas MR, Matsumoto S, Cain P, Scott NS: Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. Theor Appl Genet 86: 173–180 (1993).Google Scholar
  39. 39.
    Vain P, Keen N, Murillo J, Rathus C, Nemes C, Finer JJ: Development of the particle inflow gun. Plant Cell Tissue Organ Cult 33: 237–246 (1993).Google Scholar
  40. 40.
    Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M: Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220: 245–250 (1990).Google Scholar
  41. 41.
    Vilaplana M, Mullins MG: Regeneration of grapevine (Vitis spp.) in vitro: formation of adventitious buds on hypocotyls and cotyledons of somatic embryos. J of Plant Physiol 134: 413–419 (1989).Google Scholar
  42. 42.
    Weber H, Ziechmann C, Graessmann A: In vitro DNA methylation inhibits gene expression in transgenic tobacco. EMBO J 9: 4409–4415 (1990).Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Tricia Franks
    • 1
  • Ding Gang He
    • 2
  • Mark Thomas
    • 1
  1. 1.CSIRO Plant Industry Horticulture Unit, Hartley GroveUrrbraeAustralia
  2. 2.ForBio Research Pty LtdIndooroopillyAustralia

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