Plant Cell Reports

, Volume 22, Issue 2, pp 141–149 | Cite as

Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa)

  • I. Szankowski
  • K. Briviba
  • J. Fleschhut
  • J. Schönherr
  • H-J. Jacobsen
  • H. Kiesecker
Genetic Transformation and Hybridization

Abstract

The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs. Elstar and Holsteiner Cox in order to establish resistance against fungal diseases. The gene encoding the stilbene synthase (Vst1) from Vitis vinifera L., responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi (Actinidia deliciosa) were transferred into Holsteiner Cox and Elstar via Agrobacterium tumefaciens-mediated transformation. A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis. High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.

Keywords

Apple (Malus × domestica Borkh.) Transformation Stilbene synthase PGIP Agrobacterium 

Abbreviations

BAP

6-Benzylaminopurine

E

Elstar

H

Holsteiner Cox

HPLC

High performance liquid chromatography

IBA

Indole-3-butyric acid

NAA

α-Naphthaleneacetic acid

TDZ

Thidiazuron

YEP

Yeast extract broth

Notes

Acknowledgements

We thank Rüdiger Hain, Bayer AG, for providing the Vst1 gene and the Scottish Research Center for providing PSCP2. Elstar in vitro cultures were kindly provided by Anette Urbanietz, BAZ Ahrensburg.

References

  1. Becker D, Kemper E, Schell J, Masterson R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20:1195–1197Google Scholar
  2. Bertelli AA, Giovannini L, Giannessi D, Migliori M, Bernini W, Fregoni M, Bertelli A (1995) Antiplatelet activity of synthetic and natural resveratrol in red wine. Int J Tissue React 17:1-3PubMedGoogle Scholar
  3. Bolar JP, Brown SK, Norelli JL, Aldwinckle H (1999) Factors affecting the transformation of ´Marshall McIntosh´ apple by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 55:31–38Google Scholar
  4. Bolar JP, Norelli JL, Wong KW, Hayes K, Harman GE, Aldwickle H (2000) Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology 90:72–77Google Scholar
  5. Bolar JP, Norelli JL, Wong KW, Hayes K, Harman GE, Brown SK, Aldwickle HS (2001) Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants. Transgen Res 10:533–543CrossRefGoogle Scholar
  6. Cervone F, De Lorenzo G, Pressey R, Darvill AG, Albersheim P (1990) Can Phaseolus PGIP inhibit pectic enzymes from microbes and plants? Phytochemistry 29) 447–449Google Scholar
  7. Coutos-Thévenot P, Poinssot B, Bonomelli A, Yean H, Breda C, Buffard D, Esnault R, Hain R, Boulay M (2001) In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen-inducible PR 10 promoter. J Exp Bot 52:901–910CrossRefPubMedGoogle Scholar
  8. De Bondt A, Eggermont K, Penninckx I, Goderis I, Broekaert WF (1996) Agrobacterium-mediated transformation of apple (Malus × domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants. Plant Cell Rep 15:549–554CrossRefGoogle Scholar
  9. De Lorenzo G, D`Ovidio R, Cervone F (2001) The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against fungi. Annu Rev Phytopathol 39:313–335Google Scholar
  10. Dercks W, Creasy LL (1989) The significance of stilbene phytoalexins in the Plasmopara viticola-grapevine interaction. Physiol Mol Plant Pathol 34:189–202Google Scholar
  11. Desiderio A, Aracri B, Leckie F, Mattei B, Salvi G, Tigelaar H, Van Roekel JSC, Baulcombe DC, Melchers LS, De Lorenzo G, Cervone F (1997) Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris. Mol Plant-Microbe Interact 10:852–860Google Scholar
  12. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  13. Hain R, Reif HJ, Krause E, Langebartels R, Kindl H, Vornam B, Wiese W, Schmelzer E, Schreier PH, Stöcker RH, Stenzel K (1993) Disease resistance results from foreign phytoalexin expression in a novel plant. Nature 361:153–156Google Scholar
  14. Hipskind JD, Paiva NL (2000) Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis. Mol Plant Microbe Interact 13:551–562PubMedGoogle Scholar
  15. Holefors A, Xue Z-T, Welander M (1998) Transformation of apple rootstock M26 with the rolA gene and its influence on growth. Plant Sci 136:69–78Google Scholar
  16. Holefors A, Xue ZT, Zhu LH, Welander M (2000) The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26. Plant Cell Rep 19:1049–1056Google Scholar
  17. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium vectors for plant transformation. Transgen Res 2:208–218Google Scholar
  18. James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector. Plant Cell Rep 7:658–661Google Scholar
  19. James DJ, Passey AJ, Webster AD, Barbar DJ, Dandekar AM, Uratsu SL, Viss P (1993) Transgenic apples and strawberries: advances in transformation, introduction of genes for insect resistance and field studies of tissue cultured plants. Acta Hortic 336:179-184Google Scholar
  20. Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CWW, Fong HHS, Farnswoth NR, Kinghorn AD, Mehta RG, Moon RC, Pezzuto JM (1997) Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275:218–220PubMedGoogle Scholar
  21. Johnston DJ, Williamson B (1992) Purification and characterization of four polygalacturonases from Botrytis cinerea. Mycol Res 96:343–349Google Scholar
  22. Ko K, Norelli JL, Reynoird JP, Boresjza-Wysocka E, Brown SK, Aldwickle HS (2000) Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple. Biotechnol Lett 22:373–381CrossRefGoogle Scholar
  23. Ko K, Norelli JL, Reynoird JP, Aldwickle HS, Brown S (2002) T4 lysozyme and attacin genes enhance resistance of transgenic `Galaxy` apple against Erwinia amylovora. J Am Soc Hortic Sci 127:515–519Google Scholar
  24. Kobayashi S, Ding CK, Nakamura Y, Nakajima I, Matsumoto R (2000) Kiwifruit (Actinidia deliciosa) transformed with a Vitis stilbene synthase gene produce piceid (resveratrol-glucoside). Plant Cell Rep 19:904–910CrossRefGoogle Scholar
  25. Kohli A, Gahakwa D, Vain P, Laurie DA, Christou P (1999) Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing. Planta 208:88–97Google Scholar
  26. Langcake P, Pryce RJ (1976) The production of resveratrol by Vitis vinifera and other members of the Vitaceae as a response to infection or injury. Physiol Plant Pathol 9:77–86Google Scholar
  27. Leckband G, Lörz H (1998) Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance. Theor Appl Genet 96:1001–1012CrossRefGoogle Scholar
  28. Leckie F, Mattei B, Capodicasa C, Hemmings A, Nuss L, Aracri B, De Lorenzo G, Cervone F (1999) The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed ß-strand/ß-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability. EMBO J 18:2352–2363CrossRefPubMedGoogle Scholar
  29. Liswidowati, Melchior F, Hohmann F, Schwer B, Kindl H (1991) Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells. Planta 183:307–314Google Scholar
  30. Liu Q, Ingersoll J, Owens L, Salih S, Meng R, Hammerschlag F (2001) Response of transgenic Royal Gala apple (Malus × domestica Borkh.) shoots carrying a modified cecropin MB39 gene, to Erwinia amylovora. Plant Cell Rep 20:306–312CrossRefGoogle Scholar
  31. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619CrossRefPubMedGoogle Scholar
  32. Maheswaran G, Welander M, Hutchinson JF, Graham MW, Richards D (1992) Transformation of apple rootstock M26 with Agrobacterium tumefaciens. J Plant Physiol 139:560–568Google Scholar
  33. Manna SK, Mukhopadhyay A, Aggarwal BB (2000) Resveratrol suppresses TNF-induced activation of nuclear transcription factors NF-κB, activator protein-1, and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. J Immunol 164:6509–6519PubMedGoogle Scholar
  34. Maximova SN, Dandekar AM, Guiltinan MJ (1998) Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting. Plant Mol Biol 37:549–559PubMedGoogle Scholar
  35. Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJM (2000) Transcriptional silencing and promoter methylation triggered by double stranded RNA. EMBO J 19:5194–5201PubMedGoogle Scholar
  36. Mlotshwa S, Voinnet O, Mette MF, Matzke M, Vaucheret H, Ding SW, Pruss G, Vance VB (2002) RNA silencing and the mobile silencing signal. Plant Cell S289–S301Google Scholar
  37. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  38. Norelli JL, Aldwinckle HS, Beltran LD, Jaynes JM (1994) Transgenic 'Malling 26' apple expressing the attacin E gene has increased resistance to Erwininia amylovora. Euphytica 77:123–128Google Scholar
  39. Powell ALT, van Kan J, ten Have A, Visser J, Greve LC, Bennett AB, Labavitch JM (2000) Transgenic expression of pear PGIP in tomato limits fungal colonization. Mol Plant-Microbe Interact 13:942–950Google Scholar
  40. Puite KJ, Shaart JG (1996) Genetic modification of the commercial apple cultivars Gala, Golden Delicious and Elstar via an Agrobacterium tumefaciens-mediated transformation. Plant Sci 119:125–133Google Scholar
  41. Schoeppner A, Kindl H (1979) Stilbene synthase (Pinosylvine synthase) and its induction by ultraviolet light. FEBS Lett 108:349–352CrossRefPubMedGoogle Scholar
  42. Schoeppner A, Kindl H (1984) Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut. J Biol Chem 259:6806–6811PubMedGoogle Scholar
  43. Sedira M, Holefors A, Welander M (2001) Protocol for transformation of the apple rootstock Jork 9 with the rolB gene and its influence on rooting. Plant Cell Rep 20:517–524CrossRefGoogle Scholar
  44. Simpson CG, MacRae E, Gardner RC (1995) Cloning of a polygalacturonase-inhibiting protein from kiwifruit (Actinidia deliciosa). Plant Gene Register (PGR95-037). Plant Physiol 108:1748Google Scholar
  45. Sotheeswaran S, Pasupathy P (1993) Distribution of resveratrol oligomers in plants. Phytochemistry 32:1083–1092CrossRefGoogle Scholar
  46. Sriskandarajah S, Goodwin PB, Speirs J (1994) Genetic transformation of the apple scion cultivar 'Delicious' via Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 36:317–329Google Scholar
  47. Stark-Lorenzen P, Nelke B, Hänßler G, Mühlbach HP, Thomzik JE (1997) Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.) Plant Cell Rep 16:668–673Google Scholar
  48. Stotz HU, Contos JJA, Powell ALT, Bennett AB, Labavitch JM (1994) Structure and expression of an inhibitor of fungal polygalacturonases from tomato. Plant Mol Biol 25:607–617Google Scholar
  49. Subbaramaiah K, Chung WJ, Michaluart P, Telang N, Tanabe T, Inoue H, Jang M, Pezzuto JM, Dannenberg A (1998) Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 273:21875–21882PubMedGoogle Scholar
  50. Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J 6:2519–2523Google Scholar
  51. Van Nerum I, Incerti F, Keulemans J, Broothaerts (2000) Analysis of self-fertility in transgenic apple lines transformed with an S-allele in sense or antisense direction. Acta Hortic 538:625–629Google Scholar
  52. Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091PubMedGoogle Scholar
  53. Waterhouse AL, Lamuela-Raventós RM (1994) The occurrence of piceid, a stilbene glucoside, in grape berries. Phytochemistry 37:571–573CrossRefGoogle Scholar
  54. Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defense against viruses. Nature 411:834–842CrossRefPubMedGoogle Scholar
  55. Welander M, Pawlicki N, Holefors A, Wilson F (1998) Genetic transformation of the apple rootstock M26 with the RolB gene and its influence on rooting. J Plant Physiol 153:371–380Google Scholar
  56. Wong KW, Harman GE, Norelli JL, Gustafson HL, Aldwinckle HS (1999) Chitinase-transgenic lines of 'Royal Gala' apple showing enhanced resistance to apple scab. Acta Hortic 494:595–599Google Scholar
  57. Yao JL, Cohen D, Atkinson R, Richardson K, Morris B (1995) Regeneration of transgenic plants from the commercial apple cultivar Royal Gala. Plant Cell Rep 14:407–412Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • I. Szankowski
    • 1
  • K. Briviba
    • 3
  • J. Fleschhut
    • 3
  • J. Schönherr
    • 1
  • H-J. Jacobsen
    • 2
  • H. Kiesecker
    • 2
  1. 1.Abteilung ObstbauInstitut für Gemüse- und Obstbau, Universität HannoverSarstedtGermany
  2. 2.Lehrgebiet Molekulargenetik, Universität HannoverHannoverGermany
  3. 3.Bundesforschungsanstalt für ErnährungInstitut für ErnährungsphysiologieKarlsruheGermany

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