Plant Cell Reports

, Volume 25, Issue 11, pp 1166–1173

Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera)

  • A. Richter
  • H.-J. Jacobsen
  • A. de Kathen
  • G. de Lorenzo
  • K. Briviba
  • R. Hain
  • G. Ramsay
  • H. Kiesecker
Genetic Transformation and Hybridization


The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.


Agrobacterium Expression stability Pea PGIP Resveratrol 



Fresh weight


Polymerase chain reaction


Reverse transcriptase PCR


  1. Baulcombe D (2004) RNA silencing in plants. Nature 431:7006CrossRefGoogle Scholar
  2. Bean SJ, Gooding PS, Mullineaux PM, Davies DR (1997) A simple system for pea transformation. Plant Cell Rep 16:513–519Google Scholar
  3. 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(6):1195–1197PubMedCrossRefGoogle Scholar
  4. Berger DK, Oelofse D, Arendse MS, Du Plessis E, Dubery IA (2000) Bean polygalacturonase inhibitor protein-1 (PGIP-1) inhibits polygalacturonases from Stenocarpella maydis. Physiol Mol Plant Pathol 57:5–14CrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method forthe quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Brown DCW, Atanassov A (1985) Role of genetic backround in somatic embryogenesis in Medicago. Plant Cell Tissue Organ Cult 4:111–122CrossRefGoogle Scholar
  7. Confalonieri M (2004) Expression of the stilbene synthase (StSy) gene from grapevine in transgenic white poplar results in high accumulation of the antioxidant compounds resveratrol glucosides. Transgenic Res 13:203–214PubMedCrossRefGoogle Scholar
  8. Coutos-Thevenot 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 52:901–910PubMedCrossRefGoogle Scholar
  9. De Bolle MFC, Butaye KMJ, Coucke WJW, Goderis IJWM, Wouters PFJ, von Boxel N, Broekaert WF, Cammue BPA (2003) Analysis of the influence of promoter elements and a matrix attachment region on the inter-individual variation of transgene expression in populations of Arabidopsis thaliana. Plant Sci 165:169–179CrossRefGoogle Scholar
  10. De Lorenzo G, D’Ovidio R, Cervone F (2001) The role of polygalacturonase-inhibiting proteins (PGIP) in defense against pathogenic fungi. Ann Rev Phytopathol 39:313–335CrossRefGoogle Scholar
  11. De Neve M, De Buck S, De Wilde C, Van Houdt H, Strobbe I, Jacobs A, Van Montagu M, Depicker A (1999) Gene silencing results in instability of antibody production in transgenic plants. Mol Gen Genet 260:582–592PubMedCrossRefGoogle Scholar
  12. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12(1):13–15Google Scholar
  13. Faize M, Sugiyama T, Faize L, Ishii H (2003) Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab. Mol Plant-Microbe Interact 63:319–327Google Scholar
  14. Favaron F, Ovidio R, Porceddu E, Alghisi P (1994) Purification and molecular characterisation of a soybean polygalacturonase-inhibiting protein. Planta 195:80–87PubMedCrossRefGoogle Scholar
  15. Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back! Biotechnology 12:883–888CrossRefGoogle Scholar
  16. Grant JE, Cooper PA, McAra AE, Frew TJ (1995) Transformation of peas (Pisum sativum L.) using immature cotyledons. Plant Cell Rep 15:254–258CrossRefGoogle Scholar
  17. Grant JE, Thomson LMJ, Pither-Joyce MD, Dale TM, Cooper PA (2003) Influence of Agrobacterium tumefaciens strain on the production of transgenic peas (Pisum sativum L.). Plant Cell Rep 21:1207–1210PubMedCrossRefGoogle Scholar
  18. Hain R, Reif H-J, Krause E, Langebartels R, Kindl H, Vornam B, Wiese W, Schmeltzer E, Schreier PH, Stöcker RH, Stenzel K (1993) Disease resistance results from foreign phytoalexin expression in a novel plant. Nature (London) 361:153–156CrossRefGoogle Scholar
  19. Halpin C (2005) Gene stacking in transgenic plants—the challenge for 21th century plant biotechnology. Plant Biotechnol J 3:141–155CrossRefGoogle Scholar
  20. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in post-transcriptional gene silencing in plants. Science 286:290–252CrossRefGoogle Scholar
  21. 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
  22. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium vectors for plant transformation. Transgenic Res 2:208–218CrossRefGoogle Scholar
  23. Iyer LM, Kumpatla SP, Chandrasekharan MB, Hall TC (2000) Transgene silencing in monocots. Plant Mol Biol 43:323–346PubMedCrossRefGoogle Scholar
  24. James VA, Avart C, Worland B, Snape JW, Vain P (2002) The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet 104:553–561PubMedCrossRefGoogle Scholar
  25. Johnston DJ, Ramanathan V, Williamson B (1993) A protein from immature raspberry fruits which inhibits endopolygalacturonases from Botrytis cinerea and other microorganisms. J Exp Bot 44:971–976Google Scholar
  26. Keon JPR, Waksman G (1990) Common amino acid domains among endopolygalacturonases of ascomycete fungi. Appl Environ Microbiol 56:2522–2528PubMedGoogle Scholar
  27. Kobayashi S, Ding CK, Nakamura Y, Nakajima I, Matsumoto R (2000) Kiwifruits (Actinidia deliciosa) transformed with a Vitis stilbene synthase gene produce piceid (resveratrol-glucoside). Plant Cell Rep 19:904–910CrossRefGoogle Scholar
  28. 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 96:1004–1012CrossRefGoogle Scholar
  29. Lotter HC, Berger DK (2005) Anthracnose of lupins in South Africa in caused by Colletotrichum lupini var. setosum. Australas Plant Pathol 34:385–392CrossRefGoogle Scholar
  30. Matzke MA, Birchler JA (2005) RNAi-mediated pathways in the nucleus. Nat Rev Genet 6:24–35PubMedCrossRefGoogle Scholar
  31. Matzke AJM, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148PubMedCrossRefGoogle Scholar
  32. Matzke MA, Mette MF, Matzke AJM (2000) Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanism in plant and vertebrates. Plant Mol Biol 43:401–415PubMedCrossRefGoogle Scholar
  33. Meyer P, Saedler H (1996) Homology-dependent gene silencing in plants. Ann Rev Plant Physiol Plant Mol Biol 47:23–48CrossRefGoogle Scholar
  34. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  35. Nadolska-Orczyk A, Orczyk W (2000) Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.). Mol Breeding 6:185–194CrossRefGoogle Scholar
  36. Polowick PL, Quandt J, Mahon JD (2000) The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions. Plant Sci 153:161–170PubMedCrossRefGoogle Scholar
  37. 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 13:942–950PubMedGoogle Scholar
  38. Puonti-Kaerlas J, Eriksson T, Engström P (1990) Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens-mediated gene transfer. Theor Appl Genet 80:246–252CrossRefGoogle Scholar
  39. Puonti-Kaerlas J, Eriksson T, Engström P (1992) Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants. Theor Appl Genet 84:443–450CrossRefGoogle Scholar
  40. Schroeder HE, Schotz AH, Wardley-Richardson T, Spencer D, Higgins TJV (1993) Transformation and regeneration of two cultivars of pea (Pisum sativum L.). Plant Physiol 101:751–757PubMedCrossRefGoogle Scholar
  41. Senthil G, Williamson B, Dinkins RD, Ramsay G (2004) An efficient transformation system for chickpea (Cicer arietinum L.). Plant Cell Rep 23:297–303PubMedCrossRefGoogle Scholar
  42. Sotheeswaran S, Pasupathy J (1993) Distribution of resveratrol oligomers in plants. Phytochem 32:1083–1092CrossRefGoogle Scholar
  43. Stark-Lorenzen P, Nelke B, Hänbler G, Mühlbach HP, Thomzik JE (1997) Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.). Plant Cell Rep 16:668–673CrossRefGoogle Scholar
  44. Stevens L, Stoopen GM, Elbers IJW, Molthofff JW, Bakker HAC, Lommen A, Bosch D, Jordi W (2000) Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol 124:173–182PubMedCrossRefGoogle Scholar
  45. Szankowski I, Briviba K, Fleschhut J, Schönherr J, Jacobsen H-J, Kiesecker H (2003) Transformation of apple (Malus domestica Borkh.) with stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep 22:141–149PubMedCrossRefGoogle Scholar
  46. Taylor RJ, Secor GA (1988) An improved diffusion assay for quantifying the polygalacturonase content of Erwinia culture filtrates. Phytopathology 78:1101–1103Google Scholar
  47. 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 6:2519–2523PubMedGoogle Scholar
  48. Thomzik JE, Stenzel K, Stöcker R, Schreier PH, Hain R, Stahl DJ (1997) Synthesis of grapevine phytoalexin in transgenic tomatoes (Lycopersicon esculentum Mill.) conditions resistance against Phytophthora infestans. Physiol Mol Plant Pathol 51:265–278CrossRefGoogle Scholar
  49. Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789PubMedCrossRefGoogle Scholar
  50. Waterhouse AL, Lamuela-Raventos (1994) The occurrence of piceid, a stilbene glucoside, in grape berries. Phytochem 37:571–573CrossRefGoogle Scholar
  51. Williamson B, Johnston DJ, Ramanathan V, McNicol RJ (1993) A polygalacturonase inhibitor from immature raspberry fruits: a possible new approach to grey mold control. Acta Hort 352:601–605Google Scholar
  52. Zhu YJ, Agbayani R, Jackson MC, Tang CS, Moore PH (2004) Expression of the grapevine stilbene synthase gene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora. Planta 220:241–250PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • A. Richter
    • 1
  • H.-J. Jacobsen
    • 1
  • A. de Kathen
    • 2
  • G. de Lorenzo
    • 3
  • K. Briviba
    • 4
  • R. Hain
    • 5
  • G. Ramsay
    • 6
  • H. Kiesecker
    • 7
  1. 1.Department of Molecular GeneticsUniversity of HannoverHannoverGermany
  2. Kathen & Pickardt BioTechConsult GbRBerlinGermany
  3. 3.Dip. Biologia Vegetale Università “La Sapienza”RomaItaly
  4. 4.Institute for Nutritional PhysiologyFederal Research Center for NutritionKarlsruheGermany
  5. 5.Bayer CropScience GmBHGlobal Biology Herbicides BiochemistryFrankfurt-am-MainGermany
  6. 6.Scottish Crop Research InstituteInvergowrieUK
  7. 7.German Collection of Microorganisms and Cell Cultures GmbHBraunschweigGermany

Personalised recommendations