Transgenic Research

, Volume 8, Issue 3, pp 179–189 | Cite as

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

  • Sebastian Fettig
  • Dieter Hess
Article

Abstract

A chimeric stilbene synthase (sts)‐gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbene‐type phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars ‘Combi’ and ‘Hanno’ were used as target tissue for co‐transformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bar‐gene either driven by the ubiquitin‐promoter from maize (pAHC 25, also containing the uidA‐gene driven by the ubiquitin‐promoter), or by the actin‐promoter (pDM 302) from rice. The co‐transferred plasmid pStil 2 consisted of a grapevine sts‐coding region driven by the ubiquitin promoter. Eight transgenic ‘Combi’ and one ‘Hanno’ TO‐plant were obtained and, except one ‘Combi’ TO‐plant, found to be co‐transformants due to the integration of both the sts‐gene and the selectable marker or reporter genes. Expression of the sts‐gene was proven by RT‐PCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The sts‐gene was expressed in four of the seven transgenic ‘Combi’ T_o‐plants. Two of the respective T1‐progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the sts‐gene showed that in three non‐expressing progenies inactivation was paralleled by methylation.

methylation resveratrol stilbene synthase transgenic wheat 

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References

  1. Ahuja PS, Pental D and Cocking EC (1982) Plant regeneration from leaf base callus and cell suspensions of Triticum aestivum(L.). Z.Pflanzenzüchtg 89:139–144.Google Scholar
  2. Altpeter F, Vasil V, Srivastava V, Stöger E and Vasil IK (1996a) Accelerated production of transgenic wheat (Triticum aestivumL.) plants. Plant Cell Rep 16:12–17.Google Scholar
  3. Altpeter F, Vasil V, Srivastava V and Vasil IK (1996b) Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. Nature Biotech 14:1155–1159.Google Scholar
  4. Barro F, Rooke L, Békés F, Gras P, Tatham AS, Fido R, Lazzeri PA, Shewry PR and Barcelo P (1997) Transformation of wheat with high molecular weight subunit genes results in functional properties. Nature Biotech 15:1295–1299.Google Scholar
  5. Becker D, Brettschneider R and Lörz H (1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J 5:299–307.Google Scholar
  6. Bertelli AAE, Giovannini L, Giannessi D, Migliori M, Bernini W, FregoniMand Bertelli A (1995) Antiplatelet activity of synthetic and natural resveratrol in red wine. Int J Tiss React 17:1–3.Google Scholar
  7. Blechl AE and Anderson OD (1996) Expression of a novel highmolecular-weight glutenin subunit gene in transgenic wheat. Nature Biotech 14:875–879.Google Scholar
  8. Cao J, Duan X, McElroy D and Wu R (1992) Regeneration of fertile transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Rep 11: 586–591.Google Scholar
  9. Chen CK and Pace-Asciak CR (1996) Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta. Gen Pharmacol 27: 363–366.Google Scholar
  10. Cheng M, Fry JE, P ang S, Zhou H, Hironaka CM, Duncan DR, Conner TW and Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115: 971–980.Google Scholar
  11. Christou P (1995) Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment. Euphytica 85:13–27.Google Scholar
  12. Christensen AH and Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5: 213–218.Google Scholar
  13. de Vries S, Hoge H and Bisseling T (1988) Isolation of total and polysomal RNA from plant tissues. In: Gelvin SB, Schilperoort RA and Verma DP (eds.) Plant Molecular Biology Manual, (pp. B6/1-B6/13) Kluwer Academic Publishers, Dordrecht.Google Scholar
  14. Fischer R, Budde I and Hain R (1997) Stilbene synthase gene expression causes changes in flower colour and male sterility in tobacco, Plant J 11:489–498.Google Scholar
  15. Flavell RB, Sardana S, Jackson S and O'Dell M (1990) The molecular basis of variation affecting gene expression: Evidence from studies of the ribosomal RNA gene loci of wheat. In: Gustafson, J.P., (ed.) Gene Manipulation in plant ImprovementII, (pp. 419–436) 419–430 Plenum Press, New York.Google Scholar
  16. Gehm BD, McAndrews JM, Chien P-Y and Jameson JL (1997) Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc Natl Acad Sci USA 94: 14138–14143.Google Scholar
  17. Hain R, Bieseler B, Kindl H, Schröder G and Stöcker R (1990) Expression of a stilbene synthase gene in Nicotiana tabacumresults in synthesis of the phytoalexin resveratrol. Plant Mol Biol 15: 325–335.Google Scholar
  18. Hain R, Reif HJ, Krause E, Langebartels R, Kindl H, Vornam B, Wiese W, Schmelzer E, Schreier PH, Stöcker RH and Stenzel K (1993) Disease resistance results from foreign phytoalexin expression in a novel plant. Nature 361:153–156.Google Scholar
  19. Hess D (1996) Genetic transformation of wheat via pollen-25 years of plant transformation attempts II. In: Jain S, Sopory S and Veilleux R, (eds.) In vitro Haploid Production in Higher Plants2, (pp. 375–392) Kluwer Academic Publishers, Dordrecht.Google Scholar
  20. Hess D, Dressler K and Nimmrichter R (1990) Transformation experiments by pipetting Agrobacteriuminto the spikelets of wheat (Triticum aestivumL.). Plant Sci 72:233–244.Google Scholar
  21. Iser M, Fettig S, Scheyhing F, Viertel K and Hess D (1999) Genotype-dependent stable genetic transformation in german spring wheat varieties selected for high regeneration potential. J Plant Physiol 154:508–516.Google Scholar
  22. Jang MS, Cai EN, Udeani GO, Slowing KV, Thomas CF, Beecher CWW, Fong HHS, Farnsworth NR, Kinghorn AD, Mehta RG, Moon RC and Pezzuto J (1997) Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275:218–220.Google Scholar
  23. Langcake P and Pryce RJ (1976) The production of resveratrol by Vitis vinifera and other members of the Vitaceaeas a response to infection or injury. Physiol Plant Pathol 9:77–86.Google Scholar
  24. Lazzeri PA and Shewry PR (1993) Biotechnology of cereals. Biotech Genet Engin Rev 11:79–146.Google Scholar
  25. Leckband G and Lörz H (1998) Transformation and expression of a stilbene synthase gene of Vitis viniferaL. in barley and wheat for improved fungal resistance. Theor Appl Genet 96:1004–1012.Google Scholar
  26. Müller E, Lörz H and Lütticke S (1996) Variability of transgene expression in clonal cell lines of wheat. Plant Sci 114:71–82.Google Scholar
  27. Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M and Kartha KK (1994) Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. Plant J 5:285–297.Google Scholar
  28. Powell RG, TePaske MR, Plattner RD, White JF and Clement SL (1994) Isolation of resveratrol from Festuca versutaand evidence for the widespread occurrence of this stilbene in the Poaceae. Phytochemistry 35:335–338.Google Scholar
  29. Soleas GJ, Diamandis EP and Goldberg DM (1997) Resveratrol: a molecule whose time has come? And gone? Clin Biochem 30: 91–113.Google Scholar
  30. Srivastava V, Vasil V and Vasil IK (1996) Molecular characterisation of the fate of transgenes in transformed wheat (Triticum aestivum L.). Theoret Appl Genet 92:1031–1037.Google Scholar
  31. Stark-Lorenzen P, Nelke B, Hänssler G, Mühlbach HP and Thomzik JE (1997) Transfer of a grapevine stilbene synthase gene to rice (Oryza sativaL.). Plant Cell Rep 16:668–673.Google Scholar
  32. Theiss G, Schleicher R, Schimpff-Weiland G and Follmann H (1987) DNA methylation in wheat. European J Biochem 167: 89–96.Google Scholar
  33. Vasil V, Srivastava V, Castillo AM, Fromm ME and Vasil IK (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. Bio/Tech 11: 1553–1558.Google Scholar
  34. Vasil V, Castillo AM, Fromm ME and Vasil IK (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Tech 10: 662–674.Google Scholar
  35. Viertel K and Hess D (1996) Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures. Plant Cell Tiss Org Cult 44:183–188.Google Scholar
  36. Viertel K, Iser M, Schmid A and Hess D (1998) Regeneration of German spring wheat varieties from embryogenic scutellar callus. J Plant Physiol 152:167–172.Google Scholar
  37. Viertel K, IserM, Schmid A and Hess D (1997) Indirekter Gentransfer in Weizen über isolierte Sproßspitzen. Vortr Pflanzenzüchtg 38:17–39.Google Scholar
  38. Weeks JT, Anderson OD and Blechl AE (1993) Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiol 102:1077–1084.Google Scholar
  39. Woodward S and Pearce RB (1988) The role of stilbenes in resistance of Sitka spruce (Picea sitchensis(Bong.) Carr.) to entry of fungal pathogenes. Physiol Mol Plant Pathol 33:127–149.Google Scholar
  40. Yoder JI and Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. Bio/Tech 12:263–267.Google Scholar
  41. Zhuang J, Xu J and Chen G (1984) Studies on induction of plant differentiation in pollen callus of wheat. Acta Genet Sin 11: 374–381.Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Sebastian Fettig
  • Dieter Hess

There are no affiliations available

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