Skip to main content

Restoring full pollen fertility in transgenic male-sterile tobacco (Nicotiana tabacum L.) by Cre-mediated site-specific recombination

Molecular Breeding volume 15, pages 193–203 (2005)Cite this article

Abstract

Producing hybrid seed requires an efficient pollination control system to prevent unwanted self-pollination. For further breeding, it would be advantageous to restore pollen fertility in the hybrids. In this work we demonstrate the use of tapetum-specific expression of a stilbene synthase (sts) transgene to induce pollen sterility in tobacco as has been shown previously. The sts-coding region was flanked by loxP recognition sites for Cre-recombinase. From 10 T0-plants obtained, five proved to be male-sterile. They had smaller flowers with shorter stamina, but the vegetative phenotype was just as in the wild-type. Crossing male-sterile sts-plants with tobacco lines expressing the cre recombinase transgene resulted in site-specific recombination in the hybrids. GUS activity caused by fusion of the tap1-promoter with a promoterless gusA coding region indicated recombination events already in early stages of flower bud development. In all plants which had contained single or double sts-copies before crossing, these were excised, and pollen fertility was fully restored. The phenotype of these restored plants was as in wild-type controls. Contrary, from male sterile plants containing multiple copies of the sts-gene, not all copies were removed, and pollen sterility was maintained.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Abbreviations

CHS:

chalcone synthase

CMS:

cytoplasmic male sterility

Cre:

Cre recombinase

GFP:

green fluorescent protein

GUS:

β-glucuronidase

gusA :

β-glucuronidase gene

loxP :

recognition site of Cre recombinase

NMS:

nuclear male sterility

STS:

stilbene synthase

References

  1. K. Abremski R. Hoess N. Sternberg (1983) ArticleTitleStudies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination Cell 32 1301–1311

    Google Scholar 

  2. H. Albert E.C. Dale E. Lee D.W. Ow (1995) ArticleTitleSite-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome Plant J. 7 649–659

    Google Scholar 

  3. C.C. Bayley M. Morgan E.C. Dale D.W. Ow (1992) ArticleTitleExchange of gene activity in transgenic plants catalyzed by the Cre-lox-site-specific recombination system Mol. Biol. 18 353–361

    Google Scholar 

  4. D.G. Burgess E.J. Ralston W.G. Hanson M. Heckert T. Jenq J.M. Palys K. Tang N. Gutterson (2002) ArticleTitleA novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants Plant J. 31 IssueID1 113–125

    Google Scholar 

  5. H.J. Cho S. Kim M. Kim B.D. Kim (2001) ArticleTitleProduction of transgenic male sterile tobacco plants with the cDNA encoding a ribosome inactivating protein in Dianthus sinensis L Mol. Cell 11 IssueID3 326–333

    Google Scholar 

  6. E.R. Coppoolse M.J. de Vroomen D. Roelofs J. Smit F. van Gennip B.J.M. Hersmus H.J.J. Nijkamp M.J.J. van Haaren (2003) ArticleTitleCre recombinase expression can result in phenotypic aberrations in plants Plant Mol. Biol. 51 263–279

    Google Scholar 

  7. E.C. Dale D.W. Ow (1990) ArticleTitleIntra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase Gene 91 79–85

    Google Scholar 

  8. E.C. Dale D.W. Ow (1991) ArticleTitleGene transfer with the subsequent removal of the selection gene from the host genome Proc. Natl. Acad. Sci. USA 88 10558–10562

    Google Scholar 

  9. S. Fettig D. Hess (1999) ArticleTitleExpression of a chimeric stilbene synthase gene in transgenic wheat lines Transgenic Res. 8 179–189

    Google Scholar 

  10. R. Fischer I. Budde R. Hain (1997) ArticleTitleStilbene synthase gene expression causes changes in flower colour and male sterility in tobacco Plant J. 11 IssueID3 489–498

    Google Scholar 

  11. D. Gerlach (1969) Präparate von Kernteilungen D. Gerlach (Eds) Botanische Mikrotechnik Georg Thieme Verlag Stuttgart, Germany 251–262

    Google Scholar 

  12. A.P. Gleave D.S. Mitra S.R. Mudge B.A.M. Morris (1999) ArticleTitleSelectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene Plant Mol. Biol. 40 223–235

    Google Scholar 

  13. M. Goetz D.E. Godt A. Guivarc’h U. Kahmann D. Chriqui T. Roitsch (2001) ArticleTitleInduction of male sterility in plants by metabolic engineering of the carbohydrate supply Proc. Natl. Acad. Sci. USA 98 IssueID11 6522–6527

    Google Scholar 

  14. F.R. Guillen-Portal D.D. Baltensperger L.A. Nelson G. Frickel (2002) ArticleTitleAssessment of hard red winter wheat F2 and F3 hybrids for the Nebraska panhandle Comm. Soil Sci. Plant. Anal. 33 IssueID5/6 963–972

    Google Scholar 

  15. K. Haas M. Bauer E. Wollenweber (2003) ArticleTitleCuticular waxes and flavonol aglycones of mistletoe Z. Naturforsch. 58c 464–470

    Google Scholar 

  16. R.P. Hellens E.A. Edwards N.R. Leyland S. Bean P.M. Mullineaux (2000) ArticleTitlepGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation Plant Mol. Biol. 42 IssueID6 819–832

    Google Scholar 

  17. D. Hess (1978) ArticleTitleGenetic effects in Petunia hybrida induced by pollination with pollen treated with lac transducing phages Z. Pflanzenphysiol. 90 119–132

    Google Scholar 

  18. T.T.C. Hoa B.B. Bong E. Huq T.K. Hodges (2002) ArticleTitleCre/lox site-specific recombination controls the excision of a transgene from the rice genome Theor. Appl. Genet. 104 518–525

    Google Scholar 

  19. M. Huber R. Hahn D. Hess (2002) ArticleTitleHigh transformation frequencies obtained from a commercial wheat (Triticum aestivum L. cv. ‘Combi’) by microbombardment of immature embryos followed by GFP screening combined with PPT selection Mol. Breeding 10 IssueID1 19–30

    Google Scholar 

  20. A. Jagannath N. Arumugam V. Gupta A. Pradhan P.K. Burma D. Pental (2002) ArticleTitleDevelopment of transgenic barstar lines and identification of a male sterile (barnase)/restorer (barstar) combination for heterosis breeding in Indian oilseed mustard (Brassica juncea) Curr. Sci. 82 IssueID1 46–52

    Google Scholar 

  21. A.M. Koltunow J. Truettner K.H. Cox M. Wallroth R.B. Goldberg (1990) ArticleTitleDifferent temporal and spatial gene expression patterns occur during anther development Plant Cell 2 1201–1224 Occurrence Handle1:CAS:528:DyaK3MXkvVKnsr4%3D Occurrence Handle12354953

    CAS  PubMed  Google Scholar 

  22. H.A. Luetcke K.C. Chow F.S. Mickel K.A. Moss H.F. Kern G.A. Scheele (1987) ArticleTitleSelection of AUG initiation codons differs in plants and animals EMBO J. 6 IssueID1 43–48

    Google Scholar 

  23. H. Luo L.A. Lyznik D. Gidoni T.K. Hodges (2000) ArticleTitleFLP-mediated recombination for use in hybrid plant production Plant J. 23 IssueID3 423–430

    Google Scholar 

  24. C. Mariani M. De Beuckeleer J. Truettner J. Leemans R.B. Goldberg (1990) ArticleTitleInduction of male sterility in plants by a chimaeric ribonuclease gene Nature 347 737–741 Occurrence Handle10.1038/347737a0 Occurrence Handle1:CAS:528:DyaK3MXhvV2jsQ%3D%3D

    Article  CAS  Google Scholar 

  25. M.J. Mayer A. Narbad A.J. Parr M.L. Parker N.J. Walton F.A. Mellon (2001) ArticleTitleRerouting the plant phenylpropanoid pathway by expression of a novel bacterial enoyl-coa hydratase/lyase enzyme function Plant Cell 13 1669–1682

    Google Scholar 

  26. M. McConn J. Browse (1996) ArticleTitleThe critical requirement for linolenic acid is pollen developmentnot photosynthesis, in an arabidopsis mutant Plant Cell 8 403–416 Occurrence Handle10.1105/tpc.8.3.403 Occurrence Handle1:CAS:528:DyaK28XitVKmtLs%3D Occurrence Handle12239389

    Article  CAS  PubMed  Google Scholar 

  27. W.K. Nacken P. Huijser J.P. Beltran H. Saedler H. Sommer (1991) ArticleTitleMolecular characterization of two stamen-specific genes, tap1 and fil1, that are expressed in the wild typebut not in the deficient mutant of Antirrhinum majus Mol. Gen. Genet. 229 IssueID1 129–136

    Google Scholar 

  28. C.A. Napoli C. Lemieux R. Jorgensen (1990) ArticleTitleIntroduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans Plant Cell 2 279–289 Occurrence Handle10.1105/tpc.2.4.279 Occurrence Handle1:CAS:528:DyaK3cXkvFalu7c%3D Occurrence Handle12354959

    Article  CAS  PubMed  Google Scholar 

  29. J.T. Odell S.H. Russell (1994) Use of site-specific recombination systems in plants J. Paszkowski (Eds) Homologous Recombination and Gene Silencing in Plants Kluwer Academic Publishers The Netherlands 219–270

    Google Scholar 

  30. D.W. Ow S.L. Medburry (1995) ArticleTitleGenome manipulation through site-specific recombination Crit. Rev. Plant Sci. 14 239–261

    Google Scholar 

  31. S.Z. Pang D.L. DeBoer Y. Wan G. Ye J.G. Layton M.K. Neher C.L. Armstrong J.E. Fry M.A. Hinchee M.E. Fromm (1996) ArticleTitleAn improved green fluorescent protein gene as a vital marker in plants Plant Physiol. 112 893–900

    Google Scholar 

  32. E. Perez-Prat M.M. van Lookeren Campagne (2002) ArticleTitleHybrid seed production and the challenge of propagating male-sterile plants Trends Plant Sci. 7 IssueID5 199–203

    Google Scholar 

  33. Q. Que H. Wang R.A. Jorgensen (1998) ArticleTitleDistinct patterns of pigmentations suppression are produced by allelic sense and antisense chalcone synthase transgenes in petunia flowers Plant J. 13 401–409

    Google Scholar 

  34. T.G. Reeves K. Cassaday (2002) ArticleTitleHistory and past achievements of plant breeding Aust. J. Agric. Res. 53 851–863

    Google Scholar 

  35. J. Sambrook E.F. Fritsch T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York

    Google Scholar 

  36. J. Schaaf M.H. Walter D. Hess (1995) ArticleTitlePrimary metabolism in plant defence. Regulation of a bean malic enzyme gene promoter in transgenic tobacco by developmental and environmental cues Plant Physiol. 108 949–960

    Google Scholar 

  37. V. Srivastava O.D. Anderson D.W. Ow (1999) ArticleTitleSingle-copy transgenic wheat generated through the resolution of complex integration patterns Proc. Natl. Acad. Sci. USA 96 11117–11121

    Google Scholar 

  38. V. Srivastava D.W. Ow (2002) ArticleTitleBiolistic mediated site-specific integration in rice Mol. Breeding 8 IssueID4 345–349

    Google Scholar 

  39. N. Sternberg D. Hamilton (1981) ArticleTitleBacteriophage P1 site-specific recombination J. Mol. Biol. 150 467–486

    Google Scholar 

  40. A. Stintzi J. Browse (2000) ArticleTitleThe Arabidopsis male-sterile mutantopr3lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis Proc. Natl. Acad. Sci. USA 97 IssueID19 10625–10630

    Google Scholar 

  41. G. Vancanneyt R. Schmidt A. O’Connor-Sanchez L. Willmitzer M. Rocha-Sosa (1990) ArticleTitleConstruction 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. 10 667–674

    Google Scholar 

  42. A.C. Vergunst P.J.J. Hooykaas (1998) ArticleTitleCre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre Plant Mol. Biol. 38 393–406

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Plant Physiology and Biotechnology, University of Hohenheim, Emil-Wolff-Str. 25, D-70593, Stuttgart, Germany

    Martin Bayer & Dieter Hess

Authors
  1. Martin Bayer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dieter Hess
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dieter Hess.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bayer, M., Hess, D. Restoring full pollen fertility in transgenic male-sterile tobacco (Nicotiana tabacum L.) by Cre-mediated site-specific recombination. Mol Breeding 15, 193–203 (2005). https://doi.org/10.1007/s11032-004-5042-1

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-004-5042-1

Keywords

  • Cre/loxP
  • Hybrid breeding
  • Male sterility
  • Nicotiana tabacum
  • Site-specific recombination
  • Stilbene synthase