Floral Transformation of Wheat

  • Sujata Agarwal
  • Star Loar
  • Camille Steber
  • Janice Zale*
Part of the Methods in Molecular Biology™ book series (MIMB, volume 478)


A method is described for the floral transformation of wheat using a protocol similar to the floral dip of Arabidopsis. This method does not employ tissue culture of dissected embryos, but instead pre-anthesis spikes with clipped florets at the early, mid to late uninucleate microspore stage are dipped in Agrobacterium infiltration media harboring a vector carrying anthocyanin reporters and the NPTII selectable marker. T1 seeds are examined for color changes induced in the embryo by the anthocyanin reporters. Putatively transformed seeds are germinated and the seedlings are screened for the presence of the NPTII gene based on resistance to paromomycin spray and assayed with NPTII ELISAs. Genomic DNA of putative transformants is digested and analyzed on Southern blots for copy number to determine whether the T-DNA has integrated into the nucleus and to show the number of insertions. The non-optimized transformation efficiencies range from 0.3 to 0.6% (number of transformants/number of florets dipped) but the efficiencies are higher in terms of the number of transformants produced/number of seeds set ranging from 0.9 to 10%. Research is underway to maximize seed set and optimize the protocol by testing different Agrobacterium strains, visual reporters, vectors, and surfactants.


Pre-anthesis wheat spikes Agrobacterium nuclear transformation floral dip 



This material is based upon work supported by the National Science Foundation under Grant No. 0638421 and the USDA NRI Grant No. 2001-01856.


  1. 1.
    Hess, D., Dressler, K. Nimmrichter, R. (1990) Transformation experiments by pipetting Agrobacterium, into the spikelets of wheat (Triticum aestivum L) Plant Sci. 72,233–244.CrossRefGoogle Scholar
  2. 2.
    Langridge, P., Brettschneider, R., Lazzeri, P. and Lorz, H. (1992) Transformation of cereals via Agrobacterium, and the pollen pathway: a critical assessment Plant J. 2,631–638.CrossRefGoogle Scholar
  3. 3.
    Vasil, V., Castillo, A., Fromm, M. and Vasil, I. K. (1992) Herbicide resistant fertile transgenic wheat plants obtained by micro-projectile bombardment of regenerable embryogenic callus,Bio/Technology 1,667–674.CrossRefGoogle Scholar
  4. 4.
    Weeks, J. T., Anderson, O. D. and Blechl, A. E. (1993) Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum, L.) Plant Physiol. 102,1077–1084.Google Scholar
  5. 5.
    Nehra, N., Chibbar, R., Leung, N., Caswell, K., Mallard, C., Steinhauer, L., Baga, M. and Kartha, K. (1994) Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs,Plant J. 5,285–297.CrossRefGoogle Scholar
  6. 6.
    Bennett, M. D. and Leitch, I. J. (1996) Nuclear DNA amounts in Angiosperms,Ann. Bot. 76,113–176.CrossRefGoogle Scholar
  7. 7.
    Zale, J. and Scoles, G. (1999) Registration of Crocus hard red spring wheat,Crop Sci. 39,1539–1540.Google Scholar
  8. 8.
    McCormac, A. C., Elliott, M. C. and Chen, D. F. (1997) pBECKS. A flexible series of binary vectors for Agrobacterium,-mediated plant transformation Mol. Biotechnol. 8,199–213.CrossRefGoogle Scholar
  9. 9.
    McCormac, A. C., Wu, H., Bao, M., Wang, Y. Q., Xu, R., Elliot, M. C. and Chen, D. F. (1997) The use of visual marker genes as cell-specific reporters of Agrobacterium-,mediated T-DNA delivery to wheat and barley Euphytica 99,17–25.CrossRefGoogle Scholar
  10. 10.
    Murashige, T. and Skoog, F. A. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture,Physiol. Plant. 15,473–497.CrossRefGoogle Scholar
  11. 11.
    Bechtold, N., Ellis, J. and Pelletier, G. (1993) In planta Agrobacterium, mediated gene transfer by infiltration of adult Arabidopsis thaliana plants C. R. Acad. Sci. 316,1194–1199.Google Scholar
  12. 12.
    Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium,-mediated transformation of Arabidopsis thaliana Plant J. 16,735–743.CrossRefGoogle Scholar
  13. 13.
    Supartana, P., Shimizu, T., Nogawa, M., Shioiri, H., Nakajima, T., Haramoto, N., Nozue, M. and Kojima, M. (2006) Development of simple and efficient in planta, transformation method for wheat (Triticum aestivum L.) using Agrobacterium tumefaciens J. Biosci. Bioeng. 102,162–170.CrossRefGoogle Scholar
  14. 14.
    Cheng, M., Fry, J. E., Pang, S., Zhou, H., Hironaka, C. M., Duncan, D. R., Conner, T. W. and Wan, Y. (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens, Plant Physiol. 115,971–980.Google Scholar
  15. 15.
    Lamoureux, D., Boeuf, C., Regad, F., Garsmeur, O., Charmet, G., Sourdille, P., Lagoda, P. and Bernard, M. (2002) Comparative mapping of the wheat 5B short chromosome arm distal region with rice, relative to a crossability locus,Theor. Appl. Genet. 105,759–765.CrossRefGoogle Scholar
  16. 16.
    McCormac, A. C., Elliott, M. C. and Chen, D. F. (1999) pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates “clean-gene” facilities,Mol. Gen. Genet. 261,226–235.CrossRefGoogle Scholar
  17. 17.
    Cambia (2007) Center for the Application of Molecular Biology to International Agriculture in Canberra, Australia, June 15, 2007 (
  18. 18.
    Selinger, D. A. and Chandler, V. L. (2001) B-Bolivia,, an allele of the maize b1 gene with variable expression, contains a high copy retrotransposon-related sequence immediately upstream Plant Physiol. 125,1363–1379.CrossRefGoogle Scholar
  19. 19.
    Jacob, D., Lewin, A., Meister, B. and Appel, B. (2002) Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery,Transgen. Res. 11,291–303.CrossRefGoogle Scholar
  20. 20.
    Rybczynski, J. J., Simonson, R. I. and Baenziger, P. S. (1991) Evidence for microspore embryogenesis in wheat anther culture,In Vitro Cell. Dev. Biol. 27,168–174.CrossRefGoogle Scholar
  21. 21.
    Hucl, P. and Matus-Cadiz, M. (2001) Isolation distances for minimizing out-crossing in spring wheat,Crop Sci. 41,1348–1351.CrossRefGoogle Scholar
  22. 22.
    Bent, A. F. (2000) Arabidopsis in planta, transformation. Uses, mechanisms, and prospects for transformation of other species Plant Physiol. 124,1540–1547.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Sujata Agarwal
    • 1
  • Star Loar
    • 1
  • Camille Steber
    • 2
  • Janice Zale*
    • 1
  1. 1.Department of Plant Sciences, 2431 Joe Johnson DriveUniversity of TennesseeUSA
  2. 2.USDA/ARS Wheat Genetics, QualityPhysiology and Plant Disease UnitUSA

Personalised recommendations