Floral Transformation of Wheat
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.
KeywordsPre-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.
- 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.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
- 7.Zale, J. and Scoles, G. (1999) Registration of Crocus hard red spring wheat,Crop Sci. 39,1539–1540.Google Scholar
- 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
- 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.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
- 17.Cambia (2007) Center for the Application of Molecular Biology to International Agriculture in Canberra, Australia, June 15, 2007 (http://www.cambia.org/daisy/cambia/585(.