|Agarwal, S - UNIV OF TENNESSEE|
|Loar, S - UNIV OF TENNESSEE|
|Zale, J - UNIV OF TENNESSEE|
Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: November 14, 2008
Publication Date: November 14, 2008
Citation: Zale, J., Agarwal, S., Loar, S., Steber, C.M. 2008. Floral Transformation of Wheat, Chapter 6, pp. 105-113. IN: Huw D. Jones and Peter R. Shewry (eds.) Methods in Molecular Biology, Transgenic Wheat, Barley and Oats. Humana Press, New York, NY. Interpretive Summary: This paper describes a simple method for wheat transformation. Instead of using tissue culture based methods, this method involves dipping opened florets of wheat into a medium containing Agrobacterium. The Agrobacterium transfer a T-DNA plasmid into the plant genome. This method should be less expensive and less time consuming than current methods for wheat transformation.
Technical Abstract: Hexaploid wheat is one of the world’s most important staple crops but genetic transformation is still challenging. We are developing a floral transformation protocol for wheat that does not require tissue culture. Several T-DNA transformants have been produced in the high quality, hard red germplasm line, Crocus, and three transformants have been characterized thoroughly at the molecular level over four to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level and NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc/C1, and the NPTII gene, was also used to produce another Crocus transformant. Since Crocus is a red wheat, the anthocyanin regulators are not lethal. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and an ELISA assay for NPTII protein expression. This protocol has the potential to reduce the time and financial investment required for wheat transformation.