|ZALE, J - UNIV OF TENNESSEE
|AGARWAL, S - UNIV OF TENNESSEE
|LOAR, S - UNIV OF TENNESSEE
Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2009
Publication Date: 3/24/2009
Citation: Zale, J., Agarwal, S., Loar, S., Steber, C.M. 2009. Evidence for stable transformation of wheat by floraldip in Agrobacterium tumefaciens. Plant Cell Reports 28(6):903-913. DOI 10.1007/s00299-009-0696-0.
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 have developed a floral transformation protocol that does not utilize tissue culture. Three T-DNA wheat transformants have been produced in the germplasm line, Crocus, using this protocol and characterized at the molecular level for several generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in Agrobacterium infiltration medium harboring either pDs(Hyg)35S or 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. Agrobacterium harboring pBECKSred, which contains the maize anthocyanin regulators, Lc/C1, and the NPTII gene, was also used to produce another Crocus transformant. T1 seed was selected for red embryos; T1 and T2 plants were screened using sequential screens of paromomycin spray and NPTII ELISAs. There were differences in endosperm seed coat color presumably the result of the Lc/C1 reporters. The Agrobacterium strains AGL1 and C58C1 were used in these gene transfer experiments to generate low copy number transformants. The transformants showed Mendelian segregation in the T2, and stable transmission of the transgenes over several generations as determined by Southern analysis. Gene expression in progeny derived from the primary transformants has been demonstrated using Reverse Transcriptase-PCR. This protocol will be useful for independent research laboratories in the wheat community and in developing countries lacking the resources and infrastructure for plant biotechnology.