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 nonoptimized 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.
Agarwal
Sujata
S
Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA.
Loar
Star
S
Steber
Camille
C
Zale
Janice
J
D004269:DNA, Bacterial / Q000378:metabolism; D004351:Drug Resistance; D004797:Enzyme-Linked Immunosorbent Assay; D035264:Flowers / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D010303:Paromomycin / Q000494:pharmacology; D030821:Plants, Genetically Modified; D012231:Rhizobium / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000254:growth & development / Q000382:microbiology; D014170:Transformation, Genetic; D014908:Triticum / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology
D004269:DNA, Bacterial; C036483:T-DNA; D010303:Paromomycin