The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16 days post-anthesis, the embryonic axis is removed, and the immature scutella are precultured for 1-2 days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.
Keywords: Biolistics; DNA delivery; Gene gun; Genetic transformation; Immature embryo; Particle bombardment; Regeneration; Tissue culture; Transgenic plants; Wheat.