Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare)

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures. w hich had been initiated from immature embryo-derived (IED) and microspore-d erived (MSD) callus, were co-cultivated with various Agrobacterium tume-fac iens strains. The T-DNA vectors contained visually-detectable marker genes (CI/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also d rug resistance genes (hph or bar) to facilitate selection of stably transfo rmed cell lines. A set of normal binary vectors in a super-virulent Agrobac terium strain [EHA 101(pBECKS)] and also a super-binary vector [LBA4404(pTO K233)] were used in this study. Cells of the suspension cultures which rece ived T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolate d which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted i n a loss of the capacity to regenerate plants from the transgenic callus li nes. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated wit h DNA which was distinct from that of the original Agrobacterium plasmid. F urthermore, independent transgenic lines showed diverse patterns of hybridi sing bands. These data suggest that the T-DNA fragment was stably maintaine d through integration into the genomes of the barley cell lines.