T-DNA integration into the barley genome from single and double cassette vectors.

Patterns and sites of T-DNA integrations into the barley genome from single and double cassette vectors are of interest for the identification of cultivars with value added properties as well as for the production of selection marker-free transgenic lines that can be retransformed. T-DNA/Plant DNA junctions were obtained by capturing a single-stranded DNA with a biotinylated primer annealing to the vector adjacent to the border and an adaptor ligated to a restriction site overhang in the flanking barley DNA. The captured junction was converted into a double strand and sequenced. Fifty left and right border junctions from plants transgenic for one of five human genes were analyzed. Primers of 15-30 nucleotides designed from the genomic DNA at the insertion site can PCR amplify fragments that identify unequivocally any transformant. Adjacent transgene insertions with single cassette vectors were always in tandem direct repeat configuration. With regard to T-DNA integration the patterns were comparable to the variations found in dicotyledonous plants. Twelve of the 46 integrations characterized by blast searches were within different regions of the BARE-1 retrotransposon element occurring with a frequency of 2 x 10(5) copies in the barley genome. The use of border junctions to identify number of copies and loci of integrates in transformants is discussed.

Genetically engineered stem rust resistance in barley using the Rpg1 gene.

The stem-rust-susceptible barley cv. Golden Promise was transformed by Agrobacterium-mediated transformation of immature zygotic embryos with the Rpg1 genomic clone of cv. Morex containing a 520-bp 5' promoter region, 4,919-bp gene region, and 547-bp 3' nontranscribed sequence. Representatives of 42 transgenic barley lines obtained were characterized for their seedling infection response to pathotype Pgt-MCC of the stem rust fungus Puccinia graminis f. sp. tritici. Golden Promise was converted from a highly susceptible cultivar into a highly resistant one by transformation with the dominant Rpg1 gene. A single copy of the gene was sufficient to confer resistance against stem rust, and progenies from several transformants segregated in a 3:1 ratio for resistancesusceptibility as expected for Mendelian inheritance. These results unequivocally demonstrate that the DNA segment isolated by map-based cloning is the functional Rpg1 gene for stem rust, resistance. One of the remarkable aspects about the transformants is that they exhibit a higher level of resistance than the original sources of Rpg1 (cvs. Chevron and Peatland). In most cases, the Golden Promise transformants exhibited a highly resistant reaction where no visible sign of infection was evident. Hypersensitive necrotic "fleck" reactions were also observed, but less frequently. With both infection types, pathogen sporulation was prevented. Southern blot and RT-PCR analysis revealed that neither Rpg1 gene copy number nor expression levels could account for the increased resistance observed in Golden Promise transformants. Nevertheless, this research demonstrates that stem-rust-susceptible barley can be made resistant by transformation with the cloned Rpg1 gene.