co transformation with the bar gene provided bialaphos selection for the isolation of transformed callus used for regeneration 20 regenerants containing the sh2 dhar or ub dhar constructs were identified using pcr and dhar expression confirmed by activity assay and western analysis using anti wheat dhar antiserum t 0 plants were crossed with hiii transgene containing progeny identified and the t 1 plants selfed dhar was purified to homogeneity from 10 day old wheat leaves and was used to raise anti dhar antiserum in rabbits for western analysis a membrane containing the protein of interest was blocked for 30 min in tpbs 0 1 tween 20 13 7 mm nacl 0 27 mm kcl 1 mm na 2 hpo 4 0 14 mm kh 2 po 4 with 5 reconstituted dry milk and incubated with anti wheat dhar diluted 1 1000 in tpbs with 1 milk for 1 5 hrs the blots were then washed with tpbs incubated with goat anti rabbit horseradish peroxidase a antibody southern biotechnology diluted 1 5000 to 1 10 000 for 1 hr and dhar detected using chemilumensence amersham corp dhar activity was assayed as described in hossain et al plant cell physiol 25 85 92 1984 soluble protein was extracted from tobacco leaves or maize kernels ground in liquid nitrogen before grinding in extraction buffer 50.