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With the aim of determining the effect of different cytoplasms on anther culture response, we analyzed anthers from different tritordeum lines with either Hordeum chilense, Triticum aestivum or T. turgidum cytoplasms for their response in vitro. The use of these tritordeum alloplasmic lines enabled cytoplasms belonging to two different genera to be compared for the first time. The results showed that the response from anthers of the tritordeum line containing T. turgidum cytoplasm was significantly lower than the response of the other two lines. This finding was encouraging from a practical point of view, since cytoplasms from both H. chilense and T. aestivum are preferred for the breeding of tritordeum.