The cross-incompatibility barrier between 4x wheat and rye has limited the genetic base for triticale breeding. Experiments designed to improve the synthesis of wheat-rye amphihaploids were conducted. The effects of 2,4-D on crossability and 3x hybrid embryo differentiation, and the influence of one-step and two-step media on the culture of immature 3x embryos in vitro, were investigated. Application of 10 mg L-1 2,4-D slightly improved seed set but significantly reduced the frequency of normal embryos. In contrast to the reported favourable effect of 2,4-D on haploid embryo formation in wheat × maize crosses, the application of 2,4-D in the present study offers no real advantage on amphihaploid embryo formation from 4x wheat × rye crosses. For small and immature wheat-rye hybrid (3x) embryos, optimum recovery in vitro was obtained via a two-step procedure consisting of a semi-solid MN medium followed by MS medium supplemented with IAA (1 mg L-1) and BAP (1 mg L-1). For bigger and well-differentiated embryos, the use of a one-step Gamborg's B5 medium was sufficient.