Rye (Secale cereale L.) is known as one of the most recalcitrant species in tissue culture and genetic transformation. To establish a reproducible transformation system for rye, inbred lines with superior regeneration potential from tissue cultures were identified, optimal culture media composition, media preparation procedure and in-vitro growth conditions for individual, genetically divergent rye inbred lines found and interactions between genotypes and different tissue culture components estimated. Biolistic parameters, such as tissue age during bombardment and micro-projectile density, were compared in a multi-factorial experiment. Selection strategies using the selectable marker genes bar or nptII resulted in similar transformation efficiencies in the range of 2 to 4% of the bombarded explants. A total of 37 independent transgenic rye plants were produced by biolistic gene delivery. For Agrobacterium tumefaciens-mediated gene delivery, different influencing factors were combined and led to morphologically normal and fertile transgenic plants at a frequency of 3.9% of the inoculated explants. The Agrobacterium-mediated T-DNA transfer was confirmed by molecular and genetic analyses of transgenic plants and the analysis of T-DNA::rye DNA boundary sequences. A selection system for the "direct" production of transgenic plants, free of selectable marker gene, was developed. The high-molecular-weight glutenin subunit genes Ax1, Dx5 and Dy10 from wheat (Triticum aestivum L.) were introduced into rye and their stable expression in endosperm of primary transformants of rye and in their segregating progeny demonstrated. Transgene stability and levels of transgene expression were analysed with regard to the transgene copy number.