The objectives of this study were to establish transformation protocols for Lactobacillus plantarum CD033 and Lactobacillus buchneri CD034, two industrial silage strains and to test the influence of selected origins of replication on plasmid copy number, plasmid stability, and plasmid incompatibility in these strains. Electro-transformation protocols were optimized by examination of the influence of different electroporation solutions and cell wall weakening agents on transformation efficiency. Using Lithium acetate as cell wall weakening agent, we could achieve transformation efficiencies of 8 × 10(4) transformants per 1 μg DNA for L. buchneri CD034 which is to our knowledge the highest described for this species up to now. In order to test feasibility of previously described origins of replication derived from Bacillus subtilis, L. plantarum, Lactococcus lactis, and two novel L. buchneri CD034 plasmids to drive replication in our two selected Lactobacillus strains, six shuttle vectors were constructed. Results indicate that, in terms of stable propagation and high gene copy numbers (up to 238 copies/chromosome), the most suitable origins of replication for the construction of expression vectors for the selected silage strains were the ones derived from the novel L. buchneri CD034 plasmids.