Medicago sativa is a pasture and a model plant for studying the molecular genetic. The objectives of this work were optimizing in vitro regeneration and Agrobacterium-mediated transformation of Iranian alfalfa cultivars (Gharghologh, Malek kanedi and Kozrah). For induction of multiple shoots, different explants (meristem and cotyledon) and various hormone concentrations in three varieties (Gharghologh, Malek kanedi and Kouzrah) were studied. Results showed that MS culture medium supplemented with 0.05 mgl-1 NAA + 1.5 mgl-1 TDZ was the most effective regeneration medium. To study gus gene transformation, Agrobacterium tumefaciens strains; LBA4404 and AGL1 containing pBI121 plasmid were used. The plasmid containing the neomycin phosphotransferas gene, as the selectable marker under control of the nopaline synthesis promotor and the β-glucuronidase gene was used in combination with the reporter gene under control of the CaMV35s promoter. Various factors including type of explants (meristem and cotyledon), preparation method of bacteria suspension (I & II) and time of cocultivation were investigated. Meristem and cotyledonary explants were inoculated with bacteria suspension I and II andcocultivated on the regeneration medium. The highest percentage of Kanamycin-resistant shoot (50%) and the most transformation frequencies of plantlets were obtained from AGL1 strain. Expression of gus in leaf and stem transgenic plants derived from suspension II was the result of successful transformation by gus gene. PCR analysis of putative transgenic plants showed the integration of at least one copy of gus and nptII genes into the alfalfa genome.
