Abstract: Transgenic maize plant transformation by Agrobacterium tumefaciens has been widely used. However, methodology transformation efficiency is still one of the bounding aspects. In this regards, this study sought improving efficiency of transgenic maize plant transformation by Agrobacterium tumefaciens using as model the FR-28 Cuban synthetic maize variety. To fulfill this purpose, organoembriogenic callus lines were established from meristematic shoot and axillary buds. Next, calluses were infected with LBA4404 strain of Agrobacterium tumefaciens previously transformed with the pDUbar plasmid for assessing different regeneration conditions (media and osmotic treatment). As main results, the R2 treatment was the most favorable and the addition of an osmotic step, before and after infection (MInd Suc 12%), increased regeneration efficiency (p = 0.0001). The AAM, used during infection and modified MPC media were best combinations for obtaining a greater plant number. In detail, 35 plants per gram of calluses generated from one maize line with high regeneration capability were obtained with the improved transformation procedure. The T-DNA integration was molecularly confirmed by PCR and Southern-blot, while the bar gene expression was evidenced applying herbicide (Finale®) at 2% on the second leaf of plants recovered from genetic transformation. From this, about 33% of plants tolerated herbicide action. As conclusion, the combined application of an osmotic treatment, before and after co-cultivation and callus exposition to the light in presence of modified MPC medium during plant regeneration was the best experimental condition for increasing probability for obtaining transgenic maize plants able to tolerate different agronomic conditions such as cultivation in presence of herbicide.