We describe the procedures for recovering transgenic sugarcane from co-cultivation of both calli and in vitro plants with Agrobacterium tumefaciens. The correct tissue culture strategies and the use of super-binary vector or super-virulent strain are crucial for the successful sugarcane transformation. Both plant regeneration via calli culture and micropropagation strategies can be optimized to a wide spectrum of sugarcane genotypes, thus the procedures presented here could be applied to genetic engineering of Saccharum spp. after minor modifications. For the case of sugarcane transformation using in vitro plants, four selective micropropagation steps must be sufficient to eliminate chimera plants.