Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.