Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore, experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation. 6-benzylaminopurine at 0.05 mg l−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets in indole-3-aceticacid 0.5 mg l−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent.