To increase the genetic transformation efficiency of sweet orange infected by Agrobacterium, an explant derived from internode stem segments of sucarri orange seedling was adopted to optimize light regimes and co-cultivation condition for the growth. Meantime, seedless gene was transferred to succari orange. The results revealed that explant from seedling had higher regeneration and transformation rate under the condition of cultured 20 d in darkness first and then exposed 10 d in lighting environment. Acetosyringone (AS) concentration was a key factor impacting transformation rate, and the optimal performance of transformation was under 20 mg/L concentration of co-cultivated medium with a temperature 19 ℃ and pH 5.4–5.7. The positive rate could reach 29.4% through the analysis of GUS staining, which were further proved by PCR analysis that the foreign gene was indeed integrated into the host genome.