Abstract: Endosperm at late stage of cell formation, excised from open-pollinated fruit of sweet orange (Citrus sinensis Osbeck. cv.‘Hongjiang’), was suitable for culture in vitro. The results indicated that 2,4-D was necessary for callus' induction, and supplemented with BA, CH in medium was more effective: the percentage of induction was as high as 33.3%. Endosperm tissues, excised from the fruits treated with low temperature (4–7℃) for 16 and 19 days, and from the young seeds precuhured on MT basal medium respectively with NAA, GA3, BA for 2–6 days, also stimulated to callus formation. When endosperm callus was transfered to the differentiation medium, embryoids and shoot-buds only developed in a sequence of culture conditions. Callus was first cultured on MT+BA/GA3, then transfered to different media with various nitrogen or hormone concentration, and finally transferred back to the first culture medium. Shoots were regenerated from shoot-buds in the medium in presence of hormones with only BA or with GA3. The whole plants were regenerated from embryoids in presence of GA3 or with BA. Analysis of endosperm plantlets showed that 79.4% of the observed celld have chromosome number of 2n=26–27, nearly the triploid number of 2n=3x=27. Through grafting on lemon seedlings as rootstocks in vitro, plantlets were growing successfully in soil.