The lower frequency of spontaneously obtained autotetraploid plants from apomictic seeds ofPoncirus trifoliata promoted us to exploit the colchicine application to arise tetraploid.In this study, we attempted to identify a suitable colchicine concentration based on in vitro treatment to induce the polyploidy by in vivo colchicine application.Gradient concentrations of colchicine (0％, 0.025％, 0.05％, 0.1％, and 0.2％) that prepared either in water (CW) or in MT liquid medium (CM) were exposed for 24 h and 72 h, respectively for in vitro treatment.The colchicine concentrations with high permeability, which inhibit callus formation and shoot regeneration, were used for in vivo application.Three colchicine concentrations (0.1％, 0.2％, and 0.3％) were dropped twice daily for a week on the apical buds of one-month-old P.trifoliata seedlings.The ploidy level was then examined via flow cytometer after 70 days of in vivo colchicine treatment.The obtained autotetraploids were then confirmed after 6 months.　　The results of this study were summarized as following:　　1.The higher concentrations of colchicine (0.1％ and 0.2％) decreased the callus formation and the survival rate of the in vitro treated segments.　　2.The in vivo treatment successfully produced 12 tetraploids out of 78 survived plants after 6 months of treatment.The effective colchicine concentration was 0.1％ with 26.6 ％ of polyploidization efficiency rate.　　3.The stomatal and anatomic analysis showed that tetraploids have larger stomatal size, smaller stomatal density, and bigger cellular size compared to diploids.　　4.The morphological characterization of tetraploids manifested that they have thicker, broader and darker leaves, and less growth rate relative to the diploids.　　5.The photosynthesis analysis revealed that the tetraploids have higher net photosynthesis rate, more gas exchange that was in parallel with better chlorophyll a imaging performance, higher Fv/Fm and more chlorophyll content compared to the diploids.Exposing the tested plants to different light intensity exhibited that the tetraploids have more ETR, ΦPSⅡ, qP, NPQ over the 2x plants.