Abstract: Eucalyptus plays a great role in ecological environment and chemical industry.As a hybrid of Eucalyptus urophylla and E.grandis,E.urophylla×E.grandis has good heterosis and is more synthetical than other Eucalyptus species.In order to meet the requirements of cultivating superior Eucalyptus cultivars with different uses by genetic engineering technology,it is of great significance to establish an efficient in vitro regeneration system of E.urophylla×E.grandis and study its callus differentiation mechanism.The growth and differentiation of E.urophylla×E.grandis callus is affected by its endogenous phytohormones and mi RNA396 is a small RNA regulating the growth and development of plant leaves and roots,which is related to the biosynthesis of cytokinin.CKX is responsible for regulating cytokinin oxidase gene.In order to explore the regulatory effects of mi RNA396 and CKX genes on the growth and development of E.urophylla×E.grandis callus,in this study,the genome of E.urophylla×E.grandis was used as a template for PCR amplification and sequencing to analyse the mi RNA396 sequence of E.urophylla×E.grandis gene.The c DNA reverse transcribed by RNA from E.urophylla×E.grandis callus cultured on media with different PBU cytokinin concentrations was used as a template by fluorescence quantitative PCR and the expression differences of mi RNA396 and CKX in E.urophylla×E.grandis callus treated with different PBU concentrations were determined.The results showed that compared with the E.urophylla×E.grandis callus treated with 0.5 mg·^L（-1）PBU,the expression of mi RNA396,CKXA,CKXB and CKXF in E.urophylla×E.grandis callus treated with 1 mg·^L（-1）PBU was significantly decreased,and the difference reached a very significant level.CKXC,CKXD and CKXE were up-regulated,but only the relative expression of CKXC reached a very significant level.The expression levels of mi RNA396A,CKXD,CKXE and CKXF in E.urophylla×E.grandis callus treated with 2 mg·^L（-1）PBU were down-regulated,and the difference reached a very significant level.The rest of gene expression levels were up-regulated.The relative expression levels of CKXA,CKXB and CKXC reached a very significant level.This study preliminarily established the regulation and expression differences of mi RNA396 and CKX gene in the callus of E.urophylla×E.grandis,laid a foundation for the subsequent analysis of the mi RNA regulation network of E.urophylla×E.grandis,and provided a certain reference for the establishment of an efficient regeneration system of E.urophylla×E.grandis.