In order to establish an efficient transient expression system based on grapevine protoplasts,we used the mesophyll and callus of grape ‘Heixiangjiao’ to analyze the key factors related to isolating effectively protoplasts,such as cellulose and macerozyme enzyme composition,concentration of mannitol in enzyme solution,duration of enzyme dissolve,and so on.The protoplast was used as a vehicle to explore the establishment of a stable,efficient grape protoplast isolation and transient transformation system,and lay the foundation for building a transient expression system.The results showed that:(1)the optimal enzyme solution for leaf protoplast isolation was 3.0% cellulase onozuka R-10+0.75% macerozyme R-10+0.6 mol/L mannitol.The digestion was conducted in the dark under 28 ℃ for 14 h,and the protoplasts yield was 4.09×10⁶ per gram,the vitality was 83.12%.(2)The optimal enzyme solution for callus protoplast isolation was 2.0% cellulase onozuka R-10+0.5% macerozyme R-10+0.5 mol/L mannitol.The digestion was conducted in the dark under 28 ℃ for 14 h,and the protoplasts yield was 6.05×10⁶ per gram,the vitality was 84.13%.(3)The transient expression vector pEZS-NL with reported gene coding green fluorescent protein (GFP) was transferred into protoplasts by 40% PEG-4000 method.The GFP protein expressed stably and clearly in all over the protoplast.We establish grape protoplast isolation and transformation system in this paper.The gene can be expressed efficiently in grape protoplasts with a small amount of plasmid DNA,which provides technical support for grape functional genomics studies.