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The presence of inferior bowl seedlings in hole tray seedling cultivation would affect the survival rate of later seeding transplantation. It is urgent to clean the substrate of these holes. The existing mechanical removal methods often have the phenomenon of particle scattering and omission, while the air suction removal method can effectively compensate for this defect. In order to analyze the mechanism of air suction removal of bowl seedling substrate, parameter calibration experiments for discrete element simulation research were conducted. Totally 100g of matrix was selected for particle size distribution detection and then a funnel was used to stand still. Based on image processing, the actual stacking angle on both sides of the matrix was obtained, the average value was calculated which was used as the response value. Through the Plackett-Burman experiment to screen for four significant factors that affected the stacking angle of the matrix. The maximum response area of significant factors was determined through the steepest climbing experiment. A second-order regression model was establish based on the Box-Behnken experiment and solved for the optimal parameter combination. The results showed that on the premise of taking the intermediate value of insignificant factors, when the matrix particle-particle collision coefficient of restitution A was 0.142, the matrix particle-particle rolling friction coefficient C was 0.097, the matrix particle-stainless steel static friction factor E was 0.223 and the matrix JKR surface energy G was 2.325J/m2, the simulated stacking angle was 33.4°, and the relative error with the actual stacking angle of 34.19° was 2.31%. The research result can meet the experimental requirements, and the obtained calibration parameters can be used for discrete element simulation of bowl seedling matrix.