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Callose contributes to many biological processes of higher plants including pollen development, cell plate and vascular tissue formation, as well as regulating the transport function of plasmodesmata. The functions of callose synthase genes in maize have been little studied. We describe a maize male-sterile mutant 39 (ms39) characterized by reduced plant height. In this study, we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909 (ZmCals12), encoding a callose synthase, is responsible for the male sterility of the ms39 mutant. Compared with male-fertile plants, callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced. Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells, leading to collapse of the anther wall structure. Disordered glucose metabolism in ms39 may have intensified autophagy in anthers. Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production. [ABSTRACT FROM AUTHOR]