Interspecific cross breeding introduces superior agronomic traits into cultivated species; 12 however, problematic pollen sterility occurs in the hybrids. Our previous study obtained interspecific 13 hybrids from the cross between a cytoplasmic male sterility line of Nicotiana tabacum and Nicotiana 14 alata, and some of the hybrids were pollen sterile. Here, we conducted an in-depth cellular study to 15 understand the cytological mechanism of pollen abortion in these hybrids (F1-D sterile) compared with 16 pollen development in the fertile hybrids (F1-S sterile) from the same cross. The ultrastructure 17 observation showed that the membrane of microspore in F1-D sterile hybrid was deficient at all 18 represented developmental stages. Chromosome behavior during meiosis was studied by carbol fuchsin 19 staining, which indicated that cytomixis, chromosome leakage and asymmetric callose wall deposition 20 occurred with high frequency in the microsporocyte of F1-D sterile. The results of the ultrastructure and 21 6-diamidino-2-phenylindole (DAPI) analyses also showed that the cytoplasm and nucleus were unstable 22 and extruded from F1-D sterile microspore during the developmental process, leading to mature pollen 2 1 grains that were vacuous and collapsed in the aperture region. In addition, delayed tapetum degradation 2 was also detected in the anther of F1-D sterile, and might be associated with irregular sporopollenin 3 deposition in the aperture region of F1-D sterile pollen. Genetic unbalance and cytomembrane deficiency 4 might both be responsible for the instability of the chromosome, nuclear and cytoplasm, and resulted in 5 pollen abortion in F1-D sterile hybrids, and irregular tapetum degradation might also be related with 6 pollen sterility. 7