Nicotiana alata is resistant to Tomato spotted wilt virus (TSWV) and of great value in breeding. However, hybrid sterility constrains the application of interspecific genetic resources. Previously, we obtained interspecific hybrids between a cytoplasmic male sterility (CMS) line of Nicotiana tabacum and N. alata, some of which were pollen sterile. In the present research, we studied the cytological abnormalities during pollen development in sterile hybrids (F₁-D) by comparing pollen development with that in fertile hybrids (F₁-S) from the same cross. Transmission electron microscopy and DiI staining showed that the membrane structures of microspores and pollen in F₁-D sterile hybrids were impaired. Carbol fuchsin staining revealed that cytomixis, chromosome loss and asymmetric callose wall formation occurred with high frequency in the microsporocytes and microspores of the sterile hybrids. The cytoplasm and nucleus were lost in the microspores and pollen of sterile hybrids, leading to mature pollen grains that were vacuous and collapsed in the aperture region. In addition, delayed tapetum degradation was detected in the anther of sterile hybrids, and sporopollenin was deposited in the aperture region. Impaired membrane structures of microspores and pollen in F₁-D sterile hybrids affected the integrity of the cells, and might be associated with chromosome, nuclear and cytoplasm loss, vacuous pollen, and sterility in F₁-D hybrids. Abnormal tapetum degradation in the anther and irregular sporopollenin deposition in the pollen wall of the F₁-D sterile hybrids might also be related to the pollen sterility. This study deepens our understanding of the cytological mechanisms of hybrid sterility, and may facilitate the application of TSWV-resistant resources in cultivated Nicotiana species through hybrid fertility restoration and backcross breeding.

烟草（Nicotiana alata）对番茄斑萎病毒（TSWV）具有抗性，在育种中具有重要价值。然而，杂种不育限制了种间遗传资源的应用。以前，我们获得了烟草和N. alata的胞质雄性不育（CMS）系之间的种间杂种，其中一些是花粉不育的。在本研究中，我们研究了细胞学在无菌杂种花粉发育期间的异常（F ₁通过花粉发育与在可育杂种比较-D）（F ₁从相同的横-S）。透射电镜和DiI染色显示F _1中的小孢子和花粉的膜结构-D不育杂种受损。Carbol Fuchsin染色显示，在无菌杂种的微孢子细胞和微孢子中，细胞混合，染色体丢失和不对称call壁的形成频率很高。细胞质和细胞核在无菌杂种的小孢子和花粉中丢失，导致成熟的花粉粒呈空泡状并在孔区域塌陷。另外，在无菌杂种的花药中检测到绒毡层延迟降解，并且孢粉蛋白沉积在孔区域中。F ₁ -D无菌杂种中小孢子和花粉的膜结构受损，影响细胞的完整性，并可能与F _1中的染色体，核和细胞质损失，空泡花粉和不育有关。-D杂种。花药中绒毡层的异常降解和F ₁ -D无菌杂种的花粉壁中孢子粉的不规则沉积也可能与花粉的不育有关。这项研究加深了我们对杂交不育的细胞学机制的理解，并可能通过杂交育性的恢复和回交育种而促进了对TSWV抗性资源在栽培烟种中的应用。