GhCYP704B1 is essential for pollen Exine and anther Cutin biosynthesis and plays a critical role in cotton male fertility.

Cotton hybrids offer significant advantages, the application of male sterile lines in cotton hybrid breeding can reduce the cost of artificial castration and ensure hybrid seed purity. Pollen and anther development are a crucial aspect of plant fertility, sporopollenin synthesis provides the major component of the outer walls in pollen (exines) for preserving pollen grains activity, mutations in the genes involved in sporopollenin synthesis affect pollen development and fertility formation. The differentially expressed genes (DEGs) between the developing anthers of genic male sterile mutant (ms1) and its genetic background Coker 312 were identified, the genes related to pollen exine and anther cutin biosynthesis were screened from the DEGs. GhCYP704B1 (Gh_D12G2768) was the DEGs with a significantly down-regulated expression level in ms1 anthers, kept very low expression level in ms1 developing anthers. At the same time, we also screened 20 homologies of GhCYP704B1 from DEGs data, and the results showed that only GhCYP704B1 was predominantly expressed in cotton anthers, while other homologies did not show significant expression changes. We used VIGS technology the expression level of GhCYP704B1 in cotton C312, resulting in disrupted callose formation during the tetrad formation of microspore development, partial defect of the pollen exine, weakened pollen activity, low pollen germination rate, and poor plant fertility. The expression levels of genes related to pollen exine and anther cutin synthesis changed significantly, the composition and content of cutin monomers in cotton anthers were significantly reduced in GhCYP704B1-silenced lines. Abnormalities in callose caused blockage of sporopollenin synthesis and failure to synthesize the pollen exine properly. The findings indicate that GhCYP704B1 affects cotton fertility and is involved in pollen exine biosynthesis, thus providing a candidate gene for creating new male sterile lines in G. hirsutum.

Supplementary information: The online version contains supplementary material available at 10.1007/s11032-025-01608-7.