Polyploidization is a key pathway for species formation and genetic innovation; approximately 70% of angiosperms have undergone at least one whole-genome duplication event during their evolutionary history. To determine the genetic and phenotypic stability of artificially induced autotetraploids across generations, this study utilized a colchicine-induced autotetraploid of Gossypium herbaceum as experimental material and conducted systematic comparative analyses of morphological, cytological, and molecular marker characteristics in the S3 and S4 generations. The results showed that, compared with the 2×, seed weight in the S3 generation increased by 59.4% (to 89.22 mg), and in the S4 generation increased by 65.0% (to 92.40 mg), while there was no significant difference in fiber length. The leaf area of tetraploids decreased significantly during the flower-bell stage. Observation of pollen mother cell meiosis revealed that the proportions of normal tetrads in the S3 and S4 generations were 73.80% and 81.80%, respectively, and the proportions of normal pollen grains were 79.60% and 80.60%, respectively. Cytological stability was markedly improved in the S4 generation. A total of 34 alleles were amplified by SSR molecular marker analysis, of which 23 (67.60%) were polymorphic. The primers NBRI_G1015 and NAU1164 exhibited the highest polymorphism rates, at 87.50% and 83.30%, respectively. The average genetic diversity index (He) was 0.1411, indicating a highly inbred genetic background. The banding patterns of S3 and S4 are highly consistent, with strong signal intensity; not only do they amplify bands consistent with those of diploids, but they also exhibit specific new bands and band deletions. In summary, this autotetraploid material exhibits stable morphological advantages and genetic uniformity. As generations progress, its meiotic behavior and genetic structure tend to stabilize. The S4 generation exhibits greater cytological stability and genetic uniformity than the S3 generation, making it a highly promising new germplasm resource for cotton polyploid breeding.

Background: Information on the autopolyploid of Gossypium herbaceum remains limited until now. Previously, the autotetraploid of G. herbaceum was successfully generated via colchicine-induced chromosome doubling from the diploid cultivar ‘Hongxing’ in our lab. Methods: To investigate the drought stress response mechanism of this tetraploid, the autotetraploid S4 was used as the experimental material. The plants were subjected to drought stress during the flowering stage, followed by measurements of physiological and biochemical indicators and transcriptomic sequencing analysis. Results: Under drought stress, MDA content increased, and cell membranes sustained oxidative damage. Photosynthetic parameters, such as net photosynthetic rate (Pn), were significantly suppressed, while the activity of osmotic regulators and key antioxidant enzymes increased significantly. After rehydration, all of the above physiological indicators showed varying degrees of recovery. Transcriptome analysis revealed that, when comparing the treatment group with the control group, a total of 5530 differentially expressed genes (DEGs) were identified, with 2714 up-regulated and 2816 down-regulated. Furthermore, this study investigated the drought resistance mechanism involving the interaction between the MAPK signaling pathway and other metabolic pathways in the autotetraploid. Nine drought-resistant genes, including MAPK3, bHLH47, GaRbohD, RIBA1, PIP1-3, RCA1, RbohD, CYP707A and HSP70, were selected and analyzed using real-time quantitative PCR; the results were generally consistent with the transcriptomic data. Conclusions: These findings substantially enhance our understanding of the molecular mechanisms underlying drought responses in autotetraploids. This novel autotetraploid genotype expands the available cotton germplasm resources and is expected to hold significant value for research on polyploidy evolution.