Limitations of Flow Cytometry in Detecting Aneuploids: Insights From Chromosome Doubling in Hemerocallis citrina

ABSTRACT Flow cytometry is a commonly used method to test for chromosome doubling, but during the doubling of Hemerocallis citrina , it was found that flow cytometry sometimes failed to accurately detect aneuploidy and mixploidy in doubled seedlings. In this study, a total of 8481 germinated seeds were treated and 47.75% of the seedlings survived, of which 20.17% were doubled successfully. Chromosome counting revealed that flow cytometry had limited resolution for minor aneuploidy, and analysis of ploidy levels throughout the development of the artificially induced tetraploidy reveals that there was a reversion to mixoploidy or diploidy during this process. Confirmed stable tetraploids (2 n = 4 x = 44) exhibited distinct morphological characteristics, including a 50% reduction in stomatal density, larger stomatal size and pollen grains, and flowers that were 40.6% larger in diameter compared to diploids. The experimental results provide practical guidelines for polyploid screening in daylily breeding programmes and emphasize the importance of long‐term ploidy stability assessment for successful tetraploid development.