Reliable assessment of pollen quality in Chrysanthemum × morifolium: comparative evaluation of viability tests and germination media

Reliable evaluation of pollen quality is essential for successful hybridization and breeding programs in Chrysanthemum × morifolium. However, commonly used staining-based pollen viability tests do not always reflect functional pollen performance, and the relationship between pollen viability estimates and actual germination capacity remains insufficiently clarified in chrysanthemum.

In this study, the reliability of pollen viability and germination tests was evaluated using three chrysanthemum cultivars (Chic, Swan, and Barolo). Five pollen viability tests [iodine-potassium iodide (IKI), 2,3,5-triphenyl tetrazolium chloride (TTC), lactophenol blue (LCB), safranin (SFN), and acetocarmine (AST)] and eight in vitro germination assays consisting of four liquid and four semi solid media were compared using hanging-drop and Petri dish agar methods. In total, thirteen tests were evaluated under both fresh and heat-treated (80 °C for 48 h) pollen conditions, with 1.000 pollen grains analyzed per cultivar. Staining-based tests such as SFN, IKI, LCB, and AST produced high apparent viability values (76–98%), whereas the TTC test yielded markedly lower values (4–8%). Despite these high viability estimates, pollen germination percentages remained generally low across all media (0.1–7.3%). The highest germination performance was observed in polyethylene glycol (PEG)-based liquid media, particularly S-4 and S-1. PCA revealed an association between TTC viability results and germination success.

The results demonstrate a clear discrepancy between staining-based viability estimates and actual pollen germination capacity in chrysanthemum. The TTC test appeared to be the viability assay most closely associated with functional pollen performance under the tested conditions. TTC may provide a more functionally relevant estimate of pollen viability than staining methods such as IKI. The combined use of TTC-based viability testing and PEG-supported germination assays may therefore represent a promising approach for selecting highly functional pollen in chrysanthemum breeding programs.