Pollen viability and germination performance in Gerbera: a comparative evaluation of assessment methods

Accurate and reliable assessment of pollen fertility is a critical prerequisite for selecting suitable pollen parents in ornamental plant breeding programs. This study aimed to evaluate pollen viability and in vitro germination performance in three commercial Gerbera jamesonii cultivars and to identify a biologically meaningful method for functional pollen fertility assessment. Five staining assays, including iodine potassium iodide (IKI), 2,3,5-triphenyl tetrazolium chloride (TTC), acetocarmine stain (AST), safranin (SFN), and lactophenol cotton blue (LCB), and eight germination media were tested using both agar plate (semi-solid; K-1–K-4) and hanging drop (liquid; S-1–S-4) techniques. Structural staining assays (IKI, LCB, AST, and SFN) consistently produced high viability estimates (approximately 80–92%), although these values were not always associated with actual germination performance. In contrast, the metabolically based TTC assay generated lower viability estimates (approximately 43–53%) and showed a stronger correspondence with functional germination capacity. Heat-treated pollen (80 °C for 48 h) exhibited complete loss of germination and absence of TTC staining, confirming the metabolic specificity of TTC. Among the tested germination media, PEG-containing liquid formulations, particularly S-4 and S-2, produced the highest and most consistent germination rates, reaching 58.7% in the cultivar ‘Yeliz’, whereas semi-solid media resulted in very low or no germination. Principal component analysis (PCA) and hierarchical clustering further demonstrated the close relationship between TTC viability estimates and germination performance. The findings indicate that TTC staining provides a more functionally relevant estimate of pollen fertility than structural staining methods and can serve as a reliable indicator of germination potential in gerbera. Furthermore, PEG-enriched liquid media were identified as the most suitable conditions for in vitro pollen germination. Together, these results establish a standardized and biologically meaningful framework for evaluating pollen fertility and selecting pollen parents in gerbera breeding programs.