Abstract: To identify a rapid method for assessing pollen viability in field-based cross-breeding of pine wilt disease-resistant Pinus massoniana, three resistant clones were used as materials, with the pollen germination (PG) method as the standard. Five rapid staining methods including iodine-potassium iodide (I₂-KI), blue ink (BI), peroxidase (POD), MTT, and triphenyltetrazolium chloride (TTC) were evaluated for their applicability. The results showed that POD and BI staining failed to achieve specific staining of Masson pine pollen; I₂-KI and TTC staining yielded values significantly different from those of the PG method (P<0.001), indicating no practical application value. The MTT method showed no significant difference from the PG method in detection values, although a significant proportional bias existed between them. In the high-viability range (>90%), the deviation ranged from 0.7% to 10.5%, while the deviation increased when pollen viability was below 90%. The MTT method was simple to operate, required only 10 min to complete, and exhibited acceptable deviation from the PG method in the high-viability range, making it a suitable rapid alternative for field-based cross-breeding of resistant P. massoniana. Although less accurate for precise quantification in the low-viability range, it still retained value for rapid preliminary screening. This study provided a practical and reliable technical approach for on-site pollen quality assessment in disease resistance breeding programs.

Key words: Pinus massoniana, pine wilt disease resistance, pollen viability, rapid detection, MTT method, field applicability