Morphological characteristics and optimized protocols for in vitro germination and viability testing of Idesia...

Idesia polycarpa Maxim. is a premier woody oil species in Guizhou Province, China, whose fruit yield and oil quality largely depend on effective pollination and fertilization. However, limited research on pollen viability and germination has hindered industrial progress. To address this gap, a comprehensive evaluation framework for elite I. polycarpa germplasm was developed, integrating micromorphological analysis, optimized staining protocols, and in vitro germination assay. Scanning electron microscopy (SEM) revealed that I. polycarpa pollen, while genetically conserved at the genus level-characterized by prolate shapes, tricolporate apertures, and reticulate exine ornamentation-exhibits notable micromorphological variation among genotypes. Of the nine staining protocols tested (2,3,5-triphenyl tetrazolium chloride [TTC], carbol fuchsin, acetocarmine, methylene blue, Alexander, peroxidase, 2,5-diphenylmonotetrazolium bromide [MTT], I2-KI, and red ink), TTC and red ink were the most effective, offering clear chromatic distinction between viable and non-viable pollen. Through orthogonal experimental designs, genotype-specific optimal media for in vitro germination were identified: 0.40 g/L H3BO3, 0.01 g/L KNO3, 0.02 g/L Ca(NO3)2·4H2O, and 0.20 g/L KH2PO4 for STZ-6; and 0.20 g/L H3BO3, 0.02 g/L KNO3, 0.02 g/L Ca(NO3)2·4H2O, and 0.10 g/L KH2PO4 for STZ-9. Regression analysis confirmed a highly significant positive correlation (P < 0.01) between in vitro germination rates and the staining results from both TTC and red ink across various concentrations. Notably, 5% TTC and 30% red ink exhibited the highest coefficients of determination. A hierarchical evaluation strategy is thus proposed: the 5% TTC method is recommended for precise laboratory quantification due to its stability, while the 30% red ink method, due to its ease of use, is suited for rapid field-based screening. This study provides valuable insights into the morphological characteristics of I. polycarpa pollen and establishes a standardized evaluation framework, supporting germplasm innovation and optimizing pollination management.