Optimization of in vitro pollen germination and pollen viability tests for Castanea mollissima and Castanea henryi

•Benzidine-H2O2 and MTT dyes were determined to be suitable for the pollen viability test.•Sucrose and boric acid levels strongly influenced pollen germination.•MTT staining has a significant correlation with the in vitro germination, and the R2 was higher than 0.8.•The proportion of dark staining by MTT was close to the germination rate. The Castanea Miller (Fagaceae) family are commercially important trees in the word, grown for their nuts that are rich in starch and nutrients. The production of nuts is related to pollen fertility, but little is known about pollen viability and germination of the family. In this paper, we investigated the staining and in vitro germination traits of Chestunt (Castanea mollissima Blume) and Chinese Chinquapin (Castanea henryi (Skam) Rehder & Wilson), which were widely cultivated in China. We aimed to determine the optimal media for pollen germination and to develop a method to estimate viability using a series of correlation analyses based on staining and germination results. Pollen viability was assessed using 2,3,5-triphenyl tetrazolium chloride (TTC), 2,5-diphenyl monotetrazolium bromide (MTT), acetic carmine, benzidine-H2O2, blue ink dyeing, methylene blue, I2-KI, inorganic acid, and peroxide staining methods. For germination tests, different concentrations of sucrose, boric acid (H3BO3), calcium chloride (CaCl2), and gibberellin (GA3) were used. Benzidine-H2O2 and MTT dyes were determined to be suitable for the pollen viability test. The optimal media compositions for C. mollissima and C. henryi both had 5% sucrose, 0.01% H3BO3, and 0.005% GA3, but their amounts of CaCl2 varied at 0.01 and 0.005%, respectively. Assessments of staining methods found that MTT staining exhibited a linear relationship with pollen germination (***p