Staining Germinating Pollen and Pollen Tubes

Germinating pollen on stigmas and pollen tubes in styles of Antirrhinum, Brassica, Oenothera, Raphanus, Rosa, solatium and Tagetes spp. were prepared for examination as follows: The styles were fixed in ethyl alcohol-acetic acid 3:1 for 1 hr, and hydrolyzed at 60°C for 5 to 60 min (depending on the species) in 45% acetic acid. The stigma with its attached strand(s) of stigmatoid tissue was then dissected out under a stereoscopic microscope, placed in a few drops of a staining solution made by dissolving 150 mg of safranin O and 20 mg of aniline blue in 25 ml of hot 45% acetic acid. After 5-15 min in this stain, the tissue was placed in a fresh drop of stain on a microscope slide and gently squashed under a cover glass. Because of a gradual precipitation of the aniline blue component, the stain had to be filtered regularly before use. However, a staining solution could be kept at room temperature for several weeks.