An improved pollen number counting method using a cell counter and mesh columns

BACKGROUND: The determination of pollen number is important in evolutionary, agricultural, and medical studies. Tree species of the Cupressaceae family cause serious pollinosis worldwide. Although Japanese cedar (Cryptomeria japonica) is the most important forestry species in Japan, it is also the biggest cause of pollinosis in the country. Japanese cedar trees have been selected for growth speed and superior morphological traits and then cloned. These clones may vary in their pollen production, but there has been little research on how many pollen grains are produced by a single male strobilus (flower). A recently reported method for counting pollen number with a cell counter was applicable to Arabidopsis species and wheat, but was not suitable for Japanese cedar because the strobilus does not open with heating (e.g. 60 °C, overnight). RESULTS: Here, we report an improved pollen counting method for Japanese cedar using a precise and rapid cell counter in combination with home-made mesh columns. The male strobilus was gently crushed using a pestle. Large and small debris were then removed using 100- and 20-μm mesh columns, respectively. We successfully detected pollen sizes and numbers that differed between two clones using this method. CONCLUSIONS: This improved method is not only suitable for counting pollen from Japanese cedar, but could also be applied to other species of the Cupressaceae family with hard scale tissue covering the pollen. Moreover, this method could be applied to a broader range of plant species, such as wheat, because there is no need to wait for anthesis and debris can be removed efficiently.