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). 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. 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.

中文翻译：

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一种使用细胞计数器和网状柱的改进的花粉数计数方法。

花粉数量的测定在进化、农业和医学研究中很重要。柏科树种在世界范围内引起严重的花粉病。虽然日本雪松（Cryptomeria japonica）是日本最重要的林业树种，但它也是该国花粉病的最大原因。已经根据生长速度和优良的形态特征选择了日本雪松树，然后进行了克隆。这些克隆的花粉产量可能有所不同，但很少有人研究单个雄性 strobilus（花）会产生多少花粉粒。最近报道的一种用细胞计数器计数花粉数量的方法适用于拟南芥属物种和小麦，但不适用于日本雪松，因为 strobilus 在加热时不会打开（例如 60 °C，过夜）。这里，我们报告了一种改进的日本雪松花粉计数方法，该方法使用精确快速的细胞计数器与自制的网状柱相结合。用杵轻轻压碎雄性 strobilus。然后分别使用 100- 和 20-μm 目柱去除大小碎片。我们使用这种方法成功地检测了两个克隆之间不同的花粉大小和数量。这种改进的方法不仅适用于杉木花粉的计数，还可以应用于花粉覆盖有坚硬鳞片组织的柏科其他树种。此外，这种方法可以应用于更广泛的植物物种，例如小麦，因为不需要等待开花，并且可以有效地去除碎片。