Kakui et al. Plant Methods (2020) 16:124 https://doi.org/10.1186/s13007-020-00668-4 Plant Methods METHODOLOGY Open Access An improved pollen number counting method using a cell counter and mesh columns Hiroyuki Kakui1* , Eriko Tsurisaki2, Hidenori Sassa3 and Yoshinari Moriguchi1 Abstract 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 pro- duced by a single male strobilus (fower). 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 difered 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 efciently. Keywords: Pollen number, Pollen size, CASY cell counter, Japanese cedar (Cryptomeria japonica) Background breeding [7]. A high pollen number is also a desired trait Te determination of pollen grain number is important for crops that require artifcial pollination because artif- in evolutionary, agricultural, and medical studies. From cial pollination require a lot of pollen [8, 9]. From a medi- an evolutionary perspective, selfng plant species tend to cal perspective, pollen is relevant because it can lead to produce lower pollen numbers than closely related out- an allergic reaction called pollinosis [10, 11]. crossing plant species [1–5]. Te reduced pollen number Te recent development of next-generation sequenc- in selfng plants is thought to decrease the cost of pollen ing techniques has enabled the genomic sequences of production. From an agricultural perspective, domesti- almost 600 plant species to be determined [12]. Tese cated species such as rice tend to have low pollen num- sequenced genomes include those of plant species that bers [6]; however, the production of large numbers of have huge genome sizes, such as sugar pine (27.6 GB) or pollen grains is one of the desired traits in hybrid wheat wheat (16 GB) [13, 14]. Combined analyses of sequence and phenotype data is a powerful tool for the identi- *Correspondence: [email protected] fcation of new genes. We recently identifed a gene 1 Graduate School of Science and Technology, Niigata University, Niigata, controlling pollen number using a genome-wide associa- Niigata 950-2181, Japan Full list of author information is available at the end of the article tion study in Arabidopsis thaliana [5]. To estimate the © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kakui et al. Plant Methods (2020) 16:124 Page 2 of 13 pollen number from A. thaliana accessions, we devel- Preparation of home‑made mesh columns oped a high-throughput method to count pollen grains To remove large and small debris, two types of polyester efciently [15]. Pollen numbers showed large variation mesh [20-μm opening size (MEDIFAB 07-20/13, Sefar within individuals and between species; therefore, the AG, Switzerland) and 100-μm opening size (PETEX phenotyping step should be optimized based on the plant PET105, Sefar AG)] were used. Te concept of using species and pollen number variation determined in a pre- mesh columns was derived from a protein experimen- liminary experiment. tal protocol [26]. Te bottom of a 0.6-mL sample tube Te Cupressaceae family is conifer species with a was removed around the 100-μL line using scissors and world-wide distribution and it contains species causing bound with the polyester mesh by heating (for details see serious pollinosis such as cypress (Cupressus species, Fig. 1). Te pieces of mesh were 1 × 1 cm (Fig. 1). Chamaecyparis obtusa), Japanese cedar (Cryptomeria japonica), and mountain cedar (Juniperus ashei) [16]. Jap- Preparation of pollen suspension anese cedar is an evergreen tree and is the most impor- A fowchart of the pollen suspension preparation tant forestry species in Japan because it has excellent method is provided in Fig. 2. Te male strobilus was gen- properties for use in Japanese architecture [17]. However, tly crushed using a pestle in a 1.5-mL tube and 250 μL the pollen of Japanese cedar is the most serious allergen of distilled water (DW) was added to the 1.5-mL tube in Japan. Although the pollen grain number of Japanese (Figs. 3a–d). Te pollen-containing suspension was cedar has been reported previously [18, 19], sample num- moved to a new 1.5-mL tube. To collect almost all of the bers were limited. Traditionally, the number of pollen pollen, an additional 250 μL of DW was added to the frst grains has been counted using a hemocytometer under a tube and remaining pollen was suspended. Tis suspen- microscope [20–22], but this method is time-consuming sion was then added to the frst pollen suspension for a and laborious. Recently, efcient pollen counting meth- total volume of 500 μL. Te suspension was transferred ods using cell counter were developed [CASY cell coun- to a 100-μm mesh column and centrifuged at 2000g for ter (OMNI Life Science, Germany [15]) or Ampha Z32 5 s. Te column retaining the large male strobilus tissues (Amphasys, Switzerland [23, 24])]. Anthers from these was discarded and the fowthrough was transferred to a plants were forcibly opened by heating (60 °C, overnight) 20-μm mesh column. Tis suspension was centrifuged at from A. thaliana and wheat [15]. To count pollen grains 2000g for 1 min. Particles > 20 μm that were trapped by of Japanese cedar using a cell counter, we attempted to the mesh were suspended with 500 μL of DW and trans- apply the same protocol; however, the male strobilus ferred to a new 1.5-mL tube. Te 20-μm mesh column (fower) of Japanese cedar consists of a hard scale struc- was washed with another 500 μL of DW to collect almost ture and it did not open with heating. Here, we report an all of the remaining particles and transferred to the 1.5- improved pollen counting protocol using a cell counter mL tube for a total volume of 1 mL. and home-made mesh columns. We confrmed that Jap- anese cedar pollen can be counted efciently using this Pollen counting using a CASY cell counter method. Te cell counter (CASY cell counter) parameters were set as described in Table 1. We chose the size range from 27.75 to 45 μm as pollen particle because this range cov- Methods ered pollen peak from all samples without contamination Plant materials from small/large particles. A 200-μL pollen suspension Trees of Japanese cedar (Cryptomeria japonica) were was mixed with 10 mL of CASYton (OMNI Life Science). grown in the Niigata prefectural forest research institute. Particle numbers were counted using a cell counter by Tree clones, ‘Iwafune-9’, ‘Iwafune-15’, and ‘Nishikan- sampling 400 μL three times. Viable cells were calculated bara-1’ were used. Male strobili were collected in Febru- as the total pollen number per strobilus using the follow- ary 2020. Pollen grains are already matured during this ing equation: period [25]. (See fgure on next page.) Fig. 1 Home-made mesh columns. a Left to right: 0.6-mL tube, 0.6-mL tube with cut tip, mesh attached to 0.6-mL tube (column), and polyester meshes (20-μm and 100-μm opening sizes). b–g Column preparation. The bottom of a 0.6-mL sample tube was removed around the 100-μL line using scissors (b and c). The cut surface was heated using a cigarette lighter (d and e). The heated surface was bound with 1 1 cm pieces of × polyester mesh (f and g).