Crushing the male strobilus using a pestle to collect pollen grains
To count the number of pollen grains per strobilus precisely, it is important to collect all of the pollen grains from the strobilus. The model plant A. thaliana has thin anthers and opens easily at high temperatures (60°C, overnight [15]). We attempted to open the male strobili of Japanese cedar by heating or drying, but this approach was not successful. The male strobilus of Japanese cedar has a hard scale structure, unlike the anthers from Arabidopsis species or wheat [15] (Fig. 3a). We then attempted to crush the male strobilus with a pestle to break the scale structure. Pollen grains are physically stronger than other plant tissues because they have an external wall layer called the exine, which is a physically and chemically resistant structure [28]. A male strobilus was gently crushed using a pestle (Fig. 3b and 3c), and then suspended in DW using a pipette (Fig. 3d). The scale structure was separated after pipetting and we successfully collected all of the pollen from the pollen-containing suspension (Fig. 3e and 3f).
Removing large debris from the pollen-containing suspension using a 100-μm mesh column
The pollen suspension contained not only pollen grains but also large amounts of large and small debris (e.g., scale tissue, anther wall, etc.). Because the cell counter cannot count particles >150 μm due to the capillary becoming blocked, we made a 100-μm mesh column to remove the large debris. Japanese cedar pollen typically has a round shape with a 35-μm diameter as determined from microscope observations so it was unlikely to be retained by the 100-μm mesh column. The pollen-containing suspension was loaded onto the 100-μm mesh column and centrifuged. Large scale structures were retained in the column without stopping the passage of the pollen grains. Pollen grains moved to the flowthrough area (Fig. 3g). The capillary of the cell counter remained unblocked throughout this study, suggesting that the 100-μm mesh column successfully removed large debris.
Removing small debris from the pollen-containing suspension using a 20-μm mesh column
The cell counter can count a maximum of 20,000 particles in a single measurement. The flowthrough suspension passing through the 100-μm mesh column contained many small particles. Fig. 4 shows the particle distribution with or without passage of the same sample through a 20-μm mesh column. More than 20,000 particles were detected in a pollen suspension without passage through a 20-μm mesh column (total of 21,097 particles; Fig. 4a). In contrast, many small debris particles were removed after using the 20-μm mesh column (total of 17,185 particles remained in suspension; Fig. 4b and 4c). In the 27.75- to 45-μm particle size range, almost the same number of pollen grains remained with or without the use of the 20-μm mesh column (6,454 vs 6,463 particles, respectively; Fig. 4d). Table 2 and Fig. 5 shows the efficiency of the 20-μm mesh column for removing small debris. Approximately, 32% of the debris was removed using the 20-μm mesh column. To check whether pollen grains leaked through the 20-μm mesh column, the pollen number was counted for the pollen retained by the 20-μm mesh (normal pollen suspension) and for the pollen in the flowthrough after using the 20-μm mesh column. The particle distribution pattern revealed a clear pollen peak from the pollen sample retained by the mesh, whereas there was no peak observed in the flowthrough (Fig. 6). Table 3 shows the numbers of particles between 27.75 and 45 μm in the 20-μm mesh column-trapped samples and flowthrough samples. Less than 3% of the particles were detected in flowthrough samples for all samples. These results suggest that the use of the 20-μm mesh column reduced the amount of small debris in the pollen suspension with no loss of pollen grains.
Table 2 Particle numbers with or without passage through the 20-μm mesh column
Sample No.
|
Clone name
|
Sample with 20 μm mesh column
|
|
Sample without 20 μm mesh column
|
|
with/without 20μm mesh column
|
|
|
Total counts
|
Viable cells
|
Debris
|
|
Total counts
|
Viable cells
|
Debris
|
|
Reduced debris number
|
Reduced ratio (%)
|
Viable cell ratio (%)
|
|
|
1
|
Iwa-9
|
15,237
|
6,094
|
9,143
|
|
19,072
|
6,806
|
12,266
|
|
3,123
|
25.5%
|
89.5%
|
2
|
Iwa-15
|
17,526
|
5,797
|
11,729
|
|
20,266
|
6,040
|
14,226
|
|
2,497
|
17.6%
|
96.0%
|
3
|
Iwa-15
|
16,235
|
6,222
|
10,013
|
|
28,594
|
6,165
|
22,429
|
|
12,416
|
55.4%
|
100.9%
|
4
|
Iwa-15
|
17,451
|
7,633
|
9,818
|
|
23,346
|
6,786
|
16,560
|
|
6,742
|
40.7%
|
112.5%
|
5
|
Iwa-15
|
17,185
|
6,463
|
10,722
|
|
21,097
|
6,454
|
14,643
|
|
3,921
|
26.8%
|
100.1%
|
6
|
Iwa-15
|
14,232
|
6,394
|
7,838
|
|
17,065
|
6,433
|
10,632
|
|
2,794
|
26.3%
|
99.4%
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Average
|
|
5,249
|
32.0%
|
99.7%
|
Table 3 Pollen numbers from 20-μm mesh column-trapped sample and flowthrough sample
Sample No.
|
Viable cells counts
|
|
Viable cell ratio
|
20 μm column
|
Flow through
|
|
Flow through / Total counts
|
1
|
4597
|
133
|
|
2.81%
|
2
|
4563
|
121
|
|
2.58%
|
3
|
4381
|
118
|
|
2.62%
|
4
|
5378
|
130
|
|
2.36%
|
5
|
3826
|
44
|
|
1.14%
|
6
|
6233
|
179
|
|
2.79%
|
7
|
6578
|
171
|
|
2.53%
|
8
|
5446
|
154
|
|
2.75%
|
9
|
7421
|
193
|
|
2.53%
|
10
|
7147
|
171
|
|
2.34%
|
|
|
|
|
|
|
|
Average
|
|
2.45%
|
Numbers and sizes of pollen grains from two Japanese cedar clones
Pollen number and size for ‘Iwafune-9’ and ‘Nishikanbara-1’ were measured by the cell counter. Twenty-two strobili from ‘Iwafune-9’ and 30 strobili from ‘Nishikanbara-1’ were analyzed. Fig. 7a shows the pollen number and size distribution of 52 samples. The two clones had clearly different pollen sizes. ‘Iwafune-9’ had a low pollen grain number (mean pollen number = 196,754) but a larger pollen size (mean pollen diameter = 34.59 μm) compared to ‘Nishikanbara-1’ (mean pollen number = 304,429, mean pollen diameter = 31.79 μm). Even among clones, there was more than a two-fold difference in pollen number. Such a large variation in pollen number from the same plant has also been reported in Arabidopsis species and in wheat [5,15]. Fig. 7b shows the particle size distribution for representative samples from ‘Iwafune-9’ (magenta) and ‘Nishikanbara-1’ (green). Both samples showed a clear single peak in the viable cell range, which is the size range we expected based on microscopy observations. There was typically a 10-μm variation in pollen size within the same strobilus (e.g., 30 to 40 μm from ‘Iwafune-9’ and 26 to 37 μm from ‘Nishikanbara-1’; Fig. 7b).
Detection of pollen cells released from exines using a cell counter
Pollen of Japanese cedar has some unique features compared with angiosperm plant species. For example, mature Japanese cedar pollen cells include generative cells and tube cells [29]. When mature pollen attaches to the nucellus (pollination), the intine structure, including the pollen cell, is released from the exine structure and the germinated pollen tube grows through the nucellus [29]. This process is important for pollinosis patients because Cry j1 and Cry j2, which are the major allergenic proteins of Japanese cedar, are localized in the intine and intine is also released from the exine in the human eye [30-32]. In this study, most of the pollen grains were not released from exines after 24 h in DW (data not shown). On the other hand, we found that many pollen grains were released from the exine structure in CASYton after 30 min (Fig. 8a and 8b). The cell counter displayed an additional small peak after the pollen suspension was mixed with CASYton (Fig. 8c). The original pollen peak remained almost the same between 0 and 30 min. Although the two peaks derived from the exine and pollen cells were indicated by different particle diameters, we recommend determining the pollen number immediately after mixing with CASYton for species which have similar traits because a small overlap of the two peaks was detected (Fig. 8c, around 30 μm diameter).