Pollen Viability of Fraxinus excelsior in Storage Experiments and Investigations on the Potential Effect of Long-Range Transport

Abstract

Fragmented ash populations due to ash dieback may lead to a limited gene flow and pollination success. Therefore, the viability of ash pollen plays a major role for the survival of the species. The extent to which the long-distance transport of pollen affects pollen viability was investigated with experiments in a climate chamber using ash pollen samples from a seed orchard in Emmendingen, Germany. Furthermore, experiments with a volumetric pollen trap were conducted. A suitable storage temperature for ash pollen was determined by using four viability tests; TTC test, pollen germination, Alexander’s stain and Acetocarmine. An optimization of the germination medium was performed. We found a strong influence of prevailing temperatures on pollen viability, which decreased faster under warmer conditions. At moderate temperatures, viable pollen could still be observed after 28 days. Thus, a possible successful pollination can also be associated to long-range transported pollen. Storage experiments showed that pollen viability could be maintained longer at temperatures of −20 °C and −80 °C than at 4 °C. In particular, the TTC test has proven to be suitable for determining viability. Therefore, properly stored pollen can be used for breeding programs to support the survival of Fraxinus excelsior.

Full text



Citation: Buchner, L.; Eisen, A.-K.;
Šikoparija, B.; Jochner-Oette, S. Pollen
Viability of Fraxinus excelsior in
Storage Experiments and
Investigations on the Potential Effect
of Long-Range Transport. Forests
2022,13, 600. https://doi.org/
10.3390/f13040600
Academic Editor: Herminia
García-Mozo
Received: 23 February 2022
Accepted: 8 April 2022
Published: 11 April 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Pollen Viability of Fraxinus excelsior in Storage Experiments
and Investigations on the Potential Effect of
Long-Range Transport
Lisa Buchner 1,*, Anna-Katharina Eisen 1, Branko Šikoparija 2and Susanne Jochner-Oette 1
1Physical Geography/Landscape Ecology and Sustainable Ecosystem Development, Catholic University
of Eichstätt-Ingolstadt, 85072 Eichstätt, Germany; anna-katharina.eisen@ku.de (A.-K.E.);
susanne.jochner@ku.de (S.J.-O.)
2BioSense Institute—Research Institute for Information Technologies in Biosystems, University of Novi Sad,
21102 Novi Sad, Serbia; sikoparijabranko@biosense.rs
*Correspondence: lbuchner@ku.de; Tel.: +49-8421-93-21578
Abstract:
Fragmented ash populations due to ash dieback may lead to a limited gene flow and
pollination success. Therefore, the viability of ash pollen plays a major role for the survival of
the species. The extent to which the long-distance transport of pollen affects pollen viability was
investigated with experiments in a climate chamber using ash pollen samples from a seed orchard in
Emmendingen, Germany. Furthermore, experiments with a volumetric pollen trap were conducted.
A suitable storage temperature for ash pollen was determined by using four viability tests; TTC
test, pollen germination, Alexander’s stain and Acetocarmine. An optimization of the germination
medium was performed. We found a strong influence of prevailing temperatures on pollen viability,
which decreased faster under warmer conditions. At moderate temperatures, viable pollen could still
be observed after 28 days. Thus, a possible successful pollination can also be associated to long-range
transported pollen. Storage experiments showed that pollen viability could be maintained longer at
temperatures of
−
20
◦
C and
−
80
◦
C than at 4
◦
C. In particular, the TTC test has proven to be suitable
for determining viability. Therefore, properly stored pollen can be used for breeding programs to
support the survival of Fraxinus excelsior.
Keywords:
Acetocarmine; Alexander’s stain; ash dieback; pollen germination; TTC; volumetric
pollen trap
1. Introduction
The common ash (Fraxinus excelsior L.) was formerly thought of as a suitable tree
species for Europe’s forests in the course of climate change [
1
,
2
]. Nowadays, this tree
species is severely threatened by ash dieback, caused by the fungus Hymenoscyphus frax-
ineus (T. Kowalski) Baral, Queloz and Hosoya [
3
]. Despite the current lack of effective
conservation measures or opportunities to control ash dieback, there is hope for Frax-
inus excelsior populations. According to the current literature [
4
–
6
], there is a natural
variability in the susceptibility of ash trees to H. fraxineus. Different degrees of damage
were attributed to a genetically determined resistance of ash tree individuals [
7
–
9
]. Less
susceptible ash trees may have the potential to counter ash dieback through successful
reproduction within breeding programs [
10
]. The proportion of ash trees that is (partially)
resistant to ash dieback is estimated to be low, between 1% and 5% [
11
–
14
]. In addition,
the natural regeneration of ash trees is believed to be of importance in fighting ash dieback
since those seedlings, which are able to withstand competition, might constitute resistant
phenotypes [15].
Within the next 30 years, an ash tree decline of 75% is expected in mixed stands in
Europe [
16
]. A sharp decline in the ash populations is associated with stand fragmenta-
tion [
13
], probably resulting in the loss of genetic diversity and an increase in inbreeding
Forests 2022,13, 600. https://doi.org/10.3390/f13040600 https://www.mdpi.com/journal/forests

Forests 2022,13, 600 2 of 16
among ash individuals [
17
]. Studies of fragmented ash stands in Scotland have shown
that gene flow between isolated ash trees can be maintained by pollen transport from
distances of up to 2900 m [
18
,
19
]. However, it is known that pollen from anemophilous
plants can be dispersed at mesoscales, i.e., 10–200 km from the source [
20
,
21
] and even
synoptic scales [
22
,
23
]. These events are not frequent since they require specific environ-
mental conditions including the characteristics of the landscape [
19
] and meteorological
condition [
24
], however, they can be an important mean for geneflow providing that the
pollen viability is maintained.
The success of atmospheric pollen transport depends crucially on pollen viability. At
the same time, breeding programs with genetically more resistant ash trees can only be
carried out successfully if viable pollen are available. The determination of pollen viability
can be achieved using different methods. Besides the assessment of pollen germination, via-
bility can also be studied via pollen staining tests [
25
,
26
]. The TTC test is a commonly used
agent and proved successful determination of pollen viability for many species
[27–31]
,
although not all pollen species yielded successful staining [
32
,
33
]. For pollen of Fraxinus ex-
celsior, this test was successfully applied by Castiñeiras et al. [
34
]. Furthermore, in our study
viability was also tested using Acetocarmine, Alexander’s stain and pollen germination.
So far, no application of Alexander’s stain has been documented for Fraxinus excelsior. For
pollen germination, difficulties in achieving reliable results were reported [
34
]. In addition,
no systematic optimization of the nutrient medium for pollen germination of ash pollen
has been published as of yet.
We hypothesize that the viability of ash pollen decreases after their release from the
flower and strongly depends on meteorological conditions during pollen flight. Thus,
the aim of this study was to investigate pollen viability under different environmental
conditions and to experimentally evaluate storage temperatures using different methods.
In addition to climate chamber experiments that simulated the conditions pollen can face
during atmospheric transport, the viability of pollen sampled from the atmosphere has
been tested.
2. Materials and Methods
2.1. Study Material
For the experiments conducted in this study, ash pollen collected in an ash seed
orchard were used. The seed orchard near Emmendingen (48
◦
6
0
38.50
00
N, 7
◦
52
0
20.49
00
E,
209 m NHN) is located in Baden-Württemberg, Germany. In 1995, the plantation was
established on a previously agriculturally used area as a first afforestation on an area of
about 2.7 ha. A total of 49 clones were planted, and in the summer of 2021 there were 84
ash trees on the plantation. The annual average temperature is 10.2
◦
C and the average
precipitation sum is 882 mm (DWD station “Emmendingen-Mundingen”, 1981–2010).
For the preparation of a pollen composite sample, shoots of male ash trees were cut
shortly before flowering in March 2021. The shoots were then placed in vessels with water
at room temperature in the laboratory. After approximately four days, the flowers of the
shoots opened and pollen were released. These were then collected on paper underneath,
mixed together and immediately used for the subsequent experiments.
2.2. Viability Tests
2.2.1. TTC Test
The TTC test is an enzymatic test and detects the presence of enzyme activity in pollen,
according to which the viability of the pollen is determined. For this purpose, a colorless,
water-soluble tetrazolium salt was used, which is reduced to red formazan in the presence
of dehydrogenase in the pollen. The red colored pollen thus signals the viability and is
only produced when there is active enzyme activity in the pollen [
26
]. To perform the TTC
test, a 1% TTC solution of 0.2 g 2,3,5-triphenyl tetrazolium chloride and 12 g sucrose in
20 mL distilled water was produced. A thin layer of the pollen from the mixed sample
was dusted with a brush onto a microscope slide. Afterwards, two drops of the TTC

Forests 2022,13, 600 3 of 16
solution were added to the slide and covered with a coverslip. The slide was then kept
in a petri dish with moist filter paper. To achieve the maximum intensity of coloring, an
incubation period of 24 h was set. After 24 h, the stained pollen were counted under a light
microscope (Olympus CX23, magnification
×
40). Red stained pollen were categorized as
“viable” and colorless or yellowish stained pollen were categorized as “non-viable”. We
counted 400 pollen located on a random central row of the microscope slide. The evaluation
of the TTC test was sometimes affected by a color gradient of the stained pollen, which
made it difficult to distinguish clearly between viable and non-viable pollen. For this
reason, we additionally included the classification “semi-viable” for pollen that were only
colored slightly reddish. The coloration of pollen is often assessed very subjectively, and
in particular, the boundaries according to which pollen are assigned to a certain category
can vary depending on the processor [
26
]. Hence and in order to increase the accuracy, the
microscope slides were only analyzed by one trained person in our study.
2.2.2. Alexander’s Stain
Malachite green, a component of Alexander’s stain is used to stain the cellulose of
pollen walls green, while acid fuchsin and orange G are used to stain the cytoplasm pink.
This test of pollen viability is based on the assumption that pollen containing cytoplasm can
be regarded as viable [
35
]. Since Alexander’s stain contains toxic components, an adapted
solution according to Peterson et al. [
36
] was used. The pollen were again finely spread
on a microscope slide using a brush and then two drops of the solution were added. After
covering the slide, it was heated on a hot plate for a few seconds until visible bubbles
formed under the coverslip. Viable, semi-viable and non-viable pollen were counted after
10–15 min under the light microscope.
2.2.3. Acetocarmine
Acetocarmine stains existing chromosomes in the pollen [
25
]. Viable pollen are stained
red, while non-viable pollen remain colorless. Again, when performing this test, pollen
were first dusted onto a slide using a brush. Two drops of Acetocarmine were then
applied to the slide and covered with a coverslip. The counting of pollen was conducted
immediately after application using light microscopy.
2.2.4. Pollen Germination
Finally, the viability of the pollen was tested by pollen germination on a solid nutrient
medium. For the storage experiments, a solution of 10% sucrose, 1% agar and 20 ppm boric
acid in distilled water was used. After boiling the solution shortly, and allowing 14 mL to
set in a 90 mm diameter petri dish, pollen were finely dusted onto the solid culture medium.
Germination of the pollen then took place in the dark at room temperature for 24 h. After
24 h, pollen were counted under a light microscope and categorized as germinated or not
germinated. Pollen were scored as germinated once the pollen tube was equal or larger
than the diameter of the pollen grain [
26
]. As the germination values differed greatly from
those of the other viability tests, an optimization of the culture medium was initiated.
For this purpose, a multi-stage experimental set-up was designed, according to which
the ideal concentrations of the components of the culture medium for the pollen of Fraxi-
nus excelsior were determined step by step. Different concentrations of sucrose, boric acid
(H
3
BO
3
), calcium nitrate (Ca(NO
3
)
2
) and agar were investigated and three tests were con-
ducted for each concentration. Five different culture media were prepared with different
sucrose concentrations (0%, 5%, 10%, 15% and 20%). Since findings from our previous
pollen germination experiments could be used, 20 ppm boric acid and 1% agar were also
used for this experiment. The subsequent germination phase of 24 h took place in a reg-
ulated climate chamber (growth cabinet KBWF, Binder GmbH, Germany) at a constant
temperature of 25
◦
C and 80% relative humidity in the dark. Then, the best sucrose con-
centration determined in the previous step was used and 20 ppm, 100 ppm, 200 ppm,
500 ppm and 1000 ppm boric acid were added to the prepared culture media with 1%

Forests 2022,13, 600 4 of 16
agar, respectively. The best concentration determined was further used in the third step
of the optimization, which focused on calcium nitrate concentration. Calcium nitrate was
investigated as an additional component of the nutrient medium, a substance that can often
lead to a more successful pollen germination [
37
]. For this purpose, the concentrations
0 ppm, 100 ppm, 300 ppm, 500 ppm and 1000 ppm were tested. In the last step of the
optimization, the concentration of agar was examined. Sucrose, boric acid and calcium
nitrate concentrations, which were already determined, were used with 1% and 2% agar.
Three repetitions were performed for each concentration for all components.
For the pollen viability derived from the different concentrations of sucrose, boric
acid and calcium nitrate, a one-factor analysis of variance (ANOVA) and a subsequent
post hoc analysis, the paired t-test, were performed in R (version 3.6.3). The suitability of
the ANOVA was determined in advance by testing the variables for normal distribution
(Shapiro–Wilk test) and variance homogeneity using Levene’s test. A two-sided t-test was
used for the results of the two different agar concentrations.
Finally, a control with non-viable pollen was carried out according to Rodriguez-Riano
and Dafni [
38
]. For this purpose, ash pollen were heated for approximately 15 minutes on
a hot plate at 100
◦
C on a microscope slide. Afterwards, the non-viable pollen were tested
for viability with the four viability tests, and thus, also for the validity of the tests used.
The TTC test, Alexander’s stain, Acetocarmine and pollen germination were used
for the storage experiments, however, for experiments in the climate chamber and for the
volumetric pollen trap, only the TTC test was used. For each experiment, we repeated each
test three times.
2.3. Storage Experiments
Immediately after the preparation of the composite sample, the first viability tests were
performed and the initial viability value was determined. Subsequently, the composite
sample was stored at 4
◦
C in a fridge, at
−
20
◦
C in a common freezer and at
−
80
◦
C in a
blast freezer (Fryka Cold box B 35-85). After one, two and three months, respectively, the
viability of the samples was tested again. Please note, that results are not available for all
investigated times due to delays in the delivery of individual chemicals and difficulties in
carrying out pollen germination.
2.4. Experiments on Pollen Viability after Potential Atmospheric Transport
2.4.1. Climate Chamber
For the investigation of pollen viability under simulated natural conditions, three
experiments were executed with different settings in a climate chamber. Each trial covered
a period of 28 days. The viability of ash pollen was tested with the TTC test initially, after
24 h, 3 days, 7 days, 14 days, 21 days and 28 days.
For experiment 1 (Table 1), we firstly determined the mean April temperature and
relative humidity for Bavaria since the flowering of ash predominately occurs in this month.
Hourly data of 17 weather stations from the German Weather Service (DWD) (https://
opendata.dwd.de/climate_environment/CDC/observations_germany/climate/hourly/
accessed on 15 November 2021) were averaged for the reference period 1961–1991. Data
were divided into day (from 7 a.m. to 8 p.m.; 10
◦
C, 65% relative humidity) and night
means (from 8 p.m. to 7 a.m.; 5 ◦C, 80% relative humidity).
Table 1.
Climate chamber settings (temperature, relative humidity and UV radiation) for day and
night conditions within experiments 1, 2 and 3 to test the influence of meteorology on the viability of
ash pollen.
Experiment 1 Experiment 2 Experiment 3
Day Night Day Night Day Night
Temperature 10 ◦C 5 ◦C 12 ◦C 7 ◦C 20 ◦C 12 ◦C
Relative humidity 65% 80% 65% 80% 60% 80%
UV radiation on off on off on off

Forests 2022,13, 600 5 of 16
Experiment 2 represents a climate change scenario of +2
◦
C warming (Table 1), which
can be expected according to the SSP2-4.5 scenario, by the middle of the 21st century for
global means [39].
Experiment 3 investigated the influence of extreme temperatures on the viability
of ash pollen with temperatures of 20
◦
C during the day and 12
◦
C during nighttime.
These temperatures can obviously occur in April, although not constantly over a 28-day
period. However, with these settings, the influence of higher temperatures can be studied
in more detail.
The corresponding relative humidity for experiment 2 and 3 was estimated based
on its relationship to the temperature using the hourly meteorological data of the DWD.
UV radiation for all experiments was adjusted to the average potential sunshine duration
prevailing in April (day 13 h) (Table 1).
2.4.2. Volumetric Pollen Trap
To investigate the viability of pollen under partially natural occurring transport condi-
tions, samples of pollen suspended in the atmosphere during four weeks of the common
ash pollen season in 2021 were collected using a volumetric pollen trap of the Hirst type [
40
].
Pollen were caught within the trap for a duration of seven days and for four weeks in total.
The viability resulting from these data was assessed on a daily basis. Therefore, the pollen
that were adhered at the last day were exposed to ambient air and the air inside the sampler
either for a very short time (hours or minutes in the case of a quick impaction after pollen
release) or probably also even longer when prior transport took place. In turn, the pollen
of the first sampling day of one week was exposed to the influence of surrounding air for
seven days within the trap, and probably for an additional time in the ambient air.
The sampling was performed on the roof of a university building in Ingolstadt
(48
◦
76
0
58.06
00
N, 11
◦
41
0
56.23
00
E), Bavaria, at 13 m a. g. l. There are numerous ash pollen
sources surrounding the sampler, as there are parks and green areas close to the pollen trap.
The drum of the pollen trap was changed weekly, starting on 15 April 2021. The
silicone-coated foil strip attached to the drum was covered with a TTC solution shortly
after each change and split into seven 48 mm long sections, each section corresponds to a
24 h sample, which was placed on the glass slide and examined under a light microscope
at
×
40 magnification. Ash pollen were checked for their viability along horizontal lines
across the full range of the microscope slides and sorted into the categories of viable and
non-viable until the number of 100 was reached. In total, on ten of 26 days the total count
of all pollen on the slide was less than 100, due to a generally low pollen flight intensity
on these days. The pollen concentration was calculated, after counting ash pollen of four
random lines of the slide (representing approx. 9% of the impaction area), according to
Galán et al. [41].
The air temperature was recorded throughout the study period, with a measuring
device (Davis Vantage Pro 2) next to the pollen trap.
3. Results
3.1. Viability Tests
The applicability of the four viability tests for ash pollen was firstly evaluated on
the basis of the quality related to distinction and recognition: The TTC test enabled a
classification of the pollen into three categories (Figure 1a) based on the colors from dark
red to pale yellow or colorless. Within the experiment of pollen germination, pollen
tubes formed after 24 h, and could easily be recognized under the microscope (Figure 1b).
Alexander’s stain colored the pollen in clearly distinguishable pink and green colors
(Figure 1c). In contrast to the TTC test and Alexander’s stain, Acetocarmine led to a faint
color, although viable and non-viable pollen could be differentiated based on pale reddish
and colorless pollen. In addition to a coloration of the pollen, an increase in the size of the
colored pollen was also observed after approx. 2 h when using Acetocarmine (Figure 1d),
which decreased again after approx. 24 h.

Forests 2022,13, 600 6 of 16
Forests 2022, 13, x FOR PEER REVIEW 6 of 17
colorless pollen. In addition to a coloration of the pollen, an increase in the size of the
colored pollen was also observed after approx. 2 h when using Acetocarmine (Figure 1d),
which decreased again after approx. 24 h.
Figure 1. Exemplary pictures of the conducted viability tests, (a) TTC test, (b) Pollen germination,
(c) Alexander’s stain, (d) Acetocarmine. The green arrow points to exemplarily viable pollen, the
yellow arrow to semi-viable pollen and the red arrow to non-viable pollen.
3.2. Optimization of Pollen Germination
For optimizing the nutrient medium for pollen germination, the ANOVA analysis
revealed a significant difference between the five sucrose concentrations (p = 0.020). Ac-
cording to the post hoc analysis, there is no significant difference between 5% and 15%,
and between 5% and 20%. The optimum sucrose concentration is 10%, which differs sig-
nificantly to the other concentrations and was associated with the highest percentage of
germinated pollen (Figure 2, Table 2).
The optimal boric acid concentration was less clear. Even though the highest germi-
nation value was achieved at 20 ppm, the difference between 20 ppm, 100 ppm and 200
ppm was not statistically significant. Less suitable for pollen germination are 0 ppm and
1000 ppm. These groups were significantly different from the groups 20 ppm and 200 ppm
and exhibited the lowest mean germination (Figure 2, Table 2). For the following tests, a
concentration of 20 ppm was used.
For the calcium nitrate concentration, the highest germination value was observed at
100 ppm, however, this group was not significantly different from 0 ppm and 300 ppm.
In particular, 500 ppm and 1000 ppm led to a lower germination of pollen (Figure 2, Table
2). For the following test, 100 ppm calcium nitrate were used.
Performing the two-tailed t-test on the two agar concentrations studied, no signifi-
cant difference between the germination of pollen at 1% and 2% agar (p = 0.913) could be
shown (Figure 2). For further tests 1% agar should be used.
Figure 1.
Exemplary pictures of the conducted viability tests, (
a
) TTC test, (
b
) Pollen germination,
(
c
) Alexander’s stain, (
d
) Acetocarmine. The green arrow points to exemplarily viable pollen, the
yellow arrow to semi-viable pollen and the red arrow to non-viable pollen.
3.2. Optimization of Pollen Germination
For optimizing the nutrient medium for pollen germination, the ANOVA analysis
revealed a significant difference between the five sucrose concentrations (p= 0.020). Ac-
cording to the post hoc analysis, there is no significant difference between 5% and 15%,
and between 5% and 20%. The optimum sucrose concentration is 10%, which differs sig-
nificantly to the other concentrations and was associated with the highest percentage of
germinated pollen (Figure 2, Table 2).
Forests 2022, 13, x FOR PEER REVIEW 7 of 17
Therefore, the optimum mixture yielding to the highest germination of ash pollen
consists of 10% sucrose, 20 ppm boric acid, 100 ppm calcium nitrate and 1% agar, with
calcium nitrate being an optional component.
Figure 2. Results of the optimization of the procedure for testing ash pollen germination for different
concentrations of sucrose (a), boric acid (b), calcium nitrate (c) and agar (d). The standard deviation
is visualized with error bars.
Table 2. Results (p values) of the ANOVA post hoc paired t-test for comparing different concentra-
tions of sucrose, boric acid, and calcium nitrate concentrations in the optimization of ash pollen
germination.
0% 5% 10% 15%
5% <0.001 *
Sucrose 10%
15%
20%
<0.001 * <0.001 *
<0.001 * 0.084 0.005 *
<0.001 * 0.131 <0.001 * 0.008 *
0 ppm 20 ppm 100 ppm 200 ppm
20 ppm 0.008 *
Boric acid 100 ppm
200 ppm
1000 ppm
0.059 0.554
0.034 * 0.694 0.694
0.524 0.001 * 0.007 * 0.004 *
0 ppm 100 ppm 300 ppm 500 ppm
100 ppm 0.190
Calcium nitrate 300 ppm
500 ppm
1000 ppm
0.880 0.190
<0.001 * <0.001 * <0.001 *
<0.001 * <0.001 * <0.001 * 0.720
* = significant difference with p < 0.05.
3.3. Storage Experiments
The TTC tests showed that the viability of the pollen developed differently at the
three investigated temperatures 4 °C, −20 °C and −80 °C after one, two and three months
(Figure 3). In particular, the storage at 4 °C led to a more rapid decline in viability than at
−20 °C and −80 °C and after two months, no viable pollen could be detected. The temper-
atures below freezing were associated with a decrease of 11.5% (−20 °C) and 6.7% (−80 °C)
after three months.
Since the first test with pollen germination failed, the initial value is missing here.
For pollen germination, pollen stored at −20 °C showed the highest viability values after
one, two and three months. The viability of pollen stored at −80 °C was 11.9% lower. Pol-
len germination of pollen stored at 4 °C showed lower values in each month and no viable
pollen from the second month onwards. In contrast to the expected decrease in pollen
Figure 2.
Results of the optimization of the procedure for testing ash pollen germination for different
concentrations of sucrose (
a
), boric acid (
b
), calcium nitrate (
c
) and agar (
d
). The standard deviation
is visualized with error bars.

Forests 2022,13, 600 7 of 16
Table 2.
Results
(
pvalues) of the ANOVA post hoc paired t-test for comparing different concentrations
of sucrose, boric acid, and calcium nitrate concentrations in the optimization of ash pollen germination.
0% 5% 10% 15%
5% <0.001 *
Sucrose 10%
15%
20%
<0.001 * <0.001 *
<0.001 * 0.084 0.005 *
<0.001 * 0.131 <0.001 * 0.008 *
0 ppm 20 ppm 100 ppm 200 ppm
20 ppm 0.008 *
Boric acid 100 ppm
200 ppm
1000 ppm
0.059 0.554
0.034 * 0.694 0.694
0.524 0.001 * 0.007 * 0.004 *
0 ppm 100 ppm 300 ppm 500 ppm
100 ppm 0.190
Calcium nitrate 300 ppm
500 ppm
1000 ppm
0.880 0.190
<0.001 * <0.001 * <0.001 *
<0.001 * <0.001 * <0.001 * 0.720
* = significant difference with p< 0.05.
The optimal boric acid concentration was less clear. Even though the highest ger-
mination value was achieved at 20 ppm, the difference between 20 ppm, 100 ppm and
200 ppm was not statistically significant. Less suitable for pollen germination are 0 ppm
and 1000 ppm. These groups were significantly different from the groups 20 ppm and
200 ppm and exhibited the lowest mean germination (Figure 2, Table 2). For the following
tests, a concentration of 20 ppm was used.
For the calcium nitrate concentration, the highest germination value was observed at
100 ppm, however, this group was not significantly different from 0 ppm and 300 ppm. In
particular, 500 ppm and 1000 ppm led to a lower germination of pollen (Figure 2, Table 2).
For the following test, 100 ppm calcium nitrate were used.
Performing the two-tailed t-test on the two agar concentrations studied, no significant
difference between the germination of pollen at 1% and 2% agar (p= 0.913) could be shown
(Figure 2). For further tests 1% agar should be used.
Therefore, the optimum mixture yielding to the highest germination of ash pollen
consists of 10% sucrose, 20 ppm boric acid, 100 ppm calcium nitrate and 1% agar, with
calcium nitrate being an optional component.
3.3. Storage Experiments
The TTC tests showed that the viability of the pollen developed differently at the
three investigated temperatures 4
◦
C,
−
20
◦
C and
−
80
◦
C after one, two and three months
(Figure 3). In particular, the storage at 4
◦
C led to a more rapid decline in viability than
at
−
20
◦
C and
−
80
◦
C and after two months, no viable pollen could be detected. The
temperatures below freezing were associated with a decrease of 11.5% (
−
20
◦
C) and 6.7%
(−80 ◦C) after three months.
Since the first test with pollen germination failed, the initial value is missing here. For
pollen germination, pollen stored at
−
20
◦
C showed the highest viability values after one,
two and three months. The viability of pollen stored at
−
80
◦
C was 11.9% lower. Pollen
germination of pollen stored at 4
◦
C showed lower values in each month and no viable
pollen from the second month onwards. In contrast to the expected decrease in pollen
viability from the second to the third month, a slight increase ranging between 7% (
−
80
◦
C)
and 12% (
−
20
◦
C) was recorded for all freezing temperatures (Figure 3). The documented
increase can possibly be attributed to a higher room temperature and therefore a higher
germination temperature during the incubation period (April vs. June).

Forests 2022,13, 600 8 of 16
Forests 2022, 13, x FOR PEER REVIEW 8 of 17
viability from the second to the third month, a slight increase ranging between 7% (−80
°C) and 12% (−20 °C) was recorded for all freezing temperatures (Figure 3). The docu-
mented increase can possibly be attributed to a higher room temperature and therefore a
higher germination temperature during the incubation period (April vs. June).
Figure 3. Temporal development of ash pollen viability [%] after one, two, and three months at
storage temperatures of 4 °C, −20 °C and −80 °C evaluated with TTC test and pollen germination.
The standard deviation is visualized with error bars.
Although pollen viability derived from the TTC test and from pollen germination
differed, both tests showed changes in viability over the three months studied. The Alex-
ander’s stain and Acetocarmine test, however, did not present any change in the viability
of the pollen. Even though not all values were available, it still was evident, that there
were neither remarkable differences between the different storage temperatures nor dif-
ferences between the individual months. The proportion of viable pollen in both tests was
between 95% and 97% and thus higher than in the TTC test and pollen germination.
Within the control test using heat-killed pollen, the TTC test and the pollen germination
showed no staining or germination and thus proved their reliability. The Alexander’s
stain and Acetocarmine test on the other hand, yielded values between 95% and 97% of
viable pollen in the staining of the killed pollen. These values are comparable to those
from the storage experiments.
3.4. Atmospheric Transport
3.4.1. Simulations in Climate Chamber
The viability of the pollen exposed to the settings of climate chamber experiment 1
(10/5 °C, 65/80% relative humidity) was increased by 4% after the third day; then there
was a decrease in viable pollen (−15%) after seven days. After 21 days, about half of the
pollen were still viable, compared to the initial value. Even after 28 days, viable pollen
(8%) could still be detected.
Experiment 2 differed by only +2 °C from the settings of experiment 1 (12/7 °C 65/80%
relative humidity). An increase in the viability of ash pollen could be observed until the
third day; after seven days the viability of the pollen decreased by 9%. On day 14, the
viability of the pollen was only 10% below the documented initial value, and on day 21, it was
only less than half of the initial value. After 28 days, 15% viable pollen could be detected.
The results of experiment 3 (20/12 °C, 60/80%) also show an increase in the viability
until day three. At day seven, a rapid drop in the viability of the pollen was documented
(−80%). After 14 days, no more viable pollen were observed under the microscope.
The comparison of experiments shows, that experiment 3 in particular differs from
the two previous experiments (Figure 4). While viable pollen were still documented after
Figure 3.
Temporal development of ash pollen viability [%] after one, two, and three months at
storage temperatures of 4
◦
C,
−
20
◦
C and
−
80
◦
C evaluated with TTC test and pollen germination.
The standard deviation is visualized with error bars.
Although pollen viability derived from the TTC test and from pollen germination
differed, both tests showed changes in viability over the three months studied. The Alexan-
der’s stain and Acetocarmine test, however, did not present any change in the viability
of the pollen. Even though not all values were available, it still was evident, that there
were neither remarkable differences between the different storage temperatures nor dif-
ferences between the individual months. The proportion of viable pollen in both tests
was between 95% and 97% and thus higher than in the TTC test and pollen germination.
Within the control test using heat-killed pollen, the TTC test and the pollen germination
showed no staining or germination and thus proved their reliability. The Alexander’s stain
and Acetocarmine test on the other hand, yielded values between 95% and 97% of viable
pollen in the staining of the killed pollen. These values are comparable to those from the
storage experiments.
3.4. Atmospheric Transport
3.4.1. Simulations in Climate Chamber
The viability of the pollen exposed to the settings of climate chamber experiment 1
(10/5
◦
C, 65/80% relative humidity) was increased by 4% after the third day; then there
was a decrease in viable pollen (
−
15%) after seven days. After 21 days, about half of the
pollen were still viable, compared to the initial value. Even after 28 days, viable pollen (8%)
could still be detected.
Experiment 2 differed by only +2
◦
C from the settings of experiment 1 (12/7
◦
C 65/80%
relative humidity). An increase in the viability of ash pollen could be observed until the
third day; after seven days the viability of the pollen decreased by 9%. On day 14, the
viability of the pollen was only 10% below the documented initial value, and on day 21,
it was only less than half of the initial value. After 28 days, 15% viable pollen could
be detected.
The results of experiment 3 (20/12
◦
C, 60/80%) also show an increase in the viability
until day three. At day seven, a rapid drop in the viability of the pollen was documented
(−80%). After 14 days, no more viable pollen were observed under the microscope.
The comparison of experiments shows, that experiment 3 in particular differs from
the two previous experiments (Figure 4). While viable pollen were still documented after
28 days in both experiment 1 and 2, no viable pollen were present in experiment 3 after
only two weeks.

Forests 2022,13, 600 9 of 16
Forests 2022, 13, x FOR PEER REVIEW 9 of 17
28 days in both experiment 1 and 2, no viable pollen were present in experiment 3 after
only two weeks.
In all experiments, an increase in viability was observed up to day three. The initial
viability value was determined using the TTC test, immediately after the pollen were re-
moved from the blast freezer (−80 °C). These higher viability values can probably be ex-
plained by an increase in thawed pollen. This assumption is supported by the fact that in
preliminary experiments, in which fresh pollen were used, no initial increase in viability
was observed. However, it is also possible that the observed increases are due to counting
inaccuracies, as the difference was only small (+4% to +8%).
Figure 4. Temporal development of ash pollen viability [%] in the climate chamber for experiments
1 (10/5 °C, 65/80% relative humidity), 2 (12/7 °C 65/80% relative humidity) and 3 (20/12 °C, 60/80%
relative humidity) within a total of 28 days. The standard deviation is visualized with error bars.
3.4.2. Samples from Atmosphere
Within the pollen trap experiments, different viability values could be observed for
the four investigated weeks. In week one, high viability values between 75% and 97%
were recorded on all seven days (Figure 5). Lower viability values (17%) were recorded
on day one in week two. That means that pollen from this day were captured in the inte-
rior of the pollen trap for at least seven days. On day seven, 62% viable pollen were sam-
pled. That means that sampled pollen on this day were one day old or older in the case of
long-range transported pollen. The mean pollen concentration was the highest of all four
investigated weeks in week two and the aspired sample size of 100 ash pollen was
achieved on all seven days. Viability values ranged between 17% and 90%. Week three is
characterized by a low pollen concentration in the first half of the week and high viability
values from day three onwards. In week four, only on one day 100 ash pollen could be
counted. Since the number of counted ash pollen was low in week four, the calculated
viability values are limited in their reliability. Overall, the viability of the pollen varied
greatly between 0% and 97% over the course of the four weeks investigated (Figure 5).
However, it can be seen than especially in week two (and week three), viability values were
lower in the first days of sampling, i.e., for pollen that have a minimum age of seven days.
Meterological conditions show lower air temperatures, especially in week one, and
higher temperatures at the end of week four (Figure 5).
Figure 4.
Temporal development of ash pollen viability [%] in the climate chamber for experiments 1
(10/5
◦
C, 65/80% relative humidity), 2 (12/7
◦
C 65/80% relative humidity) and 3 (20/12
◦
C, 60/80%
relative humidity) within a total of 28 days. The standard deviation is visualized with error bars.
In all experiments, an increase in viability was observed up to day three. The initial
viability value was determined using the TTC test, immediately after the pollen were
removed from the blast freezer (
−
80
◦
C). These higher viability values can probably be
explained by an increase in thawed pollen. This assumption is supported by the fact that in
preliminary experiments, in which fresh pollen were used, no initial increase in viability
was observed. However, it is also possible that the observed increases are due to counting
inaccuracies, as the difference was only small (+4% to +8%).
3.4.2. Samples from Atmosphere
Within the pollen trap experiments, different viability values could be observed for
the four investigated weeks. In week one, high viability values between 75% and 97% were
recorded on all seven days (Figure 5). Lower viability values (17%) were recorded on day
one in week two. That means that pollen from this day were captured in the interior of the
pollen trap for at least seven days. On day seven, 62% viable pollen were sampled. That
means that sampled pollen on this day were one day old or older in the case of long-range
transported pollen. The mean pollen concentration was the highest of all four investigated
weeks in week two and the aspired sample size of 100 ash pollen was achieved on all seven
days. Viability values ranged between 17% and 90%. Week three is characterized by a low
pollen concentration in the first half of the week and high viability values from day three
onwards. In week four, only on one day 100 ash pollen could be counted. Since the number
of counted ash pollen was low in week four, the calculated viability values are limited in
their reliability. Overall, the viability of the pollen varied greatly between 0% and 97% over
the course of the four weeks investigated (Figure 5). However, it can be seen than especially
in week two (and week three), viability values were lower in the first days of sampling, i.e.,
for pollen that have a minimum age of seven days.
Meterological conditions show lower air temperatures, especially in week one, and
higher temperatures at the end of week four (Figure 5).

Forests 2022,13, 600 10 of 16
Forests 2022, 13, x FOR PEER REVIEW 10 of 17
Figure 5. Viability of ash pollen [%] captured with a volumetric pollen trap (blue bars) in Ingolstadt,
Germany in 2021, the respective pollen concentration [pollen grains/m³] (yellow line) and the ambi-
ent air temperature [°C] (red line) for week one (15.04–21.04) (a), two (22.04–28.04) (b), three (29.04–
05.05. (c) and four (06.05–11.05) (d). Light blue bars indicate that the total pollen catch was <100
pollen grains (total numbers are shown below theses bars), dark blue bars indicate that 100 pollen
grains were counted and assessed for their viability.
4. Discussion
4.1. Suitability of Viability Tests for Ash Pollen
Knowledge on pollen viability is important with respect to increasingly fragmented
ash populations and therefore an adequate assessment is crucial. The first test, the TTC
test resulted in a successful staining of the pollen with a recognizable color differentiation.
In this study, the validation using a control group of killed pollen [38] showed that no
staining appeared. For ash pollen, the TTC test was already evaluated as a suitable test for
determining their viability [34].
The Alexander’s stain resulted in a clear coloring of the ash pollen in pink and green.
While we are not aware of any study related to the pollen of Fraxinus excelsior, the Alex-
ander’s stain is frequently used for other pollen species. In particular, the clearly separated
coloring of pollen is considered an advantage [42]. Good results with the Alexander’s stain
have been obtained, among others, in the assessment of the viability of pollen of Polygala
paniculata L. [43], Crotalaria juncea L. [44], Centaurium Hill and Gentianella Moench [45]. The
easy application of the stain, and the rapid results in particular, were rated as positive
[46], which can be confirmed by our study using ash pollen.
The test is based on the assumption that the presence of pink stained cytoplasm sim-
ultaneously signals the viability of the pollen. While pollen containing no cytoplasm is
certainly not viable, conversely, it does not necessarily mean that all pollen containing
cytoplasm are viable. Therefore, pollen viability can easily be overestimated [47,48]. The
results obtained in this study, which was based on adapted staining agents [46,49], also
call this issue into question: Regardless of the duration of pollen storage and the respective
temperature, we found that the average viability ranged between 95% and 97% and was
considerably higher than that determined by the TTC test. In the control test with killed
pollen, these pollen were stained just like viable pollen, which further confirms the un-
suitability of Alexander’s stain for ash pollen. The application of Alexander’s stain for the
determination of the development of the viability of Nothofagus Blume pollen under de-
fined storage conditions also proved to be not successful [50]. This was also observed in
Asparagus officinalis L. pollen after storage at 4 °C, while other viability tests resulted in a
Figure 5.
Viability of ash pollen [%] captured with a volumetric pollen trap (blue bars) in Ingolstadt,
Germany in 2021, the respective pollen concentration [pollen grains/m
3
] (yellow line) and the
ambient air temperature [
◦
C] (red line) for week one (15.04–21.04) (
a
), two (22.04–28.04) (
b
), three
(29.04–05.05) (
c
) and four (06.05–11.05) (
d
). Light blue bars indicate that the total pollen catch was
<100 pollen grains (total numbers are shown below theses bars), dark blue bars indicate that 100 pollen
grains were counted and assessed for their viability.
4. Discussion
4.1. Suitability of Viability Tests for Ash Pollen
Knowledge on pollen viability is important with respect to increasingly fragmented
ash populations and therefore an adequate assessment is crucial. The first test, the TTC
test resulted in a successful staining of the pollen with a recognizable color differentiation.
In this study, the validation using a control group of killed pollen [
38
] showed that no
staining appeared. For ash pollen, the TTC test was already evaluated as a suitable test for
determining their viability [34].
The Alexander’s stain resulted in a clear coloring of the ash pollen in pink and
green. While we are not aware of any study related to the pollen of Fraxinus excelsior, the
Alexander’s stain is frequently used for other pollen species. In particular, the clearly
separated coloring of pollen is considered an advantage [
42
]. Good results with the
Alexander’s stain have been obtained, among others, in the assessment of the viability of
pollen of Polygala paniculata L. [
43
], Crotalaria juncea L. [
44
], Centaurium Hill and Gentianella
Moench [
45
]. The easy application of the stain, and the rapid results in particular, were
rated as positive [46], which can be confirmed by our study using ash pollen.
The test is based on the assumption that the presence of pink stained cytoplasm si-
multaneously signals the viability of the pollen. While pollen containing no cytoplasm
is certainly not viable, conversely, it does not necessarily mean that all pollen containing
cytoplasm are viable. Therefore, pollen viability can easily be overestimated [
47
,
48
]. The
results obtained in this study, which was based on adapted staining agents [
46
,
49
], also
call this issue into question: Regardless of the duration of pollen storage and the respec-
tive temperature, we found that the average viability ranged between 95% and 97% and
was considerably higher than that determined by the TTC test. In the control test with
killed pollen, these pollen were stained just like viable pollen, which further confirms the
unsuitability of Alexander’s stain for ash pollen. The application of Alexander’s stain for
the determination of the development of the viability of Nothofagus Blume pollen under
defined storage conditions also proved to be not successful [
50
]. This was also observed in
Asparagus officinalis L. pollen after storage at 4
◦
C, while other viability tests resulted in a

Forests 2022,13, 600 11 of 16
change in viability. Storage conditions probably modify structures and processes within
pollen, which affect the viability of these, however, this cannot be made visible using
Alexander’s stain [51].
Alexander’s stain is suitable for studying the absence or presence of cytoplasm, and
thus, the integrity of pollen or the proportion of sterile pollen [
52
]. For the ash trees, whose
pollen were examined in this study, the percentage of sterile pollen ranged between 3%
and 5%.
The staining of ash pollen with Acetocarmine provided a less clear staining than the
TTC test or the Alexander’s stain. Nevertheless, a differentiation between slightly reddish
colored pollen and colorless pollen was possible. Good results were also obtained for pollen
of Colocasia esculenta L. [
53
], Lilium Oriental hybrids [
33
] and Ficus carica L. [
29
]. In the case
of Castanea mollissima Blume and Castanea henryi (Skan) Rehder and E. H. Wilson, it was
not possible to distinguish between viable and non-viable pollen [
32
]. For Olea europaea
L. pollen, a reliable assessment of pollen viability could also not be achieved, as shown
by a killed control [
54
]. In this study, the control test showed that killed pollen were
colored like viable pollen. Thus, the determination of the development of viability of ash
pollen with Acetocarmine is not suitable. These results are consistent with the findings
of
Castiñeiras et al.
[
34
], who reported that the staining of Fraxinus excelsior pollen with
Acetocarmine provided high viability values close to 100%, as obtained in our study.
The composition of the nutrient medium used for pollen germination and the method
of pollen germination varies greatly depending on the type of pollen examined. In general,
both a liquid, as well as a solid culture medium, can be suitable. The nutrient medium of
Brewbaker and Kwack [
37
] is often used, e.g., for determining the viability of Swainsona
formosa (G.Don) Joy Thomps pollen [
55
], for Acacia podalyriifolia A.Cunn. ex G.Don and
Acacia mearnsii De Wild pollen [
56
] or for Elaeis guineensis Jacq. pollen [
57
], however,
individual optimizations were also frequently applied [58–60].
For the storage experiments, we used a solid nutrient medium with sucrose, boric
acid and agar, and pollen germination was carried out at room temperature in the dark.
Germination at room temperature has already been successfully performed for numerous
pollen species, including pollen of various legumes (Fabaceae) [
61
] or pollen of Swain-
sona formosa [
55
]. A successful germination at room temperature was also achieved for
pollen of Fraxinus lanuginosa Koidz [
62
]. In our study, we found that germination at room
temperature was unsuitable. Although germination of the pollen was achieved and a
difference between the different storage temperatures was observed, a comparison between
the viability of pollen for different storage durations was impossible.
For this reason, a uniform temperature of 25
◦
C was selected for the subsequent
optimization of the culture medium. According to Kremer and Jemri´c [
63
], the optimal
temperature for germination of Fraxinus pennsylvanica Marshall pollen is between 20
◦
C
and 25
◦
C, with an optimum closer to 25
◦
C. Castiñeiras et al. [
34
] also used a temperature
of 25 ◦C for Fraxinus excelsior pollen germination.
According to our results, the optimum sucrose concentration for Fraxinus excelsior
pollen is 10%. For Fraxinus lanuginosa pollen, satisfactory germination was also achieved at
5% and 10% [
62
]. The highest germination values in the investigation of boric acid were
found at 20 ppm, those of calcium nitrate at 100 ppm. Due to high viability values related
to other concentrations, we conclude that a range of 20 ppm to 200 ppm for boric acid and a
range of 0 ppm to 300 ppm for calcium nitrate is suitable for ash pollen analyses. Boric acid
in particular represents an important component of the nutrient medium [
25
] and promotes
the germination of Fraxinus excelsior pollen. The enhancement of pollen germination by
the addition of boric acid has already been shown in other pollen species including Litchi
chinensis Sonn [
64
] or Cunninghamia lanceolata (Lamb.) Hook [
65
]. The concentration of agar,
however, did not reveal any remarkable differences between 1% and 2%.
Problems related to the performance of ash pollen germination were reported by
Castiñeiras et al. [
34
]. Therefore, the optimizing tests of a solid nutrient medium performed
in our study can be very helpful for future research on ash pollen.

Forests 2022,13, 600 12 of 16
Nevertheless, the evaluation of germinated pollen is associated with difficulties, as a
strong germination with long, tangled pollen tubes can hinder the counting and thus the
evaluation of the experiment. In addition, pollen germination is highly dependent on the
environmental conditions of the experiment.
4.2. Effects of Storage Conditions on Ash Pollen
Breeding programs may benefit for the availability of viable ash pollen and therefore
it is important to evaluate the optimal storage of pollen. The results of the Alexander’s
stain and the Acetocarmine test were not reliable in regards to the development of ash
pollen viability under different storage conditions, the TTC test, however, provided good
results. It was shown that a storage temperature of
−
20
◦
C and
−
80
◦
C is superior for
preserving viability compared to 4
◦
C. Despite the limited evaluability of the results of
pollen germination, pollen viability was also found to be least at 4 ◦C.
These results are consistent with studies on the viability of other pollen species under
certain storage conditions. Pollen of strawberry have been shown to maintain their viability
for 20 months at a storage temperature of
−
18
◦
C, and for only eight months at 4
◦
C [
66
].
Phoenix Dactylifera L. pollen also showed higher viability values when stored at
−
20
◦
C
compared to 4
◦
C [
67
]. Pollen viability of date palm was maintained for up to one year
at
−
20
◦
C [
68
]. A higher viability was also observed in Herbaceous peonies pollen after
storage at
−
76
◦
C compared to 4
◦
C and
−
20
◦
C [
69
]. In contrast, other studies did not
detect remarkable differences in pollen viability between storage at
−
20
◦
C and
−
80
◦
C
(e.g., for almond pollen [
70
] or for Leonurus cardiaca L. pollen [
71
]). In these cases, it can be
concluded that a storage at
−
20
◦
C is less expensive and therefore preferable. The observed
slight decrease in germination of the frozen pollen in our study can probably be attributed
to possible damage to the pollen due to the formation of intracellular ice during the freezing
process, as also observed by Shekari et al. [71].
Our study covers only a storage duration of three months. Raquin et al. [
72
] found no
decrease in viablility of pollen from Fraxinus excelsior and Fraxinus angustifolia when stored
at
−
70
◦
C for up to eleven months. Therefore, storage of frozen and viable ash pollen for
longer than three months seems to be possible. This offers the opportunity to preserve and
use ash pollen a long time after sampling for breeding programs. In the light of ash dieback,
pollen of potentially resistant trees can successfully be stored and bear the potential for
future breeding of trees that are able to cope with the disease.
4.3. Potential Effects of Long-Range Transport of Ash Pollen
We assume that long-range pollen dispersal affects pollen viability and we there-
fore evaluated the effects of meteorology on the quality of pollen in climate chamber
experiments. We observed that pollen, which were distributed unevenly in the petri dish,
probably led to shadow effects in the climate chamber experiments. That means that pollen
that were located in the middle of a larger accumulation can be protected from the effects
of UV radiation. In the first experiment, we were not aware of these shadow effects and
from experiment two onwards, the pollen were then distributed in a very thin layer. How-
ever, due to the very small size of the individual pollen grains, shadow effects caused by
surrounding pollen cannot be ruled out even in experiments two and three. Increased or
prolonged UV radiation leading to a rapid reduction in pollen viability was demonstrated
by studying maize [
73
,
74
], oak [
75
] and pine pollen [
76
]. To what extent, and in particular
how quickly, the viability of the pollen examined is reduced differs greatly depending
on the type of pollen. When interpreting our results, it has to be considered, that our
experiments were designed to assume only cloudless days with a sunshine duration of 13 h.
This is usually not the case for such a long period in the region (Emmendingen, Germany)
from which we sampled the pollen. Under cloudy conditions, pollen viability of Festuca
arundinacea Schreb. decreased more slowly [
60
]. For pollen of Panicum virgatum L., viability
decreased five times faster under sunny compared to cloudy weather conditions [77].

Forests 2022,13, 600 13 of 16
The storage experiments have already shown that pollen viability can be maintained
longer at lower temperatures. This was also confirmed by the climate chamber experiments.
While the climate chamber experiments 1 and 2 with temperatures of 5/10
◦
C and 7/12
◦
C
were associated with viable pollen that could still be observed after 28 days, in experiment
3 (12/20
◦
C) no viable pollen were present after only seven days. Experiments 1 and 2 differ
from each other in the course of the development of viability, however, no clear conclusions
can be drawn as to which of the two temperature settings ensures a longer maintenance
of viability. Therefore, small changes in mean temperature were not associated with large
effects on viability. However, long lasting heat extremes can have serious consequences for
pollen viability.
The extent, and in particular, the time for which ash pollen maintains its viability after
release thus depends on temperature (extremes) and the prevailing UV radiation.
The impact of natural environmental conditions on pollen viability was investigated
in more detail by the volumetric pollen trap experiments. In particular, we determined
whether a difference in viability could be detected between the days one to seven. Pollen
sampled on day one may have been exposed to the prevailing temperatures and other
meteorological conditions for six days longer. Since the foil band, on which the pollen were
collected, was inside the pollen trap, the pollen already picked up were then protected
from the influence of UV radiation. Meteorological conditions show lower temperatures at
the beginning of the study period, however, a correlation with the viability values cannot
be proven with the existing data.
In week two and three, lower viability values were documented for the first samplings
days, which shows that pollen captured for a longer period may be negatively affected.
With viable pollen ranging from 0% to 97%, viability of ash pollen differed greatly within
the studied period. These data may present a rough proxy on the fraction of pollen, which
was traveling a longer time before impaction.
A possible impact of the silicone adhered to the foil strip is unlikely, since we found a
high viability of pollen on certain days.
5. Conclusions
We found that the duration of long-distance transport, and the environmental condi-
tions prevailing at that time, play an important role in the successful pollination of female
ash flowers by (long-range) transported pollen. In particular, high temperatures and sunny
weather can have a negative effect on the pollination success since these meteorological
factors reduce pollen viability.
Our methodological comparison showed that a successful pollen germination could
be achieved using an optimized solid nutrient medium. Both Alexander’s stain and
Acetocarmine did not indicate the viability of the pollen correctly. However, the TTC test
has proven to be suitable for determining viability, both in the storage experiments and
in the long-distance transport investigations. The storage experiments have shown that
storage of pollen at −20 ◦C or −80 ◦C is to be preferred to a storage temperature of 4 ◦C.
Overall, the viability of Fraxinus excelsior pollen in respect to ash dieback is shown to
be of crucial importance. For future breeding programs using ash pollen, suitable methods
to determine pollen viability are necessary, as well as an adequate storage preserving
viability of the pollen. Natural reproduction of ash trees within and between prospectively
fragmented ash populations is strongly dependent on pollen viability, and therefore, on the
natural conditions during pollen dispersal. Future studies should focus on the influence
of those natural conditions, especially UV radiation, however, they should also consider
pollen production, which is potentially modified due to ash dieback.
Author Contributions:
Conceptualization, S.J.-O.; methodology, L.B., B.Š. and A.-K.E.; software, L.B.;
formal analysis, L.B.; investigation, L.B.; writing—original draft preparation, L.B.; writing—review
and editing, A.-K.E., B.Š. and S.J.-O.; visualization, L.B.; supervision, S.J.-O.; project administration,
S.J.-O.; funding acquisition, S.J.-O. All authors have read and agreed to the published version of
the manuscript.

Forests 2022,13, 600 14 of 16
Funding:
This research was funded by the Bavarian State Ministry of Food, Agriculture and Forestry
through the Bavarian State Institute for Forests and Forestry (LWF) as part of the project “P035—Quo
vadis Pollen? Untersuchungen zur (effektiven) Pollenausbreitung und Pollen- und Samenqualität als
Beitrag zur Generhaltung bei der Esche. The open access publication of this article was supported by
the Open Access Fund of the Catholic University Eichstaett-Ingolstadt.
Acknowledgments:
We thank Georgia Kahlenberg, Johanna Jetschni and Celina Riedl for tech-
nical assistance. In addition, we thank Forst Baden-Württemberg and Forstliche Versuchs- und
Forschungsanstalt Baden-Württemberg for providing the seed plantation as study site.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Scherrer, D.; Bader, M.K.-F.; Körner, C. Drought-sensitivity ranking of deciduous tree species based on thermal imaging of forest
canopies. Agric. For. Meteorol. 2011,151, 1632–1640. [CrossRef]
2.
Kölling, C.; Zimmermann, L. Die Anfälligkeit der Wälder Deutschlands gegenüber dem Klimawandel. Gefahrenst. Reinh. Luft
2007,67, 259–268.
3. Offenberger, M. Aktuelles zur Entwicklung des Eschentriebsterbens. Anl. Nat. 2017,39, 22–27.
4.
Stener, L.-G. Clonal differences in susceptibility to the dieback of Fraxinus excelsior in southern Sweden. Scand. J. For. Res.
2013
,28,
205–216. [CrossRef]
5.
McKinney, L.V.; Nielsen, L.R.; Hansen, J.K.; Kjær, E.D. Presence of natural genetic resistance in Fraxinus excelsior (Oleraceae) to
Chalara fraxinea (Ascomycota): An emerging infectious disease. Heredity 2011,106, 788–797. [CrossRef]
6.
Bakys, R.; Vasaitis, R.; Barklund, P.; Ihrmark, K.; Stenlid, J. Investigations concerning the role of Chalara fraxinea in declining
Fraxinus excelsior.Plant Pathol. 2009,58, 284–292. [CrossRef]
7.
Kirisits, T. Ash dieback caused by Hymenoscyphus pseudoalbidus in a seed plantation of Fraxinus excelsior in Austria. J. Agric. Ext.
Rural Dev. 2012,4, 184–191. [CrossRef]
8.
McKinney, L.V.; Thomsen, I.M.; Kjaer, E.D.; Nielsen, L.R. Genetic resistance to Hymenoscyphus pseudoalbidus limits fungal growth
and symptom occurrence in Fraxinus excelsior.For. Pathol. 2012,42, 69–74. [CrossRef]
9. Pliura, A.; Lygis, V.; Suchockas, V.; Bartkevicius, E. Performance of twenty four european Fraxinus excelsior populations in three
lithuanian progeny trials with a special emphasis on resistance to Chalara fraxinea.Balt For. 2011,17, 17–34.
10.
Enderle, R.; Nakou, A.; Thomas, K.; Metzler, B. Susceptibility of autochthonous German Fraxinus excelsior clones to Hymenoscy-
phus pseudoalbidus is genetically determined. Ann. For. Sci. 2015,72, 183–193. [CrossRef]
11. Enderle, R. An overview of ash (Fraxinus spp.) and the ash dieback disease in Europe. CAB Rev. 2019,14, 1–12. [CrossRef]
12.
Wohlmuth, A.; Essl, F.; Heinze, B. Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings in Austria
to ash dieback. For. Int. J. For. Res. 2018,91, 514–525. [CrossRef]
13.
McKinney, L.V.; Nielsen, L.R.; Collinge, D.B.; Thomsen, I.M.; Hansen, J.K.; Kjaer, E.D. The ash dieback crisis: Genetic variation in
resistance can prove a long-term solution. Plant Pathol. 2014,63, 485–499. [CrossRef]
14.
Kjær, E.D.; McKinney, L.V.; Nielsen, L.R.; Hansen, L.N.; Hansen, J.K. Adaptive potential of ash (Fraxinus excelsior) populations
against the novel emerging pathogen Hymenoscyphus pseudoalbidus.Evol. Appl. 2012,5, 219–228. [CrossRef] [PubMed]
15.
Enderle, R.; Fussi, B.; Lenz, H.D.; Langer, G.; Nagel, R.; Metzler, B. Ash dieback in Germany: Research on disease development,
resistance and management options. In Dieback of European Ash (Fraxinus spp.): Consequences and Guidelines for Sustainable
Management; Vasaitis, R., Enderle, R., Eds.; European Cooperation in Science & Technology (COST): Uppsala, Sweden, 2017;
ISBN 978-91-576-8696-1.
16.
Coker, T.L.R.; Rozsypálek, J.; Edwards, A.; Harwood, T.P.; Butfoy, L.; Buggs, R.J.A. Estimating mortality rates of European ash
(Fraxinus excelsior) under the ash dieback (Hymenoscyphus fraxineus) epidemic. Plants People Planet 2019,1, 48–58. [CrossRef]
17.
Semizer-Cuming, D.; Kjær, E.D.; Finkeldey, R. Gene flow of common ash (Fraxinus excelsior L.) in a fragmented landscape. PLoS
ONE 2017,12, e0186757. [CrossRef]
18.
Bacles, C.F.E.; Ennos, R.A. Paternity analysis of pollen-mediated gene flow for Fraxinus excelsior L. in a chronically fragmented
landscape. Heredity 2008,101, 368–380. [CrossRef]
19.
Bacles, C.F.E.; Burczyk, J.; Lowe, A.J.; Ennos, R.A. Historical and contemporary mating patterns in remnant populations of the
forests tree Fraxinus excelsior L. Evolution 2005,59, 979. [CrossRef]
20.
Šikoparija, B.; Smith, M.; Skjøth, C.A.; Radisi´c, P.; Milkovska, S.; Simi´c, S.; Brandt, J. The Pannonian plain as a source of Ambrosia
pollen in the Balkans. Int. J. Biometeorol. 2009,53, 263–272. [CrossRef]
21.
Hernandez-Ceballos, M.A.; Soares, J.; García-Mozo, H.; Sofiev, M.; Bolivar, J.P.; Galán, C. Analysis of atmospheric dispersion of
olive pollen in southern Spain using SILAM and HYSPLIT models. Aerobiologia 2014,30, 239–255. [CrossRef]
22.
De Weger, L.A.; Pashley, C.H.; Šikoparija, B.; Skjøth, C.A.; Kasprzyk, I.; Grewling, Ł.; Thibaudon, M.; Magyar, D.; Smith, M.
The long distance transport of airborne Ambrosia pollen to the UK and the Netherlands from Central and south Europe. Int. J.
Biometeorol. 2016,60, 1829–1839. [CrossRef] [PubMed]

Forests 2022,13, 600 15 of 16
23.
Myszkowska, D.; Piotrowicz, K.; Ziemianin, M.; Bastl, M.; Berger, U.; Dahl, Å.; D ˛abrowska-Zapart, K.; Górecki, A.;
Lafférsová, J.
;
Majkowska-Wojciechowska, B.; et al. Unusually high birch (Betula spp.) pollen concentrations in Poland in 2016 related to
long-range transport (LRT) and the regional pollen occurrence. Aerobiologia 2021,37, 543–559. [CrossRef]
24.
Šikoparija, B.; Skjøth, C.A.; Alm Kübler, K.; Dahl, A.; Sommer, J.; Grewling, Ł.; Radiši´c, P.; Smith, M. A mechanism for long
distance transport of Ambrosia pollen from the Pannonian Plain. Agric. For. Meteorol. 2013,180, 112–117. [CrossRef]
25.
Kearns, C.A.; Inouye, D.W. Techniques for Pollination Biologists; University Press of Colorado: Niwot, CO, USA, 1993;
ISBN 9780870812811.
26.
Shivanna, K.R.; Rangaswamy, N.S. Pollen Biology: A Laboratory Manual; Springer: Berlin/Heidelberg, Germany, 1992;
ISBN 3-540-55170-0.
27.
Capitani, L.C.; Rovedder, A.P.M.; Da Silva, J.C.C., Jr.; Peccatti, A. Pollen Viability and Autogamy Fitness in Bauhinia forficata Link
(Fabaceae). Floresta Ambient. 2018,25, 1–8. [CrossRef]
28.
Sulusoglu, M.; Cavusoglu, A.
In vitro
pollen viability and pollen germination in cherry laurel (Prunus laurocerasus L.). Sci. World J.
2014,2014, 657123. [CrossRef]
29.
Gaaliche, B.; Majdoub, A.; Trad, M.; Mars, M. Assessment of pollen viability, germination, and tube growth in eight tunisian
caprifig (Ficus carica L.) cultivars. ISRN Agron. 2013,2013, 1–4. [CrossRef]
30.
Abdelgadir, H.A.; Johnson, S.D.; van Staden, J. Pollen viability, pollen germination and pollen tube growth in the biofuel seed
crop Jatropha curcas (Euphorbiaceae). S. Afr. J. Bot. 2012,79, 132–139. [CrossRef]
31.
Kelen, M.; Demirtas, I. Pollen viability, germination capability and pollen production level of some grape varieties (Vitis vinifera L.).
Acta Physiol. Plant 2003,25, 229–233. [CrossRef]
32.
Luo, S.; Zhang, K.; Zhong, W.-P.; Chen, P.; Fan, X.-M.; Yuan, D.-Y. Optimization of
in vitro
pollen germination and pollen viability
tests for Castanea mollissima and Castanea henryi.Sci. Hortic. 2020,271, 109481. [CrossRef]
33.
Du, X.; Zhu, X.; Yang, Y.; Zhao, F.; Liu, H. Pollen ultra-morphology and pollen viability test of Lilium Oriental hybrids. Int. J.
Agric. Biol. 2018,20, 1903–1907. [CrossRef]
34.
Castiñeiras, P.; Vázquez-Ruiz, R.A.; Fernández-González, M.; Rodríguez-Rajo, F.J.; Aira, M.J. Production and viability of Fraxinus
pollen and its relationship with aerobiological data in the northwestern Iberian Peninsula. Aerobiologia
2019
,35, 227–241.
[CrossRef]
35. Alexander, M.P. Differential staining of aborted and nonaborted pollen. Stain Technol. 1969,44, 117–122. [CrossRef] [PubMed]
36.
Peterson, R.; Slovin, J.P.; Chen, C. A simplified method for differential staining of aborted and non-aborted pollen grains. Int. J.
Plant Biol. 2010,1, 13. [CrossRef]
37.
Brewbaker, J.; Kwack, B. The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot.
1963
,50,
859–865. [CrossRef]
38. Rodriguez-Riano, T.; Dafni, A. A new procedure to asses pollen viability. Sex. Plant Reprod. 2000,12, 241–244. [CrossRef]
39.
Masson-Delmotte, V.; Zhai, P.; Pirani, A.; Connors, S.L.; Péan, C.; Chen, Y.; Goldfarb, L.; Gomis, M.I.; Matthews, J.B.R.;
Berger, S.; et al.
(Eds.) Summary for Policymakers. In Climate Change 2021: The Physical Science Basis. Contribution of Working
Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change; Intergovernmental Panel on Climate Change;
Cambridge University Press: Cambridge, MA, USA, 2021.
40. Hirst, J.M. An automatic volumetric spore trap. Ann. Appl. Biol. 1952,39, 257–265. [CrossRef]
41.
Galán, C.; Smith, M.; Thibaudon, M.; Frenguelli, G.; Oteros, J.; Gehrig, R.; Berger, U.; Clot, B.; Brandao, R. Pollen monitoring:
Minimum requirements and reproducibility of analysis. Aerobiologia 2014,30, 385–395. [CrossRef]
42.
De Souza, M.M.; Pereira, T.N.; Viana, A.P.; Pereira, M.G.; Bernacci, L.C.; Sudré, C.P.; Silva, L.d.C. Meiotic irregularities and pollen
viability in Passiflora edmundoi Sacco (Passifloraceae). Caryologia 2003,56, 161–169. [CrossRef]
43.
Frescura, V.; Laughinghouse, H.; Cantodorow, T.; Tedesco, S. Pollen viability of Polygala paniculata L. (Polygalaceae) using different
staining methods. Biocell 2012,36, 143–145. [CrossRef]
44.
Coelho, A.; Morais, K.; Laughinghouse, H.; Giacomini, S.; Tedesco, S. Pollen grain viability in accessions of Crotalaria juncea L.
(Fabaceae). Agrociencia 2012,46, 481–487.
45.
West, W.; Baldwin, S.; Rich, T.C.G. Pollen viability and size in British Centaurium Hill and Gentianella Moench (Gentianaceae)
taxa. Grana 2014,53, 111–116. [CrossRef]
46.
Atlagi´c, J.; Terzi´c, S.; Marjanovi´c-Jeromela, A. Staining and fluorescent microscopy methods for pollen viability determination in
sunflower and other plant species. Ind. Crops Prod. 2012,35, 88–91. [CrossRef]
47.
Dafni, A.; Firmage, D. Pollen viability and longevity: Practical, ecological and evolutionary implications. In Pollen and Pollination;
Dafni, A., Hesse, M., Pacini, E., Eds.; Springer: Vienna, Austria, 2000; pp. 113–132. ISBN 978-3-7091-7248-3.
48. Nepi, M.; Franchi, G. Cytochemistry of mature angiosperm pollen. Syst. Evol. 2000,222, 45–62. [CrossRef]
49.
Alexander, L. Ploidy level influences pollen tube growth and seed viability in interploidy crosses of Hydrangea macrophylla.Front.
Plant Sci. 2020,11, 100. [CrossRef]
50.
Báez, P.; Riveros, M.; Lehnebach, C. Viability and longevity of pollen of Nothofagus species in South Chile. N. Z. J. Bot.
2002
,40,
671–678. [CrossRef]
51.
Marcellán, O.N.; Camadro, E.L. The viability of asparagus pollen after storage at low temperatures. Sci. Hortic.
1996
,67, 101–104.
[CrossRef]

Forests 2022,13, 600 16 of 16
52.
Munhoz, M.; Da Luz, C.F.P.; Meissner Filho, P.E.; Barth, O.M.; Reinert, F. Viabilidade polínica de Carica papaya L.: Uma comparação
metodológica. Rev. Bras. Bot. 2008,31, 209–214. [CrossRef]
53. Aguila, Y.; Jiménez, M.; García, Y. Pollen Viability in Taro (Colocasia esculenta (L.) Schott) in Cuba. Agrisost 2018,24, 33–38.
54.
Mazzeo, A.; Palasciano, M.; Gallotta, A.; Camposeo, S.; Pacifico, A.; Ferrara, G. Amount and quality of pollen grains in four olive
(Olea europaea L.) cultivars as affected by ‘on’ and ‘off’ years. Sci. Hortic. 2014,170, 89–93. [CrossRef]
55.
Zulkarnain, Z. Assessment of pollen viability and germination in Swainsona formosa (G. Don) J. Thomson. Biospecies
2019
,12,
49–54.
56.
Beck-Pay, S.L. Optimisation of pollen viability tests for Acacia podalyriifolia and two ploidys of Acacia mearnsii.S. Afr. J. Bot.
2012
,
78, 285–289. [CrossRef]
57.
Sousa, A.; Santos, M.; Pelacani, C.; Santos, F. Testing culture media for pollen germination of Elaeis guineensis Jacq. (oil palm,
Arecaceae). Bot. J. Linn. Soc. 2016,182, 536–542. [CrossRef]
58.
Nogueira, P.V.; Coutinho, G.; Pio, R.; Da Silva, D.F.; Zambon, C.R. Establishment of growth medium and quantification of pollen
grains and germination of pear tree cultivars. Rev. Ciê. Agron. 2016,47, 380–386. [CrossRef]
59. Burke, I.; Wilcut, J.; Allen, N. Viability and In Vitro Germination of Johnsongrass (Sorghum halepense) Pollen. Weed Technol. 2007,
21, 23–29. [CrossRef]
60.
Wang, Z.; Ge, Y.; Scott, M.; Spangenberg, G. Viability and longevity of pollen from transgenic and nontransgenic Tall Fescue
(Festuca arundinacea) (Poaceae) plants. Am. J. Bot. 2004,91, 523–530. [CrossRef]
61.
Duro, A.; Piccione, V.; Zampino, D. Air quality biomonitoring through pollen viability of Fabaceae.Environ. Monit. Assess.
2013
,
185, 3803–3817. [CrossRef]
62.
Ishida, K.; Hiura, T. Pollen fertility and flowering phenology in an androdioecious tree, Fraxinus lanuginosa (Oleaceae), in Hokkaido,
Japan. Int. J. Plant Sci. 1998,159, 941–947. [CrossRef]
63. Kremer, D.; Jemri´c, T. Pollen germination and pollen tube growth in Fraxinus pennsylvanica.Biologia 2006,61, 79–83. [CrossRef]
64.
Matsuda, H.; Higuchi, H. Effects of temperature and medium composition on pollen germination of ‘Bengal’ and ‘Chakrapat’
lychee (Litchi chinensis Sonn.) in vitro. Trop. Agri. Dev. 2013,57, 120–125.
65.
Fragallah, S.; Lin, S.; Li, N.; Ligate, E.; Chen, Y. Effects of sucrose, boric acid, pH, and incubation time on
in vitro
germination of
pollen and tube growth of chinese fir (Cunnighamial lanceolata L.). Forests 2019,10, 102. [CrossRef]
66. Aslanta¸s, R.; Prilak, L. Storage of strawberry pollen. Acta Agro. 2001,567, 117–121. [CrossRef]
67.
El Kadri, N.; Ben Mimoun, M.
In vitro
germination of different date palm (Phoenix dactylifera L.) pollen sources from southern
tunisia under the effect of three storage temperatures. Int. J. Fruit Sci. 2020,20, S1519–S1529. [CrossRef]
68.
Mesnoua, M.; Roumani, M.; Salem, A. The effect of pollen storage temperatures on pollen viability, fruit set and fruit quality of
six date palm cultivars. Sci. Hortic. 2018,236, 279–283. [CrossRef]
69.
Du, G.; Xu, J.; Gao, C.; Lu, J.; Li, Q.; Du, J.; Lv, M.; Sun, X. Effect of low storage temperature on pollen viability of fifteen
herbaceous peonies. Biotechnol. Rep. 2019,21, e00309. [CrossRef] [PubMed]
70.
Martínez-Gómez, P.; Gradziel, T.M.; Ortega, E.; Dicenta, F. Low temperature storage of almond pollen. Hortic. Sci.
2002
,37,
691–692. [CrossRef]
71.
Shekari, A.; Nazeri, V.; Shokrpour, M. Pollen viability and storage life in Leonurus cardiaca L. J. Appl. Res. Med. Aromat. Plants
2016
,
3, 101–104. [CrossRef]
72.
Raquin, C.; Brachet, S.; Jeandroz, S.; Vedel, F.; Frascaria-Lacoste, N. Combined analysis of microsatellite and RAPD markers
demonstrate possible hybridization between Fraxinus excelsior L. and Fraxinus angustifolia Vahl. For. Gen. 2002,9, 111–114.
73.
Wang, S.; Xie, B.; Yin, L.; Duan, L.; Li, Z.; Eneji, A.E.; Tsuji, W.; Tsunekawa, A. Increased UV-B radiation affects the viability,
reactive oxygen species accumulation and antioxidant enzyme activities in maize (Zea mays L.) pollen. Photochem. Photobiol.
2010
,
86, 110–116. [CrossRef]
74.
Hidvégi, S.; Rácz, F.; Hadi, G.; Gesztesi, L.; Tóth, Z. Effect of UV-radiation on the pollen viability of some parental lines of hybrid
maize. Cer. Res. Commun. 2009,37, 349–352.
75.
Schueler, S.; Schlnzen, K.H.; Scholz, F. Viability and sunlight sensitivity of oak pollen and its implications for pollen-mediated
gene flow. Trees 2005,19, 154–161. [CrossRef]
76.
Bohrerova, Z.; Bohrer, G.; Cho, K.D.; Bolch, M.A.; Linden, K.G. Determining the viability response of pine pollen to atmospheric
conditions during long-distance dispersal. Ecol. Appl. 2009,19, 656–667. [CrossRef] [PubMed]
77.
Ge, Y.; Fu, C.; Bhandari, H.; Bouton, J.; Brummer, E.C.; Wang, Z.-Y. Pollen viability and longevity of switchgrass (Panicum virga-
tum L.). Crop Sci. 2011,51, 2698–2705. [CrossRef]