CONTACT A. Zahidi [email protected] Polydisciplinary Faculty Taroudant, P Box 271 Taroudant 83000 Morocco
The effects of temperature and humidity on in vitro pollen germination of Argania spinosa (L.) Skeels
A. Zahidi1,2, S. Benlahbil1, F. Bani-Aameur1, A. El Mousadik1
1Laboratory of Biotechnologies and Valorization of Natural Resources; Faculty of Sciences, Ibn Zohr University, Agadir 80000 Morocco
2Polydisciplinary Faculty Taroudant, Taroudant 83000 Morocco
Abstract
Argan [Argania spinosa (L.) Skeels] a multipurpose perennial tree endemic to south-west Morocco, grows in a wide array of arid to semi-arid environments characterized by great fluctuation in rainfall. As reported in other species, the reproductive phase is most vulnerable to temperature variations, but information on the effects of temperature and relative humidity on argan pollen germination are (is) still lacking. With the aim to fill this gap, in this work, the effect of temperature and relative humidity (RH) was evaluated in vitro at different combinations during two periods. Temperature, relative humidity and exposure time affect significantly the number of pollen grains germinated in vitro. We found a tendency to decrease for pollen germination following the low values of Temperature (10 °C) and / or relative humidity (15% and 50%). The best performance was observed at 25 °C and 90% of RH since percentage of pollen germination reached to 86.6% in some genotypes. But, the increasing of temperature, particularly 35 °C and 40 °C, had demonstrated inhibitory effects on pollen germination. In addition, among tested genotypes, even at 90% of RH, the average percentage of pollen germination decreased from 57.3% at 30 °C to 16.7% at 35 ° C and at a very low value of 0.53% at 40 °C. In argan forest, within-population variation in abiotic resource availability and in weather conditions was the rule rather than the exception. The selection of the most tolerant genotypes to natural weather conditions, especially in spring, was necessary for the within-population gene flow and the success of controlled crossings.
Keywords: Argania spinosa, exposure duration - humidity - pollen germination - genotype- temperature.
Introduction
Sexual reproduction in flowering plants is an integrated series of subsystems, each of which must function correctly to allow fertilization and seed maturation to occur, namely: pollen grain development;
adherence of pollen to the stigma surface;
pollen germination; pollen-tube growth;
double fertilization, development of the embryo and endosperm. In recent years many investigations have shown that genes imparting plant resistance to many biotic and abiotic stresses such as salinity, low temperatures are expressed in the pollen grains also (Mulcahy, 1979; Mascarenhas, 1989; Bajaj et al., 1992). The reproductive
system of plants is a potential pathway for the introduction of foreign genes. This pollen represents an important vector for transferring genetic information (Dumas, 1984; Luna et al., 2001). This has opened up new avenues for application of section pressure on pollen grains to achieve preferential transmission of adaptive genes to the progeny (Ottaviano & Mulchay, 1989). In several species, exposure of pollen grains to different degrees of temperature alone; or in combination with the relative humidity acts as an effective selection pressure, and increases the vigor of the resulting seedlings (Bajaj et al.,
A. Zahidi et al. / Moroccan J. Biol. 14 (2017): 36-46
1992; Petolino et al., 1992). According to Waines & Hegde (2003), environmental factors during reproductive development cause a variation in the genetic flow between populations of the same species.
High or low relative humidity and/or temperature can reduce the gene flow. In addition, high temperature and/or relative humidity significantly affect pollen vigor but not viable (Shivanna et al., 1991).
Results of other investigation showed that the pollen exposure to high temperatures inversely affects the quantity and quality of fruits in several species (McKee &
Richards, 1998). Optimal conditions for pollen fertility depend on the species, variety, and genotype (Yates, 1989; Bajaj et al., 1992; Loupassaki et al., 1997). It is often difficult to distinguish the reduction of pollen germination caused by low relative humidity of that induced by thermal stress since these two parameters act simultaneously.
Argan tree is endemic to the southwest of Morocco in arid and semi- arid Mediterranean climate (Maire, 1939;
Boudy, 1952; Prendergast & Walker, 1992) with a marked water deficit during late spring and summer (Peltier, 1982;
Ferradous et al., 1996; Zahidi et al., 2013).
The arid zone covers more than 520 000 ha; we denote its existence in the northeast, in the upper basin of Oued Grou and in the southeast of Oued Noun (Emberger,
1925a,b; Chevalier, 1953; Ehrig, 1974).
The multiple uses of argan, especially the interest of oil combined with tolerance of drought, make it a good candidate for domestication as a fruit tree and last defence against desertification in south Morocco (Bani-Aameur & Benlahbil, 2004). Variation in environmental factors (e.g. rainfall, temperature) contributes directly or indirectly to generate heterogeneity on flowering phenology in the studied populations of argan (Benlahbil et al., 2015a). Therefore, the temperature and precipitations affects at different levels the flowering duration, fruiting length, number of trees in bloom and a number of trees bearing fruits. The flowering peak in the studied areas occurred during March- April when temperatures are still low and do not exceed 25°C on average. Several works in other species have addressed the evaluation of pollen storage, viability, germination and pollen preservation.
However, similar information in argan was still missing, in spite of its importance in studies of sexual reproduction such as pollen behaviour under specified conditions. In this work, we evaluated pollen germinationin vitro in different combinations of temperatures and relative humidity levels, since argan pollen grains in the field, are often exposed to these climatic combinations during anthesis until maturation.
Material and methods
MethodologyIn full bloom the 1st week of April, flowers in early blooming are collected from six genotypes at random in the field at Ait Melloul site and carried to the laboratory. Pollen is collected from dehisced anthers by dissections performed under a binocular magnifier (G x 20).
Pollen grains of each genotype were exposed for 30 and 60 min to combinations of temperatures: 10 °C, 25 °C, 30 °C, 35
°C and 40 °C and relative humidity levels:
15%, 50% and 90%. According to Benlahbil & Bani-Aameur (2000), early
studies indicated that argan pollen grains in the field, are often exposed to these climatic combinations during anthesis until maturation, about 15 days before harvest (maximum duration of transformation of flower bud to open flower) (Figure 1). The relative humidity of 15% and 50% are prepared by CaCl2 and with a saturated solution of CuSO4 (Yates, 1989). Solutions of distilled water in combination with boiling saturated solution of CuSO4 are implemented to maintain the relative humidity at 90%. This moisture was controlled by a manual hygrometer with an
interval of detection between 5% and 98%
humidity. After exposure of pollen grains to these combinations of temperatures and relative humidity, pollen in vitro
germination was tested according to the method described by Benlahbil & Bani- Aameur (2003).
-A-
-B-
Figure1. Temperatures (a) and relative humidity (b) recorded during 15 days before pollen grains harvest between 07 and 21 April (duration for transformation from BF-bud flower- to FE-opening flower-).
Pollen of two flowers of each genotype was tested for each combination.
Estimation of the percentage of germinated pollen of each flower was calculated on
two samples of 200 grains each. Pollen was considered as germinated when the length of the tube was longer than the grain diameter. In vitro germination of fresh
0 5 10 15 20 25 30 35 40 45
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Minimum Maximum
Temperature°C
Days of April
0 10 20 30 40 50 60 70 80 90 100
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Minimum Maximum
Relative humidity %
Days of April
A. Zahidi et al. / Moroccan J. Biol. 14 (2017): 36-46
pollen not treated (pre-incubated) was tested immediately after harvest as a control.
Statistical analysis
For number of in vitro germinated pollen grains, data were subjected to arcsine root square transformation. An analysis of variance (ANOVA) with four factors (temperature, humidity, exposure
time and genotype) in hierarchical model was adopted to assess if there were differences among all factors (Dagnelie, 1984).The least significant difference test (LSD α= 5%) of equality of means was used to compare differences between means (Steel & Torrie, 1960; Sokal &
Rohlf, 1995). All statistical analyzes were performed with Statistix software.
Results
Effect of temperature
Temperature factor was significant for number of pollen germinated in vitro (Table 1). The maximum germination percentage was obtained at 25 °C. This percentage decreases with the increasing of temperature (Figure 2). Thus, At 10ºC the
germination percentage was less respectively eight times, six times and three times than pollen exposed to 25 °C, 30 °C and 35 °C. Pollen exposed to 40 °C presented remarkably lower germination percentage.
Table 1. Analysis of variance for number of pollen grains germinated in vitro observed in six genotypes after exposure to various combinations of temperatures and relative
humidity during two durations. DF, Degrees of freedom; ns, Not significant; * Significant at 5%; ** Significant at 1%.
Source of variation DF Mean square
Temperature 4 183660 *
Humidity 2 179950 *
Exposure duration 1 532.1 *
Genotype 5 4263.5 ns
Temperature x humidity 8 36719 **
Temperature x exposure duration 4 83.1 ns Humidity x exposure duration 2 221.3 ns Temperature x genotype 20 2451.5 **
Humidity x genotype 10 1451 ns Exposure duration x genotype 5 126 ns Temperature x humidity x
exposure duration 8 81.8 ns
Temperature x humidity x
genotype 40 1012 **
Temperature x exposure duration x
genotype 20 128.1 ns
Humidity x exposure duration x
genotype 10 100.5 ns
Temperature x humidity x
exposure duration x genotype 40 60.3 ns
Error 540 115.9
Effect of relative humidity
Relative humidity and humidity temperature interaction were significant for germination percentage (Table 1). Whatever the exposure temperature, percentage of pollen germination was greater at 90%, but lowest germination was recorded at 15% of relative humidity except at 40 °C. At this temperature, the increasing of relative humidity does not improve in vitro pollen germination (Figure 3, Table2).
Effect of exposure duration
The exposure duration was significant in the pollen germination percentage in vitro;
but, all interactions were not significant (Table 1). The extension of pollen exposure to 50% reduces the germination percentage. Indeed, the reduction of the percentage of germinated pollen was significantly enhanced during one hour of exposure to 35 °C and 50% of relative humidity (Table 2).
Figure 2. Average percentage of pollen germination in vitro according to the exposure temperature. 1=10°C, 2=25°C, 3=30°C, 4=35°C and 5=40°C.
Table 2. Average, maximum and minimum percentage of pollen germination in vitro by exposure duration and relative humidity. Letters followed by values are significantly different.
Exposure duration (min)
Relative humidity (%)
Average 15 % 50 % 90 %
30 min 5.1 20.9 a 32.3 19.4 a 60 min 4.7 19.0 b 32.1 18.6 b Average 4.9 c 20.0 b 32.2 a
Genotype effect
The genotype factor was not significant for pollen germination in vitro (Table1). Genotype x temperature and genotype x humidity x temperature interactions were highly significant. For a given temperature, the mean germination
percentage varied in great proportion according to the genotype (Figure 4). In some genotypes, percentage of pollen germination decreases slightly with the increasing of temperature (Figure 3).
This reduction was more pronounced in other genotypes. There are three genotypes which presented higher germination percentage at 25 °C and 30
°C than the others. However, at 10 °C and 40 °C, pollen germination of all genotypes was reduced to similar values and lowest. For genotype 4, identical results were observed at 25 °C and 30
°C. This percentage decreases with only 7% at 35 °C.
Figure 3. Average percentage of pollen germinationin vitro according to temperature and relative humidity.
d
a
b
c
e 0
5 10 15 20 25 30 35 40 45
1 2 3 4 5
Temperature (°C)
% of getrminated pollen
c
a ab b
bc c
a
a
b
c d
a
b
c
c
0 10 20 30 40 50 60 70 80
10 25 30 35 40
15%
50%
90%
Temperature °C
%of germinated pollen
T tolerant differen significa humidit for 15%
The B respect differen pollen
Figure 4.
Discus
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Table3. Pollen percentage germination in vitro observed before pre-incubation and depending on temperature, relative humidity and genotype. *Before incubation. Letters followed by values were significantly different.
Genotypes
Pollen*
10°C 25°C 30°C 35°C 40°C
15% 50% 90% 15% 50% 90% 15% 50% 90% 15% 50% 90% 15% 50% 90%
1 69.5
ab 1.6 3.6 c
10.7 ab
10.5 abc
52.9 a
75.2 b
6.7 b
40.4 b
67 a
2.1 cd
5.6 d
10.7
d 0.0 0.6 ab
1.2 a 2 52.5
cd 1.6 4.7 bc
13.8 a
13.1 a
57.3 a
81.4 ab
12.6 a
52.5 a
64.7
ab 7.0 b 15.2 b
28.1
a 0.0 0.1 c
0.0 b 3 71
a 2.6 6.1 b
8.7 bc
6.5 c
20.8 c
80.4 ab
5.4 b
17.7 c
57.9 bc
0.9 cd
12.4 bc
18.4
c 0.1 1.2 a
1.1 a 4 48
d 1.6 9.7 a
7.6 bcd
11 ab
38.1 b
60 c
11.3 a
39.4 b
53.3 cd
11.6 a
30.7 a
10.7
d 0.0 0.9 ab
0.7 ab 5 66
abc 2.1 5.7 bc
4.8 cd
7.1 bc
43.2 b
86.6 a
3.8 b
38.6 b
52.2 cd
4.2 c
10.5 c
15.7
cd 0.0 1.4 a
0.0 b 6 54
bcd 1.2 4.2 bc
6.8 cd
6.6 c
40 b
52 c
6.3 b
32.1 b
48.8 d
7.9 b
13.6 bc
16.4
cd 0.1 0.0 0.2 b
biosynthesis of polyamines which are essential for this process (Song et al., 1999). Exposure to low relative humidity dehydrates the pollen grains even at optimal temperatures (Sato et al., 1998;
Lisci et al., 1994). Exposure for 30 min to unfavourable combinations of temperature and relative humidity reduces significantly the percentage of pollen germinated in vitro. The impact of the two parameters was very fast (Sahar & Spiegel, 1984;
Loupassaki et al., 1997). Indeed, a few minutes of exposure to these adverse parameters induced a strong decrease in pollen germination in several species (Yates, 1989). According to Lansac et al.
(1994), pollen germination was impossible when it was subjected to 50% of relative humidity and at temperature of 25 °C during a period of 30 minutes. In addition, in this work we found that incubation of argan pollen at 25 °C and 90% of relative humidity improves its germination potential whatever the genotype. This result will reaffirm the strong influence of humidity also on pollen germination. Thus, hydration of pollen at 100% of relative humidity was crucial for pollen germination in some species (Dumont- BeBoux et al., 2000). Adhikari &
Campbell (1998) reported that exposure of
pollen at 25 °C without control of relative humidity was favourable for germination.
In other species, pollen grains did not germinate if they are not incubated before germination to optimum combination of temperature and relative humidity (Lansac et al., 1994). Loupasaki et al. (1997) reported that hydration of pollen was used to compensate the negative effect of adverse conditions. Contrary to the results of Lansac et al. (1994) and Craddock et al.
(2000) where the effect of dehydration under low relative humidity and / or high temperatures may not be reversed by rehydration. At least during the test period, the conditions of development of fresh pollen in our case did not cause irreversible negative effect on germination potential.
This potential decreased due to the direct exposure of this pollen to all combinations tested except at 25 °C and 90% of relative humidity. In this combination, gene flow appears to be improved in argan tree.
According to Loupassaki et al. (1997), the ambient temperature cannot be controlled under natural conditions. However, maintaining a favourable level of water regime can improve pollen germination and consequently improve fruit production.
Weaver & Timm (1988) reported that high soil humidity, and high amount of water in
A. Zahidi et al. / Moroccan J. Biol. 14 (2017): 36-46
leaves promote pollen germination by increasing moisture level of the reproductive tissues. Thus, harvesting of branches carrying flowers at flower bud (BF) stages and followed flowering process sunder controlled climatic conditions (Botto, 1997) promotes pollen performance and therefore the success of artificial pollination. In argan forest, within-population variation in abiotic resource availability and in weather conditions was the rule rather than the exception. The opening of flowers and mature pollen are not synchronous among all the trees. So, flower phase FE is at its population maximum for twenty days in spring, contributing with fertile pollen and receptive stigmas to within-population gene flow (Belahbil et al., 2015b).
According to Sukhvibul et al. (2000), the effects of temperature on pollen
germination are inconsistent and seem to be cultivar or species dependent.
Therefore, the differences in pollen germination observed in the present study were a reflecting of genotype variability.
The degree of sensitivity of argan pollen to different combinations of temperature and relative humidity was variable depending on genotype except for the extremely adverse conditions where the genotypic variation disappears. Some genotypes seem to be more tolerant to stress conditions.
This result confirms the genotypic variation in pollen fertility over looked natural climatic conditions (Benlahbil et al., 2015b).
The selection of the most tolerant genotypes to climatic conditions during the controlled pollination in particular the fertility of their pollen would be favourable for successful breeding.
Conclusion
Pollen performance is an important factor determining the fertilization success in plants. Temperature has been shown to affect each stages of this process. In argan, pollen germination percentage was important in all genotypes tested at 25 °C and 90% of relative humidity. The increase of temperature affects inversely in vitro pollen germination. However, the rise of the relative humidity improves this percentage. In the field, the monitoring of temperature and humidity in March for four successive years shows that the maximum temperature was between 23 °C and 35 °C and not exceed 30 °C in only four days. The minimum varied between 8
°C and 15 °C and was not lower than 10 °C in only four days. The minimum of relative
humidity varied from 35% to 74% and from 80% to 95% for the maximum of RH. These conditions coincide with the peak of bloom where more than 80% of trees bear flowers. Thus, the warm temperatures combined with relatively high humidity levels are favourable conditions for pollen fertility, pollen germination in vivo and pollen tube formation. Those conditions should be advantageous for argan tree to exhibit maximum reproductive efficiency at temperatures normally encountered during the sexual phase of its life cycle in order to obtain a sufficient fruit harvest.The low relative humidity, cold and high temperatures are unfavorable for these parameters.
Acknowledgements
We gratefully acknowledge anonymous reviewers and office journal which provided helpful comments that greatly improved the manuscript. We thank the Morocco-Germany Co-operative
Project ‘Conservation Project and Development the argan forest’ (PCDA- GTZ) and the project Pars-Agro 128 of the Moroccan Ministery of Scientific Research for financial support.
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