In Vitro Germination of Different Date Palm (Phoenix Dactylifera L.) Pollen Sources from Southern Tunisia under the Effect of Three Storage Temperatures

ABSTRACT

Date palm pollen storage is becoming more and more necessary due to the asynchronous maturation of male and female flowers that, frequently, occurs, during the last years in Tunisia. However, little information is available on the pollen viability and favorable storage conditions. The present study aims to compare date palm pollen storage at three temperatures (25 ± 2°C, 4°C and −20°C) and to evaluate the aptitude of pollens from different geographical origins to conserve their in vitro germinative potency. Results revealed that, after the fresh pollen, the overall pollen in vitro germinations were significantly improved with storage at −20°C (41.04 ± 23.84%) and 4°C (20.07 ± 22.19%) compared with storage at 25 ± 2°C (4.27 ± 4.39%). For each used temperature, the in vitro germination was not affected by the pollen origins. In fact, almost similar reactions were observed for fresh and stored pollen at 25 ± 2°C and 4°C. On the other hand, pollen stored at −20°C had reacted independently from the origin. However, the aptitude of pollens from similar origins to preserve their in vitro germination potencies under the three storage temperatures reveals some similarities. Physiologic characteristics of pollens were not related to the geographic origins and did not significantly affect their aptitude for storage

KEYWORDS:

• Phoenix dactilyfera L.
• storage temperature
• origins
• in vitro germination

Introduction

Date palm (Phoenix dactylifera L.) is a main income source and staple food for local populations in many countries (Chao and Krueger, 2007), since it’s grown in hot and arid habitats, including desert oases, river valleys and well-irrigated plantations (Hazzouri et al., 2015). In Tunisia, Date palm cultivation creates opportunities for rural employment, is a major source of revenue for farmers and related industries (Othmani et al., 2019), and is widely consumed as fresh fruit (Assadi et al., 2018).

Date palm is a dioecious species and wind pollinated in nature, but manual pollination is practiced to reach commercial yield (Rezazadeh et al., 2013). Date palm flowers are born on a compound spadix in leaf axils, inflorescences are sheathed in a bract or spathe until just prior to anthesis. Each sex produces thousands of tiny flowers per inflorescence. Male flowers are white, fragrant with six stamens each, and females are more yellowish or creamy colored (Chao and Krueger, 2007).

Therefore, pollination is one of the most important practices of date fruit production. It should occur at the right moment to get the optimal fruit setting and good yield. However, during the last years, asynchronous flowering of male and female date palms is more and more observed, in southern Tunisian oases. Obviously, under the climate change effects, which, may disrupt the overlap in the seasonal timing of flower production as observed in pistachio and almond in Tunisia (Benmoussa et al., 2017, 2018), and many other fruit trees (Memmott et al., 2007). Actually, the reproductive phase in flowering plants is, often, highly sensitive to hot or cold temperature stresses (Zinn et al., 2010).

Hence, to ensure pollination at time, palm growers in southern Tunisia, usually, bring and store pollens from different localities according to the traditional method in cardboard boxes at room temperature for short (one or two months) or long period (until the next season). However, pollen viability and potency of germination can be altered during storage, which can cause production losses when pollen is used for pollination.

Several researches have developed experimental methods for date palm pollen storage, such as cryoconservation (Anushma et al., 2018), freezing in liquid nitrogen and lyophilization (Babahani and Bouguedoura, 2009). But, these methods can be, often, difficult to adopt by farmers, since they require means and a technicality which are not within their reach.

Also the ability of different pollen sources to be stored still very little studied. In fact, at present, very little is known about the impact of pollen source characteristics on its viability (Abdel-Sattar and Mohamed, 2017). Some studies had focused on the morphology and the mineral composition of the date palm pollen (Aly, 2018) or the chemical composition such as total carbohydrates, sugars and crude protein (Abdel-Sattar and Mohamed, 2017), while very little is still known about physiologic traits of date palm pollen grains.

On the other hand, the most reliable way to assess the quality of pollen is the count of seeds obtained from crossing (Colas and Mercier, 2000). It indicates by relative comparison, the ability of pollen to properly fertilize female inflorescences (Peyron, 2000). However, this process is long and expensive (Colas and Mercier, 2000) and it is the most difficult test to set up (Peyron, 2000). Therefore, other methods have been used to assess the quality of the pollen such as the in vitro germination which was used as an important technique for testing pollen viability. In fact, the in vitro germination of pollen allows the measurement of its genuine aptitude to germinate outside any interaction between pollen and stigma (Mortazavi et al., 2010).

In Tunisia, studies regarding the optimization of date palm pollen storage methods for a long time and the effect of pollen origins and characteristics on the storage ability still very scarce. Therefore, developing simple methods of date palm pollen storage, and studying the ability of different pollen sources, with different physiologic characteristics, to conserve their potency during the storage is necessary to help farmers to store the pollen and to ensure its availability at the adequate time for pollination. This study aims to compare three simple methods of date palm pollen storage and to investigate the aptitude of different pollen from different origins of southern Tunisia to conserve their potencies by studying their in vitro germination before use for pollination after 12 months storage.

Material and Methods

Pollen Collection and Storage Conditions

The study was carried out during 2016 and 2017 seasons. Collection sites were chosen as usually used to collect pollen by Tunisian date palm growers, and pollen samples were coded by the name of their origins. Pollen samples were collected from all date palm production areas of southern Tunisia (Figure 1), and all types of oasis (island, coastal, saharan and mountainous), with different geographic (Table 1) and climatic characteristics (Table 2). Pollinizers selected in this study were all from spontaneous seedling of date palm cultivars (Figure 2).

Table 1. Pollen sources from different locations studied in the manuscript. Governorates, collection sites, site codes, type of oasis, geographic location and oases characteristics of date palm pollinizers origins used are shown

Site	Sites/Male names		Governorate	Type of oasis	Location	Oases characteristics
1		Djerba	Medenine	Island oasis	South-East	common varieties are predominant, (Sghaier, 2010); An important number of spontaneous date palms especially in the island oases (Zehdi-Azouzi et al., 2016)
2		Zarat	Gabes	Coastal oasis	South-East
3		Benghilouf	Gabes	Coastal oasis	South-East
4		Gataya	Kebili	Saharan oasis	South-West	The climate is characterized by a high temperatures and a dry atmosphere; The “Deglet Nour” variety is predominant (Sghaier, 2010).
5		Jemna	Kebili	Saharan oasis	South-West
6		Hazoua	Tozeur	Saharan oasis	South-West
7		Mides	Tozeur	Mountainous oasis	South-West	A mild winter; Predominance of common varieties (Sghaier, 2010).
8		Gtar	Gafsa	Mountainous oasis	South-West

Table 2. Climatic parameters in the collection sites from 2007 to 2017: Tmean (°C): average annual temperature; Tmax (°C): average maximum temperatures; Tmin (°C): average minimum temperatures; PP (mm): average annual total precipitation. (INM, 2017)

	Tmin	Tmax	Tmean	PP (mm)
Kébili	16,86	28,62	22,68	97,12
Tozeur	15,20	28,52	22,57	96,87
Gafsa	13,78	27,03	20,48	148,94
Gabes	14,87	26,27	20,49	105,31
Jerba	17,24	25,82	21,42	240,12

Figure 1. Geographic locations of the date palm pollinizers used in this work

Figure 2. Date palm pollen collection. (A) Male spathes of some collection sites. (B) Fresh pollen from newly opened inflorescences. (C) Strands were cut off and spread on paper sheets to dry slowly in clean rooms

Figure 3. In vitro germination of date palm pollen grain incubated at 25°C (40×) [NG = Non germinated, G = Germinated]

Three mature spathes (inflorescences) newly opened from each male tree were collected and fresh pollens from spikelets were collected. Strands were cut off and spread on paper sheets to dry slowly in clean rooms (Mohammadi et al., 2017) and prepared for storage. Pollen grains of the three male spathes were mixed together to minimize variations that might have existed between the pollens of those spathes. Three storage temperatures were used for the experiment (room temperature at 25 ± 2°C, 4°C at refrigerator and −20°C at freezer) for 12 months and fresh pollens (season 2017) were used as a control treatment. In storage at 25 ± 2°C pollens are kept inside the flowers, dried and stored in cardboard boxes according to the method adopted by most of the farmers. In storage at 4°C and −20°C, hand-extracted pollen powders were collected and separated from flower parts using fine sieves and dehydrated in an oven at 40°C for 24 hours and placed in dry transparent vials before storage (Boughediri et al., 1995). All parameters cited in this study were conducted in the Fruit trees laboratory at the National Institute of Agronomy of Tunisia (INAT).

Pollen Physiologic Characteristic

Some physiologic parameters of pollen grains were determined in order to study their eventual effect on the in vitro germination for fresh and stored pollens.

The measurement of pollen pH was carried out by mixing 0.25 g of pollen powder from each sample with distilled water (3cc) in glass tubes, and then placed in a centrifuge for 10 minutes. The electrode of the pH meter was introduced in the supernatant (Halimi, 2004).

The moisture content was obtained after desiccation of 0.5 g of fresh pollen (PMF) from each sample for 48 hours in an oven set at 110°C and then weighed to determine the dry weight (MS).Then, a calcination of 0.5 g of pollen powder (PMF) at 250°C for 4 hours allows determining the total water content (HT). Pollen grains were weighed after calcination (MS). HM and HT were calculated in percentages as follows: HM or HT = ((PMF-MS)/PMF)*100 (Halimi, 2004). Every measured characteristic is the arithmetical mean of three determinations.

Pollen in Vitro Germination

The pollen in vitro germination percentage was estimated by counting the percentage of germinated pollen grain cultivated in the modified medium of Brewbaker and Kwack (MBK). The solution composition was as the following (for 100 ml): 15% sucrose; 50 ppm H₃PO₄; 300 ppm Ca (NO₃)₂; 200 ppm MgSO₄; 100 ppm KNO₃; 100 ml of distilled water and 1 % Agar (Benamor et al., 2014). For each treatment 10 ml media was poured in 6-cm diameter Petri dishes then inoculated with pollen grain using a piece of cotton and incubated at 25°C for 24 hours (Ismail and Zohair, 2013; Nesiem et al., 2016). Then the growth of pollen tubes was stopped by spraying a 45% acetic acid solution on the medium (Mortazavi et al., 2010). Treatments were replicated three times in each treatment. Pollen germination (TG) was carried out by random counts of 100 pollen grains under 40X magnification in direct microscopic observation in each replication. Pollen grains, which produced a tube equal to their own diameter (Figure 3), were counted as germinated (Ismail and Zohair, 2013).

Statistical Analysis

The experiment was arranged in a completely randomized design (CRD) with three replications. All statistical analyzes were performed using IBM SPSS statistics software (Version 20), IBM, USA. Firstly, data were subjected to Two-way ANOVA analysis using General Linear Model (GLM) including two factors (eight pollen sources and four storage temperatures, including fresh pollen as a control) in order to check the effect of each factor and their interactions. Data were, then, evaluated by one-way ANOVA analysis in order to compare differences between variables at the same temperature of storage and to compare physiological pollen parameters. Duncan’s multiple comparison test were used to separate means at α = 0.05. Pearson’s correlation coefficients between all studied parameters were also calculated.

Results

Effect of Temperature Storage Conditions on the in Vitro Germination of Date Palm Pollens

The effect of storage temperature, pollen source, and the interaction between storage temperature and pollen source on the in vitro pollen germination was highly significant (Table 3).

Table 3. Two-way ANOVA showing the effect of storage temperatures (x4) on the in vitro germination of date palm pollen from (x8) pollen sources

Source of variation	In vitro germination (%)
	F-value	P
Storage temperature	360,75	0,00
Pollen source	7,23	0,00
Storage temperature * Pollen source	5,36	0,00

Statistical analysis showed that the pollen in vitro germination (Figure 4) rates were significantly influenced by the storage temperature. The overall comparison between storage temperatures showed that the in vitro germination of fresh pollen was significantly higher than stored pollen (78,21 ± 11,09%). On the other hand, the in vitro germination were significantly improved with storage at −20°C (41,04 ± 23,84%) and 4°C (20,07 ± 22,19%) compared with storage at 25 ± 2°C (4,27 ± 4,39%) (Figure 4).

Figure 4. Pollen in vitro germination (%) under the effect of fresh pollen (Control) and pollen stored at 25 ± 2°C, 4°C and −20°C of eight pollen sources during 12 months. Each value represents mean ± SD. Different letters indicate that means are significantly different (Duncan test, P < .05)

Higher pollen in vitro germination was registered for fresh pollen in all sites (Table 4). Results showed that for fresh pollen, the in vitro germination were not affected by the geographic origin of pollen, excepted for Jemna pollen, the in vitro germination was significantly lower than all other sites (60.49 ± 12.91%) (Table 4).

Table 4. Pollen in vitro germination (%), under the effect of fresh pollen (Control), and pollen stored at 25 ± 2°C, 4°C and −20°C of eight pollen sources during 12 months. Capital letters are for comparison between geographic origins for the same storage temperature. Lower case letters are for comparison between different storage temperatures for the same geographic origin

		Storage temperatures
Pollen sources	Fresh			25 ± 2°C		4°C		−20°C	F – Value		ANOVA P
Djerba	76.89 ± 6.95 c/B		4.22 ± 3.67 a/A		21.11 ± 18.33 b/A			28.89 ± 12.69 b/AB	62.52		0.00
Zarat	77.67 ± 9.38 c/B		4.78 ± 2.28 a/A		23.89 ± 11.40 b/A			28.89 ± 22.05 b/AB	48.88		0.00
Benghilouf	79.11 ± 10.46 c/B		2.50 ± 1.51 a/A		8.89 ± 8.21 ab/A			13.33 ± 13.69 b/A	119.46		0.00
Gataya	80.22 ± 6.83 d/B		3.67 ± 1.22 a/A		18.33 ± 6.12 b/A			68.33 ± 13.46 c/E	188.01		0.00
Jemna	60.49 ± 12.91 c/A		3.78 ± 2.22 a/A		12.50 ± 6.55 b/A			62.22 ± 8.33 c/DE	117.44		0.00
Hezoua	83.67 ± 7.97 d/B		4.44 ± 6.60 a/A		22.22 ± 32.99 b/A			42.22 ± 10.93 c/BC	31.75		0.00
Mides	84.78 ± 7.66 c/B		8.89 ± 7.75 a/B		44.44 ± 38.77 b/B			47.78 ± 18.22 b/CD	17.73		0.00
Gtar	82.89 ± 6.57 c/B		1.67 ± 1.58 a/A		8.33 ± 7.91 a/A			36.67 ± 29.90 b/BC	49.00		0.00
F-value	6.84		2.42		2.89			9.83
ANOVA P	0.00		0.03				0.01	0.00

Each value is an average of three replications ±SD. Mean values followed by the same lower case letters in the lines and capital letters on the columns are not significantly different at 5% level of probability according to Duncan test.

The in vitro germination rates for all pollens, from all origins stored at 25 ± 2°C was significantly reduced varying from 1.67 ± 1.58% (Gtar) to 8.89 ± 7.75% (Mides). Actually, Mides pollen was the only one that, significantly, improved the in vitro germination at this temperature, but with very low value (Table 4).

Similarly, Mides pollen stored at 4°C, was the only source that significantly improved the in vitro germination at this temperature (44.44 ± 38.77%) (Table 4).

Regarding storing pollen at −20°C, Gataya and Jemna pollen, showed the significant higher pollen in vitro germination (68.33 ± 13.46 and 62.22 ± 8.33%, respectively), compared with other pollens at this temperature. They represent similar locations and similar type of oases. Mides pollen stored at −20°C showed, also, relatively high rates (47.78 ± 18.22%) (Table 4). While insular and costal pollen (Djerba, Zarat and Benghilouf), showed the lowest in vitro germination rates at this temperature with the significantly, lower rate for pollen from the Benghilouf site (13.33 ± 13.69%).

Concerning the reaction of pollen from each site to the different storage temperatures, almost similar reactions of pollen from similar sites were observed. Pollens from Djerba and Zarat sites showed similar reactions to the storage temperatures with very close values (Table 4). The significant higher in vitro germinations were obtained for pollen stored at −20°C (28.89 ± 12.69 and 28.89 ± 22.05%, respectively), than at 4°C (21.11 ± 18.33 and 23.89 ± 11.40%, respectively) compared with storage at room temperature (4.22 ± 3.67 and 4.78 ± 2.28%, respectively). However, Benghilouf pollen was better conserved at −20°C, than the other temperatures (Table 4).

Pollen from south-west saharan oasis showed similar reactions to the different storage temperatures. The significant high in vitro germination was obtained from pollen stored at −20°C. Actually, Gataya pollen stored at −20°C showed higher germination (68.33 ± 13.46%) than Jemna (62.22 ± 8.33%) and Hezoua (42.22 ± 10.93%). On the other hand, at 4°C pollen from Hezoua site showed higher in vitro germination (22.22 ± 32.99%) than Gataya (18.33 ± 6.12%) and Jemna pollens (12.50 ± 6.55%).

Pollen from the mountainous oasis of Mides stored at −20°C and 4°C maintain significant higher in vitro germination (47.78 ± 18.22%, and 44.44 ± 38.77 respectively) than pollen stored at room temperature. However, pollen from the mountainous oasis of Gtar showed significant higher in vitro germination only for pollen stored at −20°C (36.67 ± 29.90%). While pollen from this site stored at 4°C lost the major part of their potency (8.33 ± 7.91%).

Physiologic Characteristics of Pollen Grains from Different Geographic Origins

Significant variations of pollen grains acidity and the moisture rate after desiccation (HM) were observed between different pollen sources (Table 5). The pH values of all pollen samples were close to neutrality, varying between 6.89 ± 0.22 and (Djerba) and 7.45 ± 0.14 (Gataya).

Table 5. Physiologic characteristics (pH, moisture of pollen grains (HM) and total water content of pollen grains (HT) of pollen grains from different geographic origins

Pollinizers	pH	HM (%)	HT (%)
Djerba	6.89 ± 0.22 a	11.20 ± 1.08 ab	50.50 ± 5.27 a
Zarat	7.11 ± 0.16 b	9.43 ± 2.56 ab	47.88 ± 9.06 a
Benghilouf	7.12 ± 0.18 b	6.22 ± 3.32 a	47.27 ± 9.92 a
Gataya	7.45 ± 0.14 c	14.05 ± 2.37 bc	49.07 ± 1.80 a
Jemna	7.16 ± 0.24 b	9.22 ± 2.49 ab	45.42 ± 16.22 a
Hazoua	7.30 ± 0.38 bc	12.56 ± 8.65 bc	46.69 ± 16.21 a
Mides	7.12 ± 0.15 b	9.07 ± 1.24 ab	56.27 ± 1.15 a
Gtar	7.15 ± 0.16 b	16.72 ± 9.60 c	56.10 ± 4.25 a
F-value	4.91	3.96	1.65
ANOVA P	0.00	0.00	0.14

Each value is an average of three replications ±SD. Mean values with the same letters in the same column are not significantly different at 5% level of probability according to Duncan test.

Pollen moistures had varied between 6.22 ± 3.32% (Benghilouf) and 16.72 ± 9.60% (Gtar). Mides pollens, which showed significant low HM (9.07 ± 1.24%), were from similar origins than Gtar (Table 5).

While the total content rates of pollen grains were ranged from 45.42 ± 16.22% (Jemna) to 56.27 ± 1.15% (Mides) with no significant differences according to the geographic origin of pollen samples (Table 5).

Correlation matrix presented in Table 6 showed very weak relationship between physiologic traits of the pollen sources and the in vitro germination for fresh and stored pollens. The pollen grains acidity was positively correlated with in vitro germination of pollen stored at −20°C (0.25), while negatively correlated with the in vitro germination of fresh pollen (−0.29). The pollen grain moisture (HM) was negatively correlated with the in vitro germination of fresh pollen. On the other hand, very high positive correlation was registered between the in vitro germination of pollen stored at 25°±2 C and 4°C (0.98) (Table 6).

Table 6. Correlation matrix between the physiological traits (pollen pH, moisture of pollen grains (HM), total water content of pollen grains (HT) and the in vitro germination rate TG (%) and of the eight pollen sources collected from Southern Tunisia analyzed in this study and stored according to four temperature: fresh (control), 25°±2 C, 4°C and −20°C

	TG fresh	TG 25 ± 2°C	TG 4°C	TG −20°C	Pollen pH	HM	HT
TG fresh	1.00
TG 25 ± 2°C	0.10	1.00
TG 4°C	0.15	0.98**	1.00
TG −20°C	−0.15	0.14	0.15	1.00
Pollen pH	−0.29*	−0.18	−0.22	0.25*	1.00
HM	−0.24*	0.01	0.00	0.04	0.32**	1.00
HT	−0.03	0.04	0.00	0.19	0.39**	0.36**	1.00

Discussion

Effect of Temperature Storage Conditions on the in Vitro Germination of Date Palm Pollens

Results showed that stored pollen had lost a part of their germination potency compared with fresh pollens. These results are in line with several other studies that reported that pollen stored at room temperature (25–30°C) or in a refrigerator (3–4°C) retained less viability compared to the fresh pollen in date palm (Maryam et al., 2015; Shaheen, 1986)

Also, our finding showed that storing pollen at −20°C as a simple method helps to maintain the pollen potency. The obtained pollen in vitro germination percentages were significantly improved for pollen stored at this temperature compared with storage at 25°±2 C and 4°C, while pollen conserved at room temperature has lost the major part of their germination potency. Same observation was reported for pollen of six male cultivars in Algeria, where the decrease of the in vitro germination was significantly faster at room temperature compared to those recorded at 4°C or −20°C (Mesnoua et al., 2018). Similar results were found by Anushma et al. (2018), where pollen kept under −20°C retained viability throughout the storage period and germinated after one year of storage. Mortazavi et al. (2010) also reported that storage in the freezer (−20°C) kept pollen grain viability significantly higher than in the refrigerator (4°C).

Our results are, also, relatively in line with another study on “Dhakki”, “Khadrawy” and “Hillawi” date palm cultivars where the maximum mean of in vitro germination was obtained at −20°C followed by storage at 4°C (Maryam et al., 2015, 2017).

Actually, pollen longevity has been reported to be extended when stored at low temperatures and low moisture content (Akond et al., 2012; Mortazavi et al., 2010). And the declined in pollen germination can be caused by physical and chemical modifications that proceed gradually at 4°C and −20°C in stored pollen (Akond et al., 2012; Mesnoua et al., 2018).

Regarding comparison of the effect of storage temperature on the in vitro germination of eight different pollen grain sources, the results of this study revealed a significant effect for the interaction between storage temperatures and pollen source origins. Cutivar’s pollen from different geographic origins reacted differently in the tested storage temperatures. Similar aptitude for most pollen sources was observed for fresh and stored pollen at 25°±2 C and 4°C. Mides pollen was the only one to improve the germination at those temperatures. At −20°C, pollen origins had reacted differently to the storage. Insular and coastal pollen origins showed less aptitude to be stored at this temperature. While sharan pollens from Gataya and Jemna, showed the best aptitudes to be stored at this temperature. But this was not the case for Hezoua pollen from similar site. This is in accordance with previous studies that reported different reaction of different pollen sources to the storage temperature (El Mardi et al., 1996; Mortazavi et al., 2010; Mesnoua et al., 2018). The variation in pollen longevity can be explained by the differences in desiccation tolerance of the pollen (Akond et al., 2012). Also, the differences of pollen germination could be due to their genetic variability (Chao and Krueger, 2007; Mortazavi et al., 2010), since high levels of genetic variation were reported for male date palm pollinizers in Tunisia (El Kadri et al., 2019; Zehdi-Azouzi et al., 2016).

On the other hand, pollen from similar locations and type of oases reacted by similar way to the storage temperature during 12 months. For instance, pollen from saharan oases reacted to the three storage temperatures in the same way. The same observations were registered for insular and coastal pollen sources.

Physiologic Characteristics of Pollen Grains from Different Geographic Origins

The chemical characteristics of studied pollen sources showed that for all cultivars the pH was close to neutrality, which was in accordance with other studies (Babahani and Bouguedoura, 2009; Halimi, 2004). Also, the water content after desiccation varying from 6.22 to 16.72% was close to the found values by Halimi (2004), varying from 3.1 and 26.82%. However, the total pollen humidity varying from 45.42 to 56.27% was lower than other findings which showed a mean of fresh pollen moisture content about 75% (Babahani and Bouguedoura, 2009) and between 39 and 96.7% (Halimi, 2004), but our results showed higher values than those found by Sebii et al. (2019) that reported moisture content of 30.31 ± 0.86% of date palm pollen. Our investigation showed, also, that physiologic parameters measured in this study seem to not be related to the geographic origins of the pollens. Also, the correlation matrix showed only a very weak correlation between physiologic traits of pollens and the in vitro germination for each storage temperature. Very weak negative correlation was showed in our study between the fresh pollen pH and the in vitro germination. Similar observations were reported by Halimi (2004) in her study of 15 pollen sources from Algeria (r = −0.3337).

To conclude, the overall results showed that most of pollens had lost an important part of their in vitro germination potency at 25 ± 2°C, whereas, storage −20°C gave the highest rates, independently of their geographic origins and their physiological characteristics. Investigation of some other physiologic parameters and chemical composition of date palm pollen can help to better understand the correlation between pollen characteristics and the viability.

Conclusion

Presented results regarding the date palm pollen storage are able to help farmers and researchers to better use pollen grains, especially, when the time between pollen harvest and deliverance or use may be days, weeks or months and can also be interesting when artificial pollination is used. Actually, it can be recommended to store pollens at low temperature, at −20°C, for one year, without losing their fertility. This is considered as a sample and an inexpensive storage method. However, since that the ability of a pollen to maintain its performance in one temperature more than another seems to be relatively related to their origins, date palm growers should select the pollen sources that can be better conserved and do not use any available male date palm found.

Acknowledgments

Authors would like to thank technicians of the Fruit Trees Laboratory in Agronomy and Plant Biotechnology Department of the National Agronomic Institute of Tunisia for their efficient collaboration.