Storing Pollen of Two Olive Cultivars: ‘Rasie’ and ‘Nabali Baladi’

Abstract

This study was conducted on ‘Rasie’ and ‘Nabali Baladi’ olive cultivars to establish short-, mid-, and long-term pollen storage technique. Pollen was stored in normal room condition, refrigerator (4°C), freezer (−5°C), and cryostorage (−80°C) for up to 52 weeks. Pollen was in vitro germinated on solid-agar media, and its viability was tested using 2, 3, 5–triphenlytetrazolium chloride. Cryopreserved pollen under −80°C gave the best results, and refrigerated and frozen pollen gave convenient results. Under room conditions, pollen of the two cultivars has lost its germinability and viability gradually, and after 52 weeks the pollen was nonviable.

KEYWORDS:

• 2, 3, 5–triphenlytetrazolium chloride
• cryopreservation
• in vitro germination
• Olea europaea L
• viability

INTRODUCTION

Olive (Olea europaea L.) is andromonoecious, that is, individual trees bear both perfect and staminate flowers. The amount of flowers and flower types on the tree is unique for each cultivar and varies considerably from year to year. Each flower has two anthers (rarely three) and produces a large amount of pollen. The ‘Nabali Baladi’ olive cultivar produced about 177,271 pollen grains while ‘Rasie’ produced about 3,000,000 pollen grains in each flower (Ateyyeh et al., 2000). ‘Manzanillo’ and ‘Swan Hill’ olive cultivars produced about 85,000 pollen grains per anther (O'Rourke and Bauchmann, 1986). The pollen has elliptical and circular shape depending on the side of observation. They are trizonocolpated with reticulate exine structure (Al–Dehadheh et al., 2004; D'hallewin et al., 1990; Lanza et al., 1996).

Pollen storage is generally required for use in plant breeding for controlled crosses (Ganeshan, 1998) and for distributing and exchanging genetic material among locations and preserving germplasm, as well as for studies in basic physiology, biochemistry, fertility, and biotechnology involving gene expression, transformation and in vitro fertilization (Towill and Walters, 1998). On the other hand, pollen is of great significance in other research areas such as petroleum exploration, archaeology, criminology, and testing the purity of honey (Shivanna, 2003).

Optimum pollen storage conditions can vary greatly from one species to another (Shivanna and Johri, 1985). Pinney and Polito (1990) tested pollen of three olive cultivars, ‘Manzanillo’, ‘Ascolano’, and ‘Mission’ and stored them at −20°C for up to one year. Their results revealed little loss in germination ability over the storage period when stored either in sealed containers without regard to control of relative humidity or at 28–33% relative humidity. While Ferri et al. (2008) found that the best results for storing olive pollen were obtained when storage temperature was +4°C in comparison with +20°C and −20°C.

Pollen of other fruit trees has also been studied. In the case of Actinidia chinensis and Actinidia deliciosa, pollen stored in a deep freeze at −80°C was still viable after 160 weeks (Bomben et al., 1999). In another case, Martinez-Gomez et al. (2002) found that storage of almond pollen at subfreezing temperatures (−20 and −80°C) maintained its viability above 70% in ‘Nonpareil’, ‘Ne Plus Ultra’, and ‘Sonora’ and above 40% in ‘Peerless’. In papaya, pollen that was cryopreserved in liquid nitrogen for 485 days retained viability profiles as high as fresh pollen when germinated in vitro (Ganeshan, 1986).

This research aims at establishing short-, mid-, and long-term pollen storage techniques for two olive cultivars: ‘Rasie’ and ‘Nabali Baladi’.

MATERIALS AND METHODS

Pollen of olive (Olea europaea L.) was collected from an olive orchard on the campus of the University of Jordan. The experiment was conducted in the laboratories of Department of Horticulture and Crop Science, Faculty of Agriculture, the University of Jordan.

Pollen Collection and Storage

Inflorescences of two olive cultivars, ‘Rasie’ and ‘Nabali Baladi’, were collected during mid-May 2006 and early May 2007. The flowers were at the balloon stage just before anthesis. The inflorescences were spread over white paper on the bench in the laboratory at room temperature for one day to allow the anthers to dehisce. Pollen of each cultivar was then collected in glass tubes. Pollen in open glass tubes was dehydrated for 4 hours in desiccators, which were partially filled with dry silica gel. Dehydrated pollen was stored for a period of twelve months under the following storage conditions (three glass tubes in each storage condition): (a) normal room condition, (b) refrigerator (4°C), (c) freezer (−5°C), and (d) cryopreservation under −80°C over twelve months. Pollen viability and germinability were tested after 0, 2, 4, 8, 18, 28, and 52 weeks of anthesis.

Cryopreservation

Dehydrated pollen in three glass tubes was transferred into another three cryotubes, which were then sealed and immersed directly in liquid nitrogen for at least 15 minutes. After dehydration and submersing in liquid nitrogen, cryotubes were stored in a freezer at –80°C.

In Vitro Pollen Germination Test

Olive pollen germination was evaluated in vitro as described by Ateyyeh et al. (2000); the germination medium consisted of 0.8% agar, 10% sucrose, and 50 ppm citric acid. Pollen was immersed in olive oil before spreading over the media. In all cases, the Petri dishes were covered with aluminum foil, and then incubated in chamber room at 27°C for 12 hours. The number of germinated pollen grains was calculated under a light microscope at 10X magnification power. Pollen was considered to be germinated when the length of pollen tube was equal to or exceeded its diameter (Pinney and Polito, 1990). Fresh pollen of each cultivar was tested in vitro as comparison for all treatments.

Pollen Viability Test

The TTC (2, 3, 5–triphenlytetrazolium chloride) stain was used to test viability. To find the best TTC-sucrose combination for olive pollen, different concentrations of TTC (0.2, 0.4, 0.6, 0.8, and 1%) and sucrose (5, 10, 20, 40, and 60%) were tested. One drop of TTC-sucrose solution was placed on a microslide, then a small amount of pollen was suspended in that drop and a cover glass was placed onto the microslide. The covered microslide was wrapped with aluminum foil and then incubated in the chamber room at 30 ± 2°C for 60 minutes.; toward the end of the incubation period, the microslides were unwrapped and observed under a light microscope. Each TTC-sucrose combination was replicated 5 times, about 200 pollen grains from each replicate from four different areas were counted under a light microscope.

Data Analysis

The Statistical Analysis System from SAS Institute Corporation (SAS Institute Inc., 1999) was used to analyze the results. Pollen germinability and viability percentages of fresh pollen were analyzed using a one way analysis of variance. The comparisons among the treatments were carried out using a LSD test. Pollen storage results were analyzed using a split-plot in time, and least squares means were sorted with the pdmix800 macro developed by Saxton (1998).

RESULTS

Many combinations of TTC and sucrose examined in the viability test were discarded because they gave no results or a very low percentage. Viability percentage of the combination (1% TTC and 60% sucrose) was significantly higher than the other combinations, and accordingly it was used to test pollen viability in the rest of the experiment (Table 1). Germination and viability percentages of fresh pollen were significantly higher for ‘Rasie’ than for ‘Nabali Baladi’ in both seasons (Table 2).

TABLE 1 Effect of Different Combinations of TTC and Sucrose on Viability of Fresh Pollen of the Two Olive Cultivars

Combinations of TTC and sucrose	Viability percentage
	‘Rasie’	‘Nabali Baladi’
0.8% TTC + 20% sucrose	36.3 E	32.5 E
0.8% TTC + 40% sucrose	45.1 D	43.7 D
0.8% TTC + 60% sucrose	80.8 B	77.4 B
1% TTC + 20% sucrose	57.3 C	59.2 C
1% TTC + 40% sucrose	81.4 B	79.9 B
1% TTC + 60% sucrose	90.5 A	86.6 A
Means in columns having the same letters are not significantly different at P = 0.05.

TABLE 2 Germination and Viability Percentage of Fresh Pollen of the Two Olive Cultivars for Both Seasons

Cultivar	2006–2007		2007–2008
	Germination percentage	Viability percentage	Germination percentage	Viability percentage
Rasie	79.9 A	88.3 A	81.5 A	91.1 A
Nabali Baladi	56.8 B	82.7 B	67.8 B	86.3 B
Means in columns having the same letters are not significantly different at P = 0.05.

‘Rasie’ Cultivar

During the first season (2006–2007) germination percentage of pollen was significantly reduced in all storage conditions after 2 weeks, while viability percentage was significantly reduced only in the normal room condition and refrigerator treatments (Table 3). Meanwhile, cryopreserved pollen had the highest germination and viability percentage but not significantly higher than frozen pollen. After 4 weeks, there was no significant reduction in germination and viability percentage, except for the viability percentage of refrigerated pollen. Frozen pollen had the highest germination and viability percentage but not significantly higher than cryopreserved pollen. After 8 weeks, there was a significant reduction in germination and viability percentage of room-stored pollen and viability percentage in the freezer treatment. Frozen pollen had the highest germination percentage but not significantly higher than cryopreserved pollen, while viability percentage of cryopreserved pollen was significantly higher than pollen in other storage conditions. After 18 weeks, there was dramatic reduction in germination percentage of room-stored pollen. Cryopreserved pollen had the highest germination and viability percentage but not significantly higher than frozen pollen. After 28 weeks, there was no reduction in germination percentage in all storage conditions, but there was significant reduction in viability percentage of frozen and refrigerated pollen. Cryopreserved pollen had the highest germination and viability percentage, but germination percentage was not significantly higher than frozen pollen. After 40 weeks, there was significant reduction in germination percentage in all storage conditions, and there was significant reduction in viability percentage only in the room-storage treatment. Cryopreserved pollen had the highest germination and viability percentage, but germination percentage was not significantly higher than frozen pollen. After 52 weeks, room-stored pollen did not germinate and lost its viability, and the germination percentage of cryopreserved pollen was significantly reduced. Cryopreserved pollen had the highest germination and viability percentage but not significantly higher than frozen pollen.

TABLE 3 Effect of Storage Conditions on Pollen Germination and Viability of ‘Rasie’ Olive During 2006–2007

Storage period	Germination percentage				Viability percentage
	Room temp.	Refrig.	Freezer	Liquid N	Room temp.	Refrig.	Freezer	Liquid N
0 weeks	79.9 A	79.9 A	79.9 A	79.9 A	88.3 AB	88.3 AB	88.3 AB	88.3 AB
2 weeks	68.5 E-H	70.8 C-G	74.1 BC	75.0 B	64.6 L	80.3 E-H	86.8 AB	88.7 A
4 weeks	68.8 E-H	68.7 E-H	72.8 B-D	72.3 B-D	66.5 L	76.0 I-K	86.8 AB	86.7 AB
8 weeks	62.1 K	68.1 F-I	71.4 B-F	70.0 D-G	53.4 M	76.6 H-J	82.1 D-F	87.9 AB
18 weeks	26.4 M	66.2 H-J	69.8 D-H	72.9 B-D	54.4 M	78.0 G-J	82.8 C-E	86.4 A-C
28 weeks	24.0 M	67.4 G-J	71.6 C-F	71.9 B-E	53.3 M	72.4 K	77.7 G-J	86.9 AB
40 weeks	17.3 N	57.2 L	64.5 I-K	67.5 G-I	29.5 N	74.4 JK	78.9 F-I	84.8 B-D
52 weeks	0 O	57.3 L	61.0 K	63.9 JK	0 O	75.8 I-K	77.8 G-J	81.2 D-G
Means in columns and rows having the same letters are not significantly different at P = 0.05.

In the second season (2007–2008) after 2 weeks, germination and viability percentage were reduced significantly in all storage conditions except germination percentage in cryostorage (Table 4). Meanwhile, cryopreserved pollen had the highest germination and viability percentage but not significantly higher than frozen pollen. After 4 weeks, there was significant reduction in germination and viability percentage in all storage conditions except viability percentage in the refrigerator. Cryopreserved pollen had the highest germination and viability percentage, but viability percentage was not significantly higher than frozen pollen. After 8 weeks, germination and viability percentage of room-stored pollen was significantly reduced. Cryopreserved pollen had the highest germination and viability percentage, but viability percentage was not significantly higher than frozen and refrigerated pollen. After 18 weeks, germination percentage of room-stored and refrigerated pollen and viability percentage of room-stored pollen were significantly reduced. Cryopreserved pollen had significantly the highest germination and viability percentage, but germination percentage was not significantly higher than frozen pollen. After 28 weeks, germination percentage of room-stored and frozen pollen and viability percentage of room-stored and refrigerated pollen were significantly reduced. Cryopreserved pollen had the highest germination and viability percentage. After 40 weeks, there was a significant reduction in the germination percentage in all storage conditions except in the freezer; and the viability percentage of room-stored pollen was significantly reduced. Cryopreserved pollen had the highest germination and viability percentage, but germination percentage was not significantly higher than frozen pollen. After 52 weeks, room-stored pollen did not germinate and lost its viability, and the viability percentage of frozen pollen was significantly reduced. Cryopreserved pollen had the highest germination and viability percentage.

TABLE 4 Effect of Storage Conditions on Pollen Germination and Viability of ‘Rasie’ Olive During 2007–2008

Storage period	Germination percentage				Viability percentage
	Room temp.	Refrig.	Freezer	Liquid N	Room temp.	Refrig.	Freezer	Liquid N
0 weeks	81.5 A	81.5 A	81.5 A	81.5 A	91.1 A	91.1 A	91.1 A	91.1 A
2 weeks	72.8 C-E	72.7 C-E	75.2 BC	78.5 AB	82.2 C-F	80.5 D-G	84.3 BC	86.4 B
4 weeks	65.6 G-J	68.8 FG	67.6 F-I	74.9 C	77.6 G-J	77.7 G-J	79.6 E-H	81.7 C-G
8 weeks	43.1 N	67.9 F-H	64.5 H-K	74.1 CD	70.6 M	78.9 F-I	79.0 F-I	81.5 C-G
18 weeks	34.7 O	63.2 J-L	67.6 F-I	71.1 D-F	65.1 N	76.6 H-J	78.3 F-J	82.7 C-E
28 weeks	25.7 P	64.2 I-K	63.0 J-L	70.0 EF	50.0 O	71.9 LM	75.0 J-L	83.6 B-D
40 weeks	8.6 Q	60.6 LM	61.7 K-M	64.6 H-K	10.9 P	72.7 K-M	75.7 I-K	81.7 C-G
52 weeks	0 R	61.8 K-M	59.2 M	63.1 J-L	0 Q	69.8 M	71.4 LM	80.4 D-G
Means in columns and rows having the same letters are not significantly different at P = 0.05.

‘Nabali Baladi’ Cultivar

In the first season (2006–2007) after 2 weeks, germination percentage was reduced significantly in all storage conditions except in cryostorage, while viability percentage was reduced significantly only in the refrigerator treatment (Table 5). Cryopreserved pollen had the highest germination percentage, and frozen pollen had the highest viability percentage but not significantly higher than cryopreserved and refrigerated pollen. After 4 weeks, germination and viability percentage of room-stored and refrigerated pollen was significantly reduced. Cryopreserved pollen had significantly the highest germination percentage, while frozen pollen had significantly the highest viability percentage but not significantly higher than cryopreserved pollen. After 8 weeks, germination and viability percentage of room-stored pollen and viability percentage of refrigerated pollen were significantly reduced. Cryopreserved pollen had the highest germination and viability percentage, but viability percentage was not significantly higher than frozen pollen. After 18 weeks, germination and viability percentage of room-stored pollen was significantly reduced. Cryopreserved pollen had the highest germination and viability percentage, but not significantly higher than frozen pollen. After 28 weeks, germination and viability percentage of room-stored pollen and viability percentage of refrigerated pollen were significantly reduced. Cryopreserved pollen had the highest germination and viability percentage, but viability percentage was not significantly higher than frozen pollen. After 40 weeks, germination and viability percentage of room-stored pollen and germination percentage of refrigerated pollen were significantly reduced. Cryopreserved pollen had the highest germination and viability percentage, but viability percentage was not significantly higher than frozen pollen. After 52 weeks, room-stored pollen did not germinate and lost its viability, also germination percentage of cryopreserved pollen and viability percentage of refrigerated and frozen pollen were significantly reduced. Cryopreserved pollen had the highest germination and viability percentage but not significantly higher than frozen pollen.

TABLE 5 Effect of Storage Conditions on Pollen Germination and Viability of ‘Nabali Baladi’ Olive During 2006–2007

Storage period	Germination percentage				Viability percentage
	Room temp.	Refrig.	Freezer	Liquid N	Room temp.	Refrig	Freezer	Liquid N
0 weeks	56.8 A	56.8 A	56.8 A	56.8 A	82.7 A	82.7 A	82.7 A	82.7 A
2 weeks	45.3 G-I	46.6 F-H	47.6 F-H	55.7 AB	80.9 AB	78.6 BC	82.0 AB	81.3 A
4 weeks	33.7 M	38.8 J-L	48.0 E-H	55.2 A-C	66.4 EF	74.0 D	79.9 AB	79.6 A-C
8 weeks	26.8 N	38.9 J-L	47.6 F-H	54.0 A-D	40.9 H	70.0 E	80.3 AB	81.7 AB
18 weeks	16.2 O	37.8 KL	48.6 E-G	51.6 C-E	24.1 I	69.0 E	81.5 AB	81.0 AB
28 weeks	10.5 P	39.0 JK	46.4 F-H	52.6 B-D	19.3 J	63.4 F	79.4 A-C	81.4 AB
40 weeks	6.7 Q	35.0 LM	44.5 HI	50.1 D-F	11.0 K	64.5 F	79.9 AB	80.6 AB
52 weeks	0 R	33.2 M	42.0 IJ	44.4 HI	0 L	50.3 G	75.8 CD	78.3 BC
Means in columns and rows having the same letters are not significantly different at P = 0.05.

In the second season, after 2 weeks, germination and viability percentage were reduced significantly in all storage conditions except in cryostorage (Table 6). Cryopreserved pollen had the highest germination and viability percentage, but viability percentage was not significantly higher than room-stored and frozen pollen. After 4 weeks, germination and viability percentage room-stored pollen and germination percentage of refrigerated pollen were significantly reduced. Cryopreserved pollen had the highest germination and viability percentage. After 8 weeks, germination and viability percentage of room-stored pollen was significantly reduced. Cryopreserved pollen had the highest germination and viability percentage, but germination percentage was not significantly higher than frozen pollen. After 18 weeks, germination percentage of room-stored pollen and viability percentage of cryopreserved pollen were significantly reduced. Cryopreserved pollen had the highest germination percentage, while refrigerated pollen had the highest viability percentage, but not significantly higher than cryopreserved and frozen pollen. After 28 weeks, germination percentage of room-stored pollen and viability percentage of room-stored and cryopreserved pollen were significantly reduced. Cryopreserved pollen had the highest germination percentage, while refrigerated pollen had significantly the highest viability percentage but not significantly higher than cryopreserved and frozen pollen. After 40 weeks, germination percentage of room-stored and cryopreserved pollen and viability percentage of room-stored pollen were significantly reduced. Refrigerated pollen had significantly the highest germination and viability percentage, except that germination percentage was not significantly higher than cryopreserved pollen, and viability percentage was not significantly higher than cryopreserved and frozen pollen. After 52 weeks, room-stored pollen did not germinate and lost its viability, and there was significant reduction in germination percentage in other storage conditions; in addition viability percentage of refrigerated pollen was significantly reduced. Cryopreserved pollen had the highest germination percentage but not significantly higher than refrigerated pollen, while frozen pollen had the highest viability percentage but not significantly higher than cryopreserved pollen.

TABLE 6 Effect of Storage Conditions on Pollen Germination and Viability of ‘Nabali Baladi’ Olive During 2007–2008

Storage period	Germination percentage				Viability percentage
	Room temp.	Refrig.	Freezer	Liquid N	Room temp.	Refrig	Freezer	Liquid N
0 weeks	67.8 A	67.8 A	67.8 A	67.8 A	86.3 A	86.3 A	86.3 A	86.3 A
2 weeks	60.9 D-G	62.0 CD	63.3 B-D	68.2 A	81.0 B-E	80.6 C-F	81.7 B-D	85.0 AB
4 weeks	40.2 K	59.1 F-H	60.0 D-F	65.2 A-C	75.8 G-I	77.4 E-H	78.1 D-H	83.2 A-C
8 weeks	38.5 L	58.7 GH	62.7 C-E	65.6 A-C	46.1 L	78.8 D-G	77.1 F-H	84.8 AB
18 weeks	15.3 M	60.6 D-G	62.0 D-F	66.0 AB	47.7 L	79.1 D-G	76.7 F-I	77.1 F-H
28 weeks	10.0 N	60.1 E-H	60.0 E-H	65.4 A-C	32.8 M	80.7 C-F	76.9 F-I	78.0 D-H
40 weeks	5.2 O	61.1 D-G	57.4 HI	60.6 D-G	17.9 N	76.7 F-I	75.3 G-I	75.5 G-I
52 weeks	0 P	54.6 I	50.7 J	57.1 HI	0 O	59.6 K	75.1 G-I	74.1 HI
Means in columns and rows having the same letters are not significantly different at P = 0.05.

DISCUSSION

Pollen viability could be measured by tests such as the tetrazolium test, but there were many limitations to its use (Shivanna, 2003). Therefore, in addition to the tetrazolium test, an in vitro germination test was used in this experiment. Shivanna (2003) considered the in vitro germination test the most commonly used and acceptable test, but this decision was restricted by the optimum germination media. Ferri et al. (2008) tested 10 media suggested by previous researches on 11 advanced selections and 4 cultivars of olive. The highest germination percentage was 28. In this experiment, germination media improved by Ateyyeh et al. (2000) was used. It was more convenient, and the highest germination percentage was for the ‘Rasie’ cultivar was 81.5%. Selection of the best in vitro germination medium is important as it can affect the results of pollen storage conditions trials.

Regarding the germinability and viability of fresh pollen, the results showed significant differences between the two cultivars in both seasons. The pollen viability and germinability of ‘Rasie’ was significantly higher than that of ‘Nabali Baladi’, which was in contrast to the results of Ateyyeh et al. (2000). This contraction was illustrated by Eti and Stosser (1988) who mentioned that the pollen viability of different species and cultivars varies depending primarily on the environmental factors and nutritional conditions of species and cultivars.

Under room conditions, the pollen of the two cultivars lost their germinability and viability gradually over time, and after 52 weeks the pollen was nonviable. The pollen longevity of different species and cultivars varies with time depending primarily on the taxonomic status of the plant and on abiotic environmental conditions (Barnabas and Kovacs, 1997). Although the pollen viability and germinability showed the same trend, the pollen viability percentage was always higher than the pollen germination percentage. This could be associated with the loss of permeability to water, hence reducing pollen hydration and growth of the pollen tube, while maintaining viability (Ferri et al., 2008).

The maintenance of pollen viability, germinability, and the capability for tube growth over time clearly depends on storage conditions. Baja (1987) mentioned that the survival, longevity, and storability of pollen depends on the following factors: moisture content and humidity, storage temperature, nuclear condition of pollen, method of freezing, oxygen/air pressure, method of thawing and rehydration, genotype, method of collection, and the physiological state of the plant. Longevity has been found to be greatest at RH between 6 and 60% for most species, with optimum conditions typically being considerably below 60% (Stanley and Linskens, 1974). Therefore, pollen of the two cultivars was dehydrated in a desiccator and filled with dry silica gel for 4 hours before all storage conditions.

Theoretically, pollen can be stored in liquid nitrogen for many years since biological activity is stopped (Withers, 1991). Cryopreserved pollen of the two cultivars had the best results in short-, mid-, and long-term storage in both growing seasons. Other species were successfully cryopreserved for long periods such as walnut (Juvenal and Polito, 1985), papaya (Ganeshan, 1986), Actinidia deliciosa (Miaja and Me, 1992), tomato (Erik and Dina, 1996), and bromeliad (Parton et al., 2002). Frozen and refrigerated pollen of both olive cultivars were conveniently stored for short-, mid-, and long-periods. Pollen of three olive cultivars, ‘Manzanillo’, ‘Ascolano’, and ‘Mission’ showed little loss in germinability over the storage period when stored at −20°C for up to one year in controlled relative humidity (Pinney and Polito, 1990). While the best results of pollen storage of 11 advanced selections and 4 cultivars of olive (‘Carolea’, ‘Frantoio’, ‘Leccino’, and ‘Moraiolo’) were obtained when storage temperature was +4°C (Ferri et al., 2008). In this study, in both seasons frozen pollen had more stable results than refrigerated pollen. The same trend was found for pollen of Malus pumila L. stored at low temperatures. Pollen stored at −20°C and −30°C showed better germination percentages as compared to pollen stored at +4°C using fresh pollen, while freeze-dried pollen showed the highest germination percentage at −60°C (Perveen and Khan, 2008). Other species like pistachio were very difficult to store in the refrigerator and freezer, since refrigerated pollen retained their germinability for at least a week, while frozen pollen irreversibly lost most of their germinability (Vaknin and Eisikowitch, 2000).

Author is grateful to the Deanship of Academic Research–The University of Jordan for financial support.