Impaginato 441 Adv. Hort. Sci., 2019 33(3): 441-446 DOI: 10.13128/ahs-23998 Pollen viability and in vitro germination of six pistachio (Pistacia vera L.) cultivars grown in northern Jordan A. Aldahadha (*), K. Al Sane, A. Bataineh, A. Abu Alloush, Z. Hamouri National Agricultural Research Center (NARC), Baq’a, Jordan. Key words: in vitro germination, Jordan, pistachio, pollen storage, pollen viability. Abstract: This study was conducted on six pistachio cultivars (Lazaourdi, Nab-El Jamal, Boundiki, Batouri, Marawhi and Aschouri) to investigate the percentage of pollen viability and in vitro pollen germination under stored and non-stored (fresh) conditions. The results indicated that there was a significant interaction between pollen viability of pistachio cultivars and storage period. This study showed that the non-stored (fresh) pollen of cultivars Batouri and Lazaourdi had significantly the highest viability (87%) and in vitro pollen germination (69.7%), respectively; indicating that such cultivars could be used as best polli- nators. On the other hand, cultivar Nab-El Jamal had the lowest viability (43.7%) and in vitro pollen germination (40.3%). It was found that pollen viabili- ty for all stored pollen cultivars were significantly reduced by 30% when com- pared with non-stored (fresh) pollen. However, stored pollen germination for one month was zero for all pistachio cultivars. In addition, the results of viabili- ty for all fresh pollen cultivars were poorly linearly correlated (r2=0.149) with the results of in vitro germination of fresh pollen. Further research is required to examine both pollen viability and in vitro pollen germination under different short and long-term storage periods and methods. 1. Introduction Pistachio (Pistacia vera L.) is a member of the family Anacardiaceae (Ak et al., 2016). Pistachio trees are dioecious, meaning that the pistillate and staminate flowers are formed on different trees. Pistachio trees are wind pollinated as flowers have no petals to attract insects (Hosseini et al., 2015). One male tree is required for every eight female trees, but this ratio is usually not observed in orchards (Bahramabadi et al., 2018). Therefore, the amount of pollen produced in each cluster and germina- tion rate of pollen must be high in male trees (Ak et al., 2016). Pistachio is mainly cultivated in warm-temperate to subtropical parts of the world for its commercially valuable and edible seeds (Tilkat and Onay, 2009). The center of origin of pistachio species in the Near East includes the Central Asia and Turkey. Jordan ranks number 13 for pistachio production in the world and has a world share of 0.1 %. Production quantity of pistachio in Jordan increased from 10 tons in 1975 to 967 tons in 2016 (FAO, 2016), (*) Corresponding author: abdallah.aldahadha@narc.gov.jo Citation: ALDAHADHA A., AL SANE K., BATAINEH A., ABU ALLOUSH A., HAMOURI Z., 2019 - Pollen viability and in vitro germination of six pistachio (Pistacia vera L.) cultivars grown in northern Jordan. - Adv. Hort. Sci., 33(3): 441-446 Copyright: © 2019 Aldahadha A., Al Sane K., Bataineh A., Abu Alloush A., Hamouri Z. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 26 September 2018 Accepted for publication 24 April 2019 AHS Advances in Horticultural Science Short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(3): 441-446 442 with an increase in harvested area up to 301 hectares in 2016. In vitro pollen germination is a very useful tech- nique because it can unravel the physiological and biochemical conditions required for the successful pollen germination and pollen tube development (Sanjay et al., 2016). In addition, in vitro pollen ger- mination is one of the most convenient and reliable methods used to test the viability of fresh or stored pollen. However, pollen grains of Pistacia vera L. have been considered to be difficult to germinate in vitro (Golan-Goldhirsh et al., 1991). On the other hand, pollen viability usually refers to the ability to distribute functional sperm cells to the embryo sac following compatible pollination (Shivanna and Ram, 1993). The quality of pollen is evaluated on the basis of viability and vigor. Actually, pollen represents a critical stage in the life cycle of plants because viable pollen is essential for effective reproduction of sexual plants. There has been recurrent interest in developing reliable methods for short and long-term storage of pistachio pollen (Ateyyeh, 2012). Such methods would be useful in storing pollen to be used in pista- chio breeding programs (Vithanage and Alexander, 1985) and supplemental pollination programs in pis- tachio production (Crane and Iwakiri, 1981) which requires collection of sufficient amounts of pollen and its storage for short (hours to weeks) or long (months to years) periods, while maintaining viabili- ty (Shivanna and Sawhney, 1997). In addition, stor- ing pollen is very important for cross-pollination, crop breeding, physiology, biotechnology, plant bio- diversity and its conservation (Polito and Luza, 1988). In this study, six pistachio cultivars were grown in Maru Agricultural Research Station, Irbid, Jordan since 1984 (all cultivars were introduced from Syria). The percentages of pollen viability and in vitro pollen germination have never been tested in our orchard and these percentages may be varied among these tested cultivars. Thus, the overall aim of this experiment was to investigate in vitro germi- nation and pollen viability in six pistachio cultivars under non-stored (fresh) and stored pollen in refrig- erator for one month. In addition, this experiment was performed to find a correlation between in vitro pollen germination and viability, to check effi- ciency of stored pollen and to determine which pis- tachio cultivar could be recommended as best polli- nator. 2. Materials and Methods Plant material and location This research was carried out on six pistachio cul- tivars: Lazaourdi, Nab-El Jamal, Boundiki, Batouri, Marawhi and Aschouri from Pistachio orchard at Maru Agricultural Research Station. This station is located in Irbid governorate at 32° 33’ mN latitude, 35° 51’ E longitude and 589 m above mean sea level ( A l - G h z a w i e t a l . , 2 0 1 8 ) . M a r u h a s t y p i c a l Mediterranean climate conditions with hot and dry summer and an average annual precipitation of about 380 mm and represents an intermediate drought area. In vitro pollen germination and viability tests were carried out at the biotechnology laborato- ry of the National Agricultural Research Center (NARC), Baqa’a, Jordan. Pollen collection Pollen collection took place during the flowering period from 3rd to 6th of April, 2018. Pollen was col- lected from six pistachio cultivars. Upon anthesis, pis- tachio clusters of each cultivar were detached and shacked to a glass square. Care was taken through this process to prevent contamination. Afterwards, pollen of each cultivar was placed in a closed vial to be used later on. Pollen storage For each pistachio cultivar, samples of pollen were placed in small glass vials and these samples of non-stored (fresh) pollen were immediately taken to laboratory for in vitro germination and viability tests. Other samples were stored in refrigerator at 4°C for 1 month to compare in vitro pollen germination and viability with those of non-stored pollen. In vitro pollen germination test The medium used for in vitro germination testing as recently described by Ateyyeh (2012) contained 1 % a g a r , 1 5 % s u c r o s e a n d 1 0 0 p p m b o r i c a c i d (H3BO3). Pollen grains were placed on medium and incubated at 24°C for 24 hours. After this time, 100 pollens from each cultivar were counted using light microscope to estimate the percentage of in vitro pollen germination. For each cultivar, 3 replicates (petri-dishes) were used. Pollen is considered to be germinated if the developed pollen tube is exceed (2- 3 times) of its diameter. Pollen viability test Pollen viability was estimated as described by Ateyyeh (2012) by using 1% TTC (2, 3, 5-triphenyl Aldahadha et al. ‐ Pollen viability and in vitro germination of Pistacia vera L 443 tetrazolium chloride) and 60% sucrose. TTC-sucrose solution was stored in brown glass bottle in a refrig- erator. One drop of solution was placed onto micro- slide then a small amount of pollen was suspended in the drop and cover glass was placed onto the micro- slide, wrapped with aluminum foil and incubated in chamber room at 28°C for 60 minutes. After incuba- tion, 100 pollens from each cultivar were counted using light microscope to estimate the percentage of pollen viability. For each cultivar, 3 replicates (micro- slides) were used. Pollen grains stained orange or bright red color were considered viable. Experimental design and statistical analysis The experiment was performed in a factorial design with six pistachio cultivars and two storage treatments (non-stored and stored pollen for 1 month) to investigate pollen viability and in vitro pollen germination separately. There were three replicates for each cultivar and storage treatment. Data were analyzed by factorial ANOVA. When there were significant interactions, one-way ANOVA was used and means were separated using least sig- nificant difference (LSD). 3. Results and Discussion In vitro pollen germination Both proper pollination and pollen vigor are essential for pistachio productivity since the mar- ketable portion is the seed. To obtain a good fruit s e t , p o l l i n a t i o n a n d f e r t i l i z a t i o n a r e r e q u i r e d . Previous studies reported that the yield and quality of nuts were influenced by pollen performance in pistachio (Acar and Kakani, 2010). It has been indi- cated that the validity of the in vitro evaluation of pollen germination is a predictor of in vivo behavior (Acar and Kakani, 2010). The percentages of in vitro pollen germination for six pistachio cultivars are summarized in Table 1. The results indicated that the germinability of pistachio pollen varies according to the cultivar under non- stored (fresh) condition (Table 1). In particular, fresh pollen of cultivar Lazaourdi had significantly the high- est in vitro germination percentage (69.7%), followed by cultivars Aschouri and Boundiki, then cultivars Batouri and Marawhi. However, fresh pollen of culti- var Nab-El Jamal had significantly (P < 0.01) the low- est germination percentage (40.3%). In addition, Acar et al. (2010) found that under in vitro conditions, pollen germination showed that Atli, Uygur and Kaska male pistachio cvs were generally better than their F1 hybrids. Contrary to the reports of Polito and Luza (1988) who found that pistachio pollen lost its germinability after several days, preliminary tests of Vaknin and Eisikowitch (2000) revealed that fresh pollen lost most of its germinability within several hours. Results of Vaknin and Eisikowitch (2000) indicated that fresh- ly collected pollen showed the highest germination rate (76.7%). In addition, Günver-Dalkılıç and Dayı- Doğru (2011) found that pollen grain germination ratio for pistachio was changed between 78.22% and 63.29% under room conditions at initial day (day 1) in pistachio. However, pollen grain germination ratios were found between 55.83% and 43.26% in pistachio at the 2nd day storage in refrigerator. After one month of pollen storage in refrigerator, in vitro pollen germination was zero for all pollen pis- tachio cultivars (Table 1). This is supported precisely with findings of Ateyyeh (2012), suggesting that the Table 1 - In vitro pollen germination and pollen viability percentage of six pistachio cultivars grown in Maru Agricultural Research Station under non-stored (fresh) and stored pollen for one month at 4°C Data with the same letter in each column are not significantly different (least significant difference at P<0.05). Cultivars In vitro pollen germination (%) Pollen viability (%) Non-stored pollen (fresh) Stored pollen (1 month) Non-stored pollen (fresh) Stored pollen (1 month) Lazaourdi 69.7 a 0 66.7 b 43.0 d Nab-El Jamal 40.3 d 0 43.7 d 34.3 e Boundiki 60.0 b 0 55.0 c 35.7 e Batouri 51.0 c 0 87.0 a 53.7 c Marawhi 47.3 c 0 65.0 b 46.0 d Aschouri 62.3 b 0 85.0 a 67.0 b Mean 55 0 67 47 Standard error 1.64 0 1.64 3.24 LSD (0.05) 5.142 0 7.547 7.547 Adv. Hort. Sci., 2019 33(3): 441-446 444 stored pollen in refrigerator for one month is not effective method for in vitro pollen germination. Furthermore, the pollen grain germination ratios for pistachio were dramatically decreased and reached to about zero starting from the 4th day of storage under room conditions and at 10th day of storage under refrigerated conditions (Günver-Dalkılıç and Dayı-Doğru, 2011). Pistachio pollen could be stored in refrigerator just for two weeks, which is enough for artificial cross pollination purpose, if the differ- ence in flowering period between males and females didn’t exceed two weeks. Vaknin and Eisikowitch (2000) found that germinability of pollen kept in the refrigerator for six days was reduced but it retained about 60%. Pollen viability The results showed that the percentages of pollen viability are significantly different in regard to pista- chio cultivar under non-stored (fresh) and stored conditions (Table 1). Specifically, non-stored pollen of cultivars Batouri (87%) and cv. Aschouri (85%) had significantly the highest pollen viability percentage, followed by cultivars (Lazaourdi and Marawhi), and then cultivar Boundiki. Nevertheless, cultivar Nab-El Jamal had significantly (P<0.01) the lowest pollen via- bility percentage (43.7%). On the other hand, stored pollen for one month significantly (P<0.01) reduced pollen viability percentage in all pistachio cultivars when compared with non-stored pollen. For exam- ple, the highest and lowest pollen viability were in cultivars Aschouri (67%) and Nab-El Jamal (34.3%); respectively under stored conditions for one month (Table 1). The mean percentage value for fresh pollen viability for all pistachio cultivars was 67%, while for those of stored pollen viability was 47%. Thus, all pis- tachio cultivars lost approximately 30% of their pollen viability when pollen stored at 4°C in refrigera- tor. However, the mean value for fresh in vitro pollen germination for all pistachio cultivars was 55%. Therefore, the percentage of pollen germination was 18% less than those for pollen viability under non- stored conditions. Günver-Dalkılıç and Dayı-Doğru (2011) found that the highest and lowest pollen grain viability ratios were obtained as 88.24% (in safranin test) and 70.18% (in TTC test), respectively, in pistachio types (4 male pistachios (Pistacia vera L.) grafted on tere- binth). Ateyyeh (2012) found that fresh pollen viabili- ty of pistachio was 87.4% during 2006/2007 season. Ateyyeh (2012) found that pollen viability of pista- chio under refrigerated conditions for 4 weeks was 0 % a n d 3 8 . 7 % f o r s e a s o n s 2 0 0 6 / 2 0 0 7 a n d 2007/2008; respectively. In fact, many factors affect pollen viability and longevity such as genetic varia- tion between species, abiotic environmental condi- tions, temperature, moisture content, oxygen pres- sure, nutritional and physiological conditions under which the plants are grown, and the methods of pollen collection and storage (Barnabas and Kovacs, 1997). It was suggested that the loss of pollen viabili- ty in the course of short-term storage is directly related to changes in the water content of the pollen grains, rather than to a deficiency of essential metabolites (Barnabas and Kovacs, 1997). Correlation between tests There was a significant (P<0.05) regression of in vitro pollen germination on viability (Fig. 1), meaning that there is a relationship (linear correlation) between in vitro germination and viability for the f r e s h o r n o n - s t o r e d p o l l e n b u t w i t h p o o r f i t (r2=0.149). Particularly, in vitro pollen germination and viability percentages were similar for cultivars (Lazaourdi, Nab-El Jamal and Boundiki), However, for cultivars (Batouri, Marawhi and Aschouri), in vitro pollen germination was less than viability percent- ages. This is why the overall correlation between pollen viability and in vitro germination is weak under non-stored pollen condition. On the other hand, there was no correlation between in vitro pollen germination and viability when pollen stored in refrigerator at 4°C as pollen germination was zero for all tested pistachio cultivars. Pollen viability has been correlated with in vitro pollen germination in Banksia and some other Proteaceae Plants (Schori et al., 1992). A positive and highly significance correla- tion between different pollen viability stains and p o l l e n g e r m i n a t i o n t e s t i n M o m o r d i c a s p e c i e s Fig. 1 - Correlation between viability and in vitro germination of fresh pollen in six pistachio cultivars grown in Maru Agricultural Research Station. Significant regression at P<0.05. n=3. Aldahadha et al. ‐ Pollen viability and in vitro germination of Pistacia vera L 445 (Rathod et al., 2018), Even though, very high percent pollen viability was reported but when the germina- tion test was performed the pollen germination was not showed more than 70 %. It has been pointed out that in vitro pollen germination rates are considered the best indicator of pollen viability (Shivanna et al., 1991). Furthermore, a good correlation was revealed between in vitro pollen germination with fruit and seed setting in three ornamental tropical tree species (Sanjay et al., 2016). 4. 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