Pollen viability of eight quince cultivars (ʻLeskovackaʼ, ʻVranjskaʼ, ʻMoravaʼ, ʻPazardzijskaʼ, ʻHemusʼ, ʻAsenicaʼ, ʻPortugalʼ and ʻTriumphʼ), was studied in the two-year period (2011-2012). Testing of pollen viability was performed using two methods: the staining of pollen with acetocarmine (indirect method) and pollen germination in vitro with sucrose and agar-agar (direct method). Studied cultivars differed significantly in terms of pollen viability. The lowest percentage of stained pollen grains was detected in ʻLeskovackaʼ cultivar (70.29%) and the highest in the cultivars ʻAsenicaʼ, ʻHemusʼ and ʻTriumphʼ (over 90%). Similarly to that, the lowest percentage of pollen germination was obtained in ʻLeskovackaʼ cultivar (62.86%) and the highest in the cultivars ʻMoravaʼ, ʻAsenicaʼ, and ʻTriumphʼ (over 80%). With the exception of ʻPortugalʼ cultivar, the values of pollen viability determined by staining with acetocarmine were higher for 3-15% compared to the pollen germination in vitro. However, values obtained using these tho methods are highly positively correlated. On the basis of obtained results, the both methods can be recommended as reliable tests for pollen viability of quince, although priority should be given to the method of pollen germination in vitro, because it is more accurate. All tested cultivars are distinguished for high pollen viability, and can be successfully used as male parents in hybridization. In addition, they also can be recommended as a good pollenisers when are planting new quince orchards

Aleksandar Radovic

Dragan Milatovic

Dragan Nikolic

Ivana Bakic

Vera Rakonjac

English

Ss. Cyril and Methodius University in Skopje: Journals / Универзитет "Св. Кирил и Методиј"-Скопје

3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 68  POLLEN VIABILITY IN QUINCE CULTIVARS  Aleksandar Radovid¹, Dragan Nikolid¹, Dragan Milatovid¹, Vera Rakonjac¹, Ivana Bakid²  ¹Faculty of Agriculture, University of Belgrade, Belgrade, Serbia ²Institute for Science Application in Agriculture, Belgrade, Serbia  Corresponding author: radovicaleksandar@yahoo.com  Abstract Pollen viability of eight quince cultivars (ʻLeskovackaʼ, ʻVranjskaʼ, ʻMoravaʼ, ʻPazardzijskaʼ, ʻHemusʼ, ʻAsenicaʼ, ʻPortugalʼ and ʻTriumphʼ), was studied in the two-year period (2011-2012). Testing of pollen viability was performed using two methods: the staining of pollen with acetocarmine (indirect method) and pollen germination in vitro with sucrose and agar-agar (direct method). Studied cultivars differed significantly in terms of pollen viability. The lowest percentage of stained pollen grains was detected in ʻLeskovackaʼ cultivar (70.29%) and the highest in the cultivars ʻAsenicaʼ, ʻHemusʼ and ʻTriumphʼ (over 90%). Similarly to that, the lowest percentage of pollen germination was obtained in ʻLeskovackaʼ cultivar (62.86%) and the highest in the cultivars ʻMoravaʼ, ʻAsenicaʼ, and ʻTriumphʼ (over 80%). With the exception of ʻPortugalʼ cultivar, the values of pollen viability determined by staining with acetocarmine were higher for 3-15% compared to the pollen germination in vitro. However, values obtained using these tho methods are highly positively correlated. On the basis of obtained results, the both methods can be recommended as reliable tests for pollen viability of quince, although priority should be given to the method of pollen germination in vitro, because it is more accurate. All tested cultivars are distinguished for high pollen viability, and can be successfully used as male parents in hybridization. In addition, they also can be recommended as a good pollenisers when are planting new quince orchards.  Key words: Cydonia oblonga, pollen staining, pollen germination in vitro, breeding.  Introduction Achieving high yields and obtaining high quality fruits are main goals in production of each fruit species, including quince. In order to obtain high yields, successful pollination and fertilization are necessary. In these processes, one of the key factors is the pollen viability. Pollen viability was primarily determined by genotype. It varies between different cultivars of the same species of fruit trees (Stösser et al., 1996), and depends on rootstock on which the cultivar is grafted (Kidman et al., 2014). In addition to genetic factors, some environmental factors may also influence pollen germination (Sorkheh et al., 2011). The external factors influencing pollen germination are temperature (Chagas et al., 2008; Radovid et al., 2016a), boric acid (Liu et al., 2013), plant hormones (Sotomayor et al., 2012; Radovid et al., 2016b), fungicides (Kargar and Imani, 2011) and the presence of heavy metals (Gür and Topdemir, 2008). Two methods are most often used to test the pollen viability: pollen staining (indirect method) and pollen germination in vitro with sucrose and agar-agar (direct method). Pollen staining is a quick and cheap method in assessing pollen viability. However, Pearson (1984) states that pollen germination in vitro is a better indicator to evaluate pollen viability than the pollen staining. Knowledge of pollen viability is very important for breeding and production of quince. For breeders it is very important to know the functional ability of pollen before crossing. For producers it is important for the choice of appropriate pollenisers when planting new quince orchards. The aim of this study was to examine pollen viability of eight quince cultivars in order to determine the cultivars with the highest pollen viability. They will be recommended as a potentially good pollenisers when planting of quince orchards and as parents for breeding work.   3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 69  Material and methods Investigations were carried out at the Experimental field “Radmilovac” of the Faculty of Agriculture, University of Belgrade (Serbia) during the two-year period (2011-2012). The subject of this research was eight quince cultivars: ʻLeskovackaʼ, ʻVranjskaʼ, ʻMoravaʼ, ʻPazardzijskaʼ, ʻHemusʼ, ʻAsenicaʼ, ʻPortugalʼ and ʻTriumphʼ. The orchard was established in the spring of 1999, with the planting space of 4.5 m × 3 m. The examined cultivars were grafted on the rootstock ʻQuince MAʼ. For examination of pollen viability branches with flower buds in the ‘balloon’ phase were taken and carried to the laboratory. In order to collect pollen anthers were isolated from the flower buds in Petri dishes. They are stored at room temperature (20ºC) for 24-48 h to dry and to release the pollen. Testing of pollen viability was performed using two methods: the staining of pollen with acetocarmine (indirect method) and pollen germination in vitro with sucrose and agar agar (direct method). Pollen viability was tested immediately after the anthers drying and pollen release. For the pollen staining assay, 1-2% acetocarmine solution was used. One to two drops of acetocarmine were placed on the microscope slide, and the pollen was added using special needles. Immediately after placing, pollen was covered with a cover slip and observed under light microscope ‘Leica DM LS’ (Leica Microsystems, Wetzlar, Germany). The experiment was done in three repetitions. In each repetition at least 300 pollen grains were analyzed. Pollen grains that were colored in pink were considered vital, while pollen grains that lack vitality were not colored at all. From the ratio of the number of colored and uncolored pollen grains, the pollen vitality was calculated. For testing of pollen germination in vitro, the pollen of each cultivar was sown with fine brushes in Petri dishes (9 cm diameter) on the previously prepared nutrient medium consisting of 15% sucrose and 0.7% agar-agar. After 24 hours at 20ºC, Petri dishes with a sowed pollen were observed under the light microscope ‘Leica DM LS’, for counting of germinated pollen grains. The experiment was done in three repetitions, each including at least 300 pollen grains. Pollen is considered as germinated if the length of pollen tube was larger than the diameter of the pollen grain. The obtained results were processed statistically using the two-factorial analysis of variance. Percentage data were subjected to arcsin square root transformation before the statistical analysis. Tukey’s test (5%) was performed for means comparison. Correlations among the parameters were determined by correlation-regresion analysis and Pearson's correlation coefficients. Data analysis was performed using the statistical software package ‘Statistica’ (StatSoft, Inc., Tulsa, Oklahoma, USA).  Results and discussion Pollen staining is a quick method in assessing its viability. Vital pollen grains are pink, unlike non-functional pollen grains which do not colored at all (Figure 1). The number of stained pollen grains was quite high and significantly varied between the investigated quince cultivars. It was in the range from 70.29% in the ‘Leskovacka’ cultivar to 94.04% in the ‘Asenica’ cultivar (Table 1). In addition to the ‘Asenica’ cultivar, high levels of pollen staining (over 85%) were also found in the cultivars ‘Morava’, ‘Pazardzijska’, ‘Hemus’ and ‘Triumph’. The high values of pollen staining (over 90%) were also found in some quince cultivars in Turkey (Dalkiliç and Mestav, 2011).  Table 1. Pollen staining with acetocarmine of quince cultivars (%).  Cultivar/Year 2011 2012 Mx Leskovacka 62.81 77.78 70.29 e Vranjska 75.57 82.43 79.00 d Morava 86.11 87.90 87.00 bc Pazardzijska 81.10 83.47 82.29 cd Hemus 95.20 89.28 92.24 ab Asenica 94.40 93.67 94.04 a Portugal 74.73 78.94 76.83 de Triumph 88.83 92.20 90.52 abc Mx 82.34 b 85.71 a -  3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 70  In addition to the cultivar, the pollen staining was varied considerably between the years studied. It was slightly higher in 2012. The ‘Leskovacka’ cultivar had the lowest value of pollen staining in both years (62.81% - 2011 and 77.78% - 2012), while the highest values in 2011 were determined for the ‘Hemus’ cultivar (95.20%), and in 2012 in the ‘Asenica’ cultivar (93.67%).           Test of pollen germination in vitro is one of the main indicators of pollen viability (Figure 2). Pollen germination was significantly different among the quince cultivars. In accordance with the staining of pollen, the highest pollen germination was found in ‘Morava’ and ‘Asenica’ cultivars (84.34% and 84.19%). In addition to the ‘Morava’ and ‘Asenica’ cultivars, high pollen germination had ‘Triumph’ cultivar (81.82%) (Table 2). On the other hand, as with the staining of pollen, pollen germination rate was the lowest in the ‘Leskovacka’ cultivar (62.86%). The variability of pollen germination in our work was in the range determined by Dalkiliç and Mestav (2011) in the quince cultivars in Turkey.  Table 2. Pollen germination in vitro of quince cultivars (%). Cultivar/Year 2011 2012 Mx Leskovacka 75.33 50.39 62.86 d Vranjska 83.78 53.36 68.57 cd Morava 90.22 78.46 84.34 a Pazardzijska 89.89 61.61 75.75 bc Hemus 88.81 64.70 76.75 abc Asenica 86.03 82.35 84.19 a Portugal 80.66 75.08 77.87 ab Triumph 91.65 71.99 81.82 ab Mx 85.80 a 67.24 b -  Pollen germination significantly differed between years. Depending on the cultivar, it was for 4-30% lower in 2012. Different meteorological conditions during the development of pollen between the years studied were most likely contributed to this. Of the meterological factors, the greatest influence on pollen germination has the temperature (Pirlak, 2002; Milatovid and Nikolid, 2014). In both examined years, the smallest pollen germination was determined in the ‘Leskovacka’ cultivar (75.33% - 2011 and 50.39% - 2012), and the highest in the cultivars ‘Triumph’ (91.65% - 2011) and ‘Asenica’ (82.35% - 2012). Pollen staining and pollen germination in vitro were in a strong positive correlation (r = 0.80). That is in line with previous studies (Khatun and Flowers, 1995). Therefore, both these methods can be recommended as reliable in testing of pollen viability. However, the pollen germination in vitro should be preferred because it is stricter and more reliable.    Figure 1. Pollen staining with acetocarmine of Portugal cultivar.  Figure 2. Pollen germination in vitro of Morava cultivar.  3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 71  Conclusions The staining of pollen with acetocarmine and pollen germination in vitro are very important methods in assessing pollen viability in quince. These two methods were highly positively correlated. Pollen viability significantly varied among the studied quince cultivars. It was the lowest in ʻLeskovackaʼ cultivar, and the highest in the cultivars ʻMoravaʼ, ʻAsenicaʼ, and ʻHemusʼ. Generally, all tested cultivars are characterized by high pollen viability, and can be successfully used as male parents in hybridization and as a good pollenisers when planting new quince orchards.  Acknowledgements This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia through the project TR 31063.  References 1. Chagas, E.A., Barbosa, W., Saito, A., Pio, R. and Feldberg, N.P. (2008). Temperature, pH and development period on in vitro pollen germination in Pyrus calleryana. Acta Horticulturae, 800: 521-526. 2. Dalkiliç, Z. and Mеstаv, O. (2011). In vitro pollen quantity, viability and germination tests in quince. African Journal of Biotechnology, 10 (73): 16516-16520. 3. Gür, N. and Topdemir, A. (2008). Effects of some heavy metals on in vitro pollen germination and tube growth of apricot (Armeniaca vulgaris Lam.) and cherry (Cerasus avium L.). World Applied Sciences Journal, 4 (2): 195-198. 4. Kargar, M.H. and Imani, A. (2011). Effects of fungicides on pollen germination peach and nectarine in vitro. African Journal of Plant Science, 5: 643-647. 5. Khatun, S. and Flowers, T.J. (1995). The estimation of pollen viability in rice. Journal of Experimental Botany, 46 (1): 151-154. 6. Kidman, C.M., Dry, P.R., McCarthy, M.G., and Collins, C. (2014). Effect of rootstock on nutrition, pollination and fertilisation in 'Shiraz' (Vitis vinifera L.). Vitis, 53: 139-145. 7. Liu, L., Huang, L. and Li, Y. (2013). Influence of boric acid and sucrose on the germination and growth of areca pollen. American Journal of Plant Sciences, 4: 1669-1674. 8. Milatovid, D. and Nikolid, D. (2014). Uticaj temperature na klijavost polena i rast polenovih cevčica sorti višnje. Journal of Agricultural Sciences, 59: 45-52. 9. Pearson, H.M. (1984). Pollen viability in Rosa. HortScience, 19 (5): 710-711. 10. Pirlak, L. (2002). The effects of temperature on pollen germination and pollen tube growth of apricot and sweet cherry. Gartenbauwissenschaft, 67: 61-64. 11. Radovid, A., Nikolid, D., Milatovid, D. and Đurovid, D. (2016a). Uticaj temperature na klijavost polena i rast polenovih cevčica sorti kruške. Journal of Agricultural Sciences, 61 (4): 333-341 12. Radovid, A., Nikolid, D., Milatovid, D., Živkovid, B. and Stevanovid, N. (2016b). The effect of plant hormones on pollen germination and pollen tube growth of almond cultivars. Acta Horticulturae, 1139: 375-380. 13. Sorkheh, K., Shiran, B., Rouhi, V., Khodambashi, M., Wolukau, J.N., and Ercisli, S. (2011). Response of in vitro pollen germination and pollen tube growth of almond (Prunus dulcis Mill.) to temperature, polyamines and polyamine synthesis inhibitor. Biochemical Systematics and Ecology, 39: 749-757. 14. Sotomayor, C., Castro, J., Velasco, N., and Toro, R. (2012). Influence of seven growth regulators on fruit set, pollen germination and pollen tube growth of almonds. Journal of Agricultural Science and Technology B, 2: 1051-1056. 15. Stösser, R., Hartman, W., and Anvari, S.F. (1996). General aspects of pollination and fertilization of pome and stone fruit. Acta Horticulturae, 423: 15-22.  

POLLEN VIABILITY IN QUINCE CULTIVARS

Pollen viability of eight quince cultivars (ʻLeskovackaʼ, ʻVranjskaʼ, ʻMoravaʼ, ʻPazardzijskaʼ, ʻHemusʼ,
ʻAsenicaʼ, ʻPortugalʼ and ʻTriumphʼ), was studied in the two-year period (2011-2012). Testing of
pollen viability was performed using two methods: the staining of pollen with acetocarmine (indirect
method) and pollen germination in vitro with sucrose and agar-agar (direct method). Studied
cultivars differed significantly in terms of pollen viability. The lowest percentage of stained pollen
grains was detected in ʻLeskovackaʼ cultivar (70.29%) and the highest in the cultivars ʻAsenicaʼ,
ʻHemusʼ and ʻTriumphʼ (over 90%). Similarly to that, the lowest percentage of pollen germination
was obtained in ʻLeskovackaʼ cultivar (62.86%) and the highest in the cultivars ʻMoravaʼ, ʻAsenicaʼ,
and ʻTriumphʼ (over 80%). With the exception of ʻPortugalʼ cultivar, the values of pollen viability
determined by staining with acetocarmine were higher for 3-15% compared to the pollen
germination in vitro. However, values obtained using these tho methods are highly positively
correlated. On the basis of obtained results, the both methods can be recommended as reliable tests
for pollen viability of quince, although priority should be given to the method of pollen germination
in vitro, because it is more accurate. All tested cultivars are distinguished for high pollen viability,
and can be successfully used as male parents in hybridization. In addition, they also can be
recommended as a good pollenisers when are planting new quince orchards

Radović, Aleksandar

Nikolić, Dragan

Milatović, Dragan

Rakonjac, Vera

Bakić, Ivana

AgroSpace

International ScientificJournal          Vol. 72, No 1           April 2018ISSN 2545-4315Special Issue: Proceedings3rd International Symposium for Agriculture and Food18-20 October 2017, Ohrid, Republic of  MacedoniaINTERNATIONAL SCIENTIFIC JOURNALJOURNAL OF AGRICULTURAL, FOOD AND ENVIRONMENTAL SCIENCEShttp://www.fznh.ukim.edu.mk/jafes/The JAFES is an International scientific peer-reviewed Open Access Journal published twice per yearJAFES On line (e-ISSN 2545-4315) offers free access to all articles at http://www.fznh.ukim.edu.mk/jafes/Published by: “Ss. Cyril and Methodius" University in Skopje,Faculty of Agricultural Sciences and Food-SkopjeEDITORIAL BOARDЕditors in ChiefVjekoslav Tanaskovikj, Skopje, MacedoniaKocho Porchu, Skopje, MacedoniaAssociate EditorsSnezana Jovanović, Belgrade, SerbiaJovica Vasin, Novi Sad, SerbiaRadmila Stikić, Belgrade, SerbiaBiljana Škrbić, Novi Sad, SerbiaAna Marjanović Jeromela¸ Novi Sad, SerbiaBojan Srdljević, Novi Sad, SerbiaZoran Rajić, Belgrade, SerbiaJasmina Havranek, Zagreb, CroatiaMirjana Herak­ Ćustić, Zagreb, CroatiaVlasta Piližota, Osijek, CroatiaIvo Turšić, Zagreb, CroatiaDarko Vončina, Zagreb, CroatiaZlatan Sarić, Sarajevo, B&HJosip Čolo, Sarajevo, B&HMuhamed Brka, Sarajevo, B&HVelibor Spalević, Podgorica, MontenegroBozidarka Marković, Podgorica, MontenegroNazim Gruda, Bonn, GermanyVenelin Roychev, Plovdiv, BulgariaNasya Tomlekova, Plovdiv, BulgariaIrena Rogelj, Ljubljana, SloveniaDrago Kompan, Ljubljana, SloveniaMichael Murković, Graz, AustriaHristaq Kume, Tirana, AlbaniaSonja Srbinovska, Skopje, MacedoniaMarjan Kiprijanovski, Skopje, MacedoniaMarina Stojanova, Skopje, MacedoniaBiljana Kuzmanovska, Skopje, MacedoniaMirjana Jankulovska, Skopje, MacedoniaDragi Dimitrievski, Skopje, MacedoniaJOURNAL OF AGRICULTURAL, FOOD AND ENVIRONMENTAL SCIENCESAddress (Editorial Board)“Ss. Cyril and Methodius" University in Skopje Faculty of Agricultural Sciences and Food-SkopjeP.O. Box 297, MK-1000 Skopje,Republic of MacedoniaE-mail: jafes@fznh.ukim.edu.mkИздава:Универзитет „Св. Кирил и Методиј“ во Скопје,Факултет за земјоделски науки и храна ­ СкопјеУРЕДУВАЧКИ ОДБОРГлавни уреднициВјекослав Танасковиќ, Скопје, МакедонијаКочо Порчу, Скопје, МакедонијаУреднициСнежана Јовановиќ, Белград, СрбијаЈовица Васин, Нови Сад, СрбијаРадмила Стикиќ, Белград, СрбијаБилјана Шкрбиќ, Нови Сад, СрбијаАна Марјановиќ Јеромелa, Нови Сад, СрбијаБојан Срдљевиќ, Нови Сад, СрбијаЗоран Рајиќ, Белград, СрбијаЈасмина Хавранек, Загреб, ХрватскаМирјана Херак­Ќустиќ, Загреб, ХрватскаИво Туршиќ, Осијек, ХрватскаВласта Пилижота, Загреб, ХрватскаДарко Вончина, Загреб, ХрватскаЗлатан Сариќ, Сарајево, БиХЈосип Чоло, Сарајево, БиХМухамед Брка, Сарајево, БиХВелибор Спалевиќ, Подгорица, Црна ГораБожидарка Марковиќ, Подгорица, Црна ГораНазим Груда, Бон, ГерманијаВенелин Ројчев, Пловдив, БугаријаНасиа Томлекова, Пловдив, БугаријаИрена Рогељ, Љубљана, СловенијаДраго Компан, Љубљана, СловенијаМихаел Мурковиќ, Грац, АвстријаХристаќ Куме, Тирана, АлбаниаСоња Србиновска, Скопје, МакедонијаМарјан Кипријановски, Скопје, МакедонијаМарина Стојанова, Скопје,  МакедонијаБилјана Кузмановска, Скопје,  МакедонијаМирјана Јанкуловска, Скопје, МакедонијаДраги Димитриевски, Скопје, МакедонијаАдреса(Редакција)Универзитет „Св. Кирил и Методиј“ во СкопјеФакултет за земјоделски науки и храна ­ СкопјеП. фах. 297, МК­1000 Скопје,Република Македонија*THE AUTHORS ARE RESPONSIBLE FOR THE CONTENT AND FOR THE LANGUAGE OF THEIR CONTRIBUTION3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 68  POLLEN VIABILITY IN QUINCE CULTIVARS  Aleksandar Radovid¹, Dragan Nikolid¹, Dragan Milatovid¹, Vera Rakonjac¹, Ivana Bakid²  ¹Faculty of Agriculture, University of Belgrade, Belgrade, Serbia ²Institute for Science Application in Agriculture, Belgrade, Serbia  Corresponding author: radovicaleksandar@yahoo.com  Abstract Pollen viability of eight quince cultivars (ʻLeskovackaʼ, ʻVranjskaʼ, ʻMoravaʼ, ʻPazardzijskaʼ, ʻHemusʼ, ʻAsenicaʼ, ʻPortugalʼ and ʻTriumphʼ), was studied in the two-year period (2011-2012). Testing of pollen viability was performed using two methods: the staining of pollen with acetocarmine (indirect method) and pollen germination in vitro with sucrose and agar-agar (direct method). Studied cultivars differed significantly in terms of pollen viability. The lowest percentage of stained pollen grains was detected in ʻLeskovackaʼ cultivar (70.29%) and the highest in the cultivars ʻAsenicaʼ, ʻHemusʼ and ʻTriumphʼ (over 90%). Similarly to that, the lowest percentage of pollen germination was obtained in ʻLeskovackaʼ cultivar (62.86%) and the highest in the cultivars ʻMoravaʼ, ʻAsenicaʼ, and ʻTriumphʼ (over 80%). With the exception of ʻPortugalʼ cultivar, the values of pollen viability determined by staining with acetocarmine were higher for 3-15% compared to the pollen germination in vitro. However, values obtained using these tho methods are highly positively correlated. On the basis of obtained results, the both methods can be recommended as reliable tests for pollen viability of quince, although priority should be given to the method of pollen germination in vitro, because it is more accurate. All tested cultivars are distinguished for high pollen viability, and can be successfully used as male parents in hybridization. In addition, they also can be recommended as a good pollenisers when are planting new quince orchards.  Key words: Cydonia oblonga, pollen staining, pollen germination in vitro, breeding.  Introduction Achieving high yields and obtaining high quality fruits are main goals in production of each fruit species, including quince. In order to obtain high yields, successful pollination and fertilization are necessary. In these processes, one of the key factors is the pollen viability. Pollen viability was primarily determined by genotype. It varies between different cultivars of the same species of fruit trees (Stösser et al., 1996), and depends on rootstock on which the cultivar is grafted (Kidman et al., 2014). In addition to genetic factors, some environmental factors may also influence pollen germination (Sorkheh et al., 2011). The external factors influencing pollen germination are temperature (Chagas et al., 2008; Radovid et al., 2016a), boric acid (Liu et al., 2013), plant hormones (Sotomayor et al., 2012; Radovid et al., 2016b), fungicides (Kargar and Imani, 2011) and the presence of heavy metals (Gür and Topdemir, 2008). Two methods are most often used to test the pollen viability: pollen staining (indirect method) and pollen germination in vitro with sucrose and agar-agar (direct method). Pollen staining is a quick and cheap method in assessing pollen viability. However, Pearson (1984) states that pollen germination in vitro is a better indicator to evaluate pollen viability than the pollen staining. Knowledge of pollen viability is very important for breeding and production of quince. For breeders it is very important to know the functional ability of pollen before crossing. For producers it is important for the choice of appropriate pollenisers when planting new quince orchards. The aim of this study was to examine pollen viability of eight quince cultivars in order to determine the cultivars with the highest pollen viability. They will be recommended as a potentially good pollenisers when planting of quince orchards and as parents for breeding work.   3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 69  Material and methods Investigations were carried out at the Experimental field “Radmilovac” of the Faculty of Agriculture, University of Belgrade (Serbia) during the two-year period (2011-2012). The subject of this research was eight quince cultivars: ʻLeskovackaʼ, ʻVranjskaʼ, ʻMoravaʼ, ʻPazardzijskaʼ, ʻHemusʼ, ʻAsenicaʼ, ʻPortugalʼ and ʻTriumphʼ. The orchard was established in the spring of 1999, with the planting space of 4.5 m × 3 m. The examined cultivars were grafted on the rootstock ʻQuince MAʼ. For examination of pollen viability branches with flower buds in the ‘balloon’ phase were taken and carried to the laboratory. In order to collect pollen anthers were isolated from the flower buds in Petri dishes. They are stored at room temperature (20ºC) for 24-48 h to dry and to release the pollen. Testing of pollen viability was performed using two methods: the staining of pollen with acetocarmine (indirect method) and pollen germination in vitro with sucrose and agar agar (direct method). Pollen viability was tested immediately after the anthers drying and pollen release. For the pollen staining assay, 1-2% acetocarmine solution was used. One to two drops of acetocarmine were placed on the microscope slide, and the pollen was added using special needles. Immediately after placing, pollen was covered with a cover slip and observed under light microscope ‘Leica DM LS’ (Leica Microsystems, Wetzlar, Germany). The experiment was done in three repetitions. In each repetition at least 300 pollen grains were analyzed. Pollen grains that were colored in pink were considered vital, while pollen grains that lack vitality were not colored at all. From the ratio of the number of colored and uncolored pollen grains, the pollen vitality was calculated. For testing of pollen germination in vitro, the pollen of each cultivar was sown with fine brushes in Petri dishes (9 cm diameter) on the previously prepared nutrient medium consisting of 15% sucrose and 0.7% agar-agar. After 24 hours at 20ºC, Petri dishes with a sowed pollen were observed under the light microscope ‘Leica DM LS’, for counting of germinated pollen grains. The experiment was done in three repetitions, each including at least 300 pollen grains. Pollen is considered as germinated if the length of pollen tube was larger than the diameter of the pollen grain. The obtained results were processed statistically using the two-factorial analysis of variance. Percentage data were subjected to arcsin square root transformation before the statistical analysis. Tukey’s test (5%) was performed for means comparison. Correlations among the parameters were determined by correlation-regresion analysis and Pearson's correlation coefficients. Data analysis was performed using the statistical software package ‘Statistica’ (StatSoft, Inc., Tulsa, Oklahoma, USA).  Results and discussion Pollen staining is a quick method in assessing its viability. Vital pollen grains are pink, unlike non-functional pollen grains which do not colored at all (Figure 1). The number of stained pollen grains was quite high and significantly varied between the investigated quince cultivars. It was in the range from 70.29% in the ‘Leskovacka’ cultivar to 94.04% in the ‘Asenica’ cultivar (Table 1). In addition to the ‘Asenica’ cultivar, high levels of pollen staining (over 85%) were also found in the cultivars ‘Morava’, ‘Pazardzijska’, ‘Hemus’ and ‘Triumph’. The high values of pollen staining (over 90%) were also found in some quince cultivars in Turkey (Dalkiliç and Mestav, 2011).  Table 1. Pollen staining with acetocarmine of quince cultivars (%).  Cultivar/Year 2011 2012 Mx Leskovacka 62.81 77.78 70.29 e Vranjska 75.57 82.43 79.00 d Morava 86.11 87.90 87.00 bc Pazardzijska 81.10 83.47 82.29 cd Hemus 95.20 89.28 92.24 ab Asenica 94.40 93.67 94.04 a Portugal 74.73 78.94 76.83 de Triumph 88.83 92.20 90.52 abc Mx 82.34 b 85.71 a -  3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 70  In addition to the cultivar, the pollen staining was varied considerably between the years studied. It was slightly higher in 2012. The ‘Leskovacka’ cultivar had the lowest value of pollen staining in both years (62.81% - 2011 and 77.78% - 2012), while the highest values in 2011 were determined for the ‘Hemus’ cultivar (95.20%), and in 2012 in the ‘Asenica’ cultivar (93.67%).           Test of pollen germination in vitro is one of the main indicators of pollen viability (Figure 2). Pollen germination was significantly different among the quince cultivars. In accordance with the staining of pollen, the highest pollen germination was found in ‘Morava’ and ‘Asenica’ cultivars (84.34% and 84.19%). In addition to the ‘Morava’ and ‘Asenica’ cultivars, high pollen germination had ‘Triumph’ cultivar (81.82%) (Table 2). On the other hand, as with the staining of pollen, pollen germination rate was the lowest in the ‘Leskovacka’ cultivar (62.86%). The variability of pollen germination in our work was in the range determined by Dalkiliç and Mestav (2011) in the quince cultivars in Turkey.  Table 2. Pollen germination in vitro of quince cultivars (%). Cultivar/Year 2011 2012 Mx Leskovacka 75.33 50.39 62.86 d Vranjska 83.78 53.36 68.57 cd Morava 90.22 78.46 84.34 a Pazardzijska 89.89 61.61 75.75 bc Hemus 88.81 64.70 76.75 abc Asenica 86.03 82.35 84.19 a Portugal 80.66 75.08 77.87 ab Triumph 91.65 71.99 81.82 ab Mx 85.80 a 67.24 b -  Pollen germination significantly differed between years. Depending on the cultivar, it was for 4-30% lower in 2012. Different meteorological conditions during the development of pollen between the years studied were most likely contributed to this. Of the meterological factors, the greatest influence on pollen germination has the temperature (Pirlak, 2002; Milatovid and Nikolid, 2014). In both examined years, the smallest pollen germination was determined in the ‘Leskovacka’ cultivar (75.33% - 2011 and 50.39% - 2012), and the highest in the cultivars ‘Triumph’ (91.65% - 2011) and ‘Asenica’ (82.35% - 2012). Pollen staining and pollen germination in vitro were in a strong positive correlation (r = 0.80). That is in line with previous studies (Khatun and Flowers, 1995). Therefore, both these methods can be recommended as reliable in testing of pollen viability. However, the pollen germination in vitro should be preferred because it is stricter and more reliable.    Figure 1. Pollen staining with acetocarmine of Portugal cultivar.  Figure 2. Pollen germination in vitro of Morava cultivar.  3rd INTERNATIONAL SYMPOSIUM FOR AGRICULTURE AND FOOD – ISAF 2017 __________________________________________________________________________________ 71  Conclusions The staining of pollen with acetocarmine and pollen germination in vitro are very important methods in assessing pollen viability in quince. These two methods were highly positively correlated. Pollen viability significantly varied among the studied quince cultivars. It was the lowest in ʻLeskovackaʼ cultivar, and the highest in the cultivars ʻMoravaʼ, ʻAsenicaʼ, and ʻHemusʼ. Generally, all tested cultivars are characterized by high pollen viability, and can be successfully used as male parents in hybridization and as a good pollenisers when planting new quince orchards.  Acknowledgements This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia through the project TR 31063.  References 1. 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Pollen viability in quince cultivars

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POLLEN VIABILITY IN QUINCE CULTIVARS

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