The effect of plant hormones on pollen germination and pollen tube growth of almond cultivars A. Radović a, D. Nikolić , D. Milatović , B. Zivković and N. Stevanović Faculty of Agriculture, University of Belgrade, Belgrade, Serbia.
Abstract The effects of two plant hormones (auxin - IAA and gibberellin – GA3) on pollen germination and pollen tube growth in vitro were investigated in five almond cultivars: ‘Tuono’, ‘Nessebar’, ‘Ferragnes’, ‘Ikar’ and ‘Exinogard’ during 2014 and 2015. Germination rate and pollen tube growth were determined on a culture medium containing 15% sucrose and 0.7% agar. Pollen germination in the control variant (without application of hormones) ranged from 23.56% (‘Ferragnes’) to 51.81% (‘Tuono’). Plant hormones mainly influenced an increase in pollen germination. The more pronounced effect of plant hormones was manifested on the length of pollen tubes. Thus, in the pollen treated with auxin, length of pollen tubes increased by 23 to 86%, and in pollen treated with gibberellins by 6 to 22%, depending on the cultivar. The exception was only the cultivar ‘Nessebar’ in which the gibberellin influenced the decrease in the length of pollen tubes. Keywords: Prunus dulcis, auxin, gibberellin, pollen germination in vitro, pollen tube length INTRODUCTION Almond is a highly appreciated fruit tree species. The almond kernel has a high nutritional, dietary, therapeutic and technical value, because it contains a large amount of oils, proteins, carbohydrates, minerals, vitamins, etc. The kernel of the almond is used as a table fruit and as a raw material in the food industry, culinary, pharmaceutical, cosmetics and chemical industries (Agunbiade and Olanlokun, 2006). For a successful fertilization and obtaining satisfactory yields of fruit trees, cultivars need to be characterized by good pollen germination. It varies between different cultivars of the same species of fruit trees (Stö sser 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 (DeCeault and Polito, 2010; Milatović and Nikolić , 2014), boric acid (Liu et al., 2013), fungicides (Yi et al., 2003), the presence of heavy metals (Gü r and Topdemir, 2008). One of the most important factors affecting pollen germination are plant hormones (Bolat and Pirlak, 2003; Tosun and Koyuncu, 2007). They have relatively low specificity because a large number act in a similar way, and depend on the type and concentration of the hormone, the physiological state of plant tissue and environmental conditions (Kastori, 2005). In addition to pollen germination, plant hormones influence the growth of pollen tubes (Sotomayor et al., 2012). Among plant hormones, auxins have particular impact on the growth of pollen tubes, such as IAA (indole-3-acetic acid). Wu et al. (2008a) found increased content of IAA in the pistil after pollination, on the basis of which it was concluded that IAA stimulates the growth of pollen tubes (Aloni et al., 2006). In addition to auxins, the influence of gibberellins on pollen germination and the growth of pollen tubes was also determined (Tosun and Koyuncu, 2007; Wu et al., 2008a). The aim of this study was to determine the effect of two plant hormones (auxin and gibberellin) on pollen germination and pollen tube growth in vitro in five almond cultivars.
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Acta Horticulturae 1139: 375-379
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MATERIALS AND METHODS Investigations were carried out at the Experimental farm ‘Radmilovac’ of the Faculty of Agriculture, University of Belgrade, Serbia. The subject of this research was five cultivars of almond: ‘Tuono’, ‘Nessebar’, ‘Ferragnes’, ‘Ikar’ and ‘Exinogard’ grafted on vineyard peach seedlings, with modified Slender Spindle training system and planting distance of 4.0×2.5 m. Researches were carried out during 2014 and 2015. For examination of pollen germination branches with flower buds in the ‘balloon’ phase were taken and carried to the laboratory. In order to collect pollen from the flower buds anthers were isolated in petri dishes. They are stored at room temperature (20°C) for 24-48 h to dry and to release the pollen. Then, 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 sowing of the pollen in petri dishes, the following plant hormones: auxin (IAA - indole-3-acetic acid at 0.3%) and gibberellin (GA3 – gibberellic acid at 0.0002%) were added. As the control, a variant without applying hormones was used. After 24 h at 20°C, petri dishes with a sowed pollen were observed under the light microscope ‘Leica DM LS’ (Leica Microsystems, Wetzlar, Germany), 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. Pollen tube length was measured from images using ‘Leica IM 1000’ program. From each cultivar 60 pollen tubes were measured. The obtained results were processed statistically using a three factorial analysis of variance. Percentage data were subjected to arcsin square root transformation before the statistical analysis. The significance of differences between the mean values was determined using the LSD test for significance level P≤0.05. Data analysis was performed using the statistical software package STATISTICA, Version 8 (StatSoft, Inc., Tulsa, Oklahoma, USA). RESULTS AND DISCUSSION Pollen germination differed significantly between the almond cultivars (Table 1). In control variant (without the use of hormones), it ranged from 23.56% in the cultivar ‘Ferragnes’ to 51.81% in the ‘Tuono’ cultivar (Table 2). The average pollen germination for all cultivars was 36.04%. Pollen germination rate was significantly different between the years. In all cultivars it was higher in 2014 compared to 2015. This can be explained by differences in weather conditions between the years during the period of pollen formation. Pollen germination in our study was lower compared to the results obtained by Sharafi (2011). He found that pollen germination ranged from 43.4 to 94.2% in almond cultivars grown in Iran. This may be due to genetic differences and differences in agro-ecological conditions. Table 1. Analysis of variance for pollen germination and pollen tube length of almond cultivars. Source of variation Cultivar (C) Year (Y) Hormone (H) C×Y C×H Y×H C×Y×H Error
Pollen germination df Mean squares 4 2113.67** 1 21384.87** 2 448.35** 4 686.34** 8 76.90ns 2 0.98ns 8 119.12** 60 37.96
** Significant at P≤0.05; ns non-significant.
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Pollen tube length df Mean squares 4 132292.18** 1 3144559.44** 2 454700.68** 4 99246.09** 8 22521.70ns 2 139447.42** 8 20555.57ns 60 18925.70
Table 2. The influence of plant hormones on pollen germination of almond cultivars (%). Cultivar Tuono Nessebar Ferragnes Ikar Exinogard Mean LSD (P≤0.05)
Control 2014 2015 68.29 35.34 42.58 17.51 29.81 17.32 54.82 16.37 62.82 15.53 51.66 20.41 Cultivar (C) Year (Y) Hormone (H)
Mean 51.81 30.05 23.56 35.60 39.17 36.04
2014 72.58 54.70 45.74 53.26 68.96 59.05 4.11 2.60 3.18
Auxin 2015 55.35 18.10 17.00 34.55 17.26 28.45
Mean 63.96 36.40 31.37 43.91 43.11 43.75 C×Y C×H Y×H C×Y×H
Gibberellin 2014 2015 66.45 42.54 43.31 22.04 42.70 20.86 45.65 17.34 75.50 17.61 54.72 24.08 5.81 10.06
Mean 54.49 32.67 31.78 31.50 46.55 39.40
Plant hormones (auxin and gibberellin) showed a statistically significant effect on pollen germination of the tested almond cultivars (Figure 1). Pollen treated by auxin had significantly higher pollen germination than the control one, and the average for all cultivars was 43.75%. The highest pollen germination was found in the cultivar ‘Tuono’ (63.96%) and the lowest in the cultivar ‘Ferragnes’ (31.37%). a
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Figure 1. Pollen germination of ‘Nessebar’ cultivar: a) without hormones (control variant), b) treated with auxin, c) treated with gibberellin. Unlike auxin, gibberellin had less impact on pollen germination. Pollen treated with gibberellin had significantly higher germination than in the control variant and amounted to 39.40% in average. It varied from 31.50% (‘Ikar’) to 54.49% (‘Tuono’). In cultivar ‘Ikar’ pollen germination treated with gibberellin was lower compared to the control. This indicates that gibberellin does not affect the increase in pollen germination in all tested almond cultivars. It is in line with results of Tosun and Koyuncu (2007) for sweet cherry. However, Sotomayor et al. (2012) studied the impact of seven growth regulators on almond pollen germination and found significantly higher pollen germination when treated with gibberellin compared to the control. Pollen tube length considerably varied, depending on the cultivar, hormone and year of study. In the control variant (without the use of hormones), it ranged from 441.6 µm in the cultivar ‘Ikar’ to 740.4 µm in the cultivar ‘Nessebar’ and averaged 571.3 µm (Table 3). Our results are consistent with those of Sharafi and Bahmani (2011). Plant hormones influenced the length of pollen tubes which is in line with the results of Wu et al. (2008b). In pollen treated by auxin, the length of pollen tubes varied from 689.3 (‘Ferragnes’) to 913.6 µm (‘Nessebar’), and was higher than in the control from 23 to 86% depending on the cultivar. Wu et al. (2008b) found that IAA is the most effective hormone that stimulates the growth of pollen tubes and this effect is even greater when used in combination with GA3.
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Table 3. The influence of plant hormones on pollen tube length of almond cultivars (μm). Cultivar Tuono Nessebar Ferragnes Ikar Exinogard Mean LSD (P≤0.05)
2014 802.3 1037.0 726.0 603.0 767.9 787.2
Control 2015 Mean 493.0 647.6 443.7 740.4 248.4 487.2 280.2 441.6 311.4 539.7 355.3 571.3 Cultivar (C) Year (Y) Hormone (H)
2014 1129.4 1240.1 935.4 942.7 902.9 1030.1 91.71 58.00 71.04
Auxin 2015 546.8 587.1 443.2 701.6 514.0 558.5
Mean 838.1 913.6 689.3 822.1 708.5 794.3 C×Y C×H Y×H C×Y×H
2014 953.2 828.5 599.5 509.0 617.2 701.5
Gibberellin 2015 476.6 397.1 440.4 574.5 528.5 483.4 129.70 100.47 -
Mean 714.9 612.8 520.0 541.8 572.9 592.5
In comparison with the auxin, gibberellin had a smaller influence on the length of pollen tubes, which is in accordance with the results of Wu et al. (2008a). With pollen treated with gibberellin, the length of pollen tubes ranged from 520.0 (‘Ferragnes’) to 714.9 µm (‘Tuono’) and it was from 6 to 22% higher than in the control variant. The exception was the cultivar ‘Nessebar’ in which the gibberellin influenced the decrease in the length of pollen tubes. Comparing to the control, the pollen tube length was significantly higher only in the cultivar ‘Ikar’. CONCLUSIONS Plant hormones (auxin and gibberellin) showed a significant effect on pollen germination and pollen tube growth of tested almond cultivars. The more pronounced effect was on the pollen tube growth. Auxin had a greater influence on pollen germination and pollen tube growth than gibberellin. It influenced the increasing of pollen germination and pollen tube length in all studied cultivars. ACKNOWLEDGEMENTS This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia through the project TR 31063. Literature cited Agunbiade, S.O., and Olanlokun, J.O. (2006). Evaluation of some nutritional characteristics of Indian almond (Prunus amygdalus) nut. Pak. J. Nutr. 5 (4), 316–318 http://dx.doi.org/10.3923/pjn.2006.316.318. Aloni, R., Aloni, E., Langhans, M., and Ullrich, C.I. (2006). Role of auxin in regulating Arabidopsis flower development. Planta 223 (2), 315–328 http://dx.doi.org/10.1007/s00425-005-0088-9. PubMed Bolat, I., and Pirlak, L. (2003). Effects of three plant growth regulators and boric acid on pollen germination and tube growth in apricot (Prunus armeniaca L.). Bangl. J. Bot. 32, 53–56. DeCeault, M.T., and Polito, V.S. (2010). High temperatures during bloom can inhibit pollen germination and tube growth, and adversely affect fruit set in the Prunus domestica cultivars ‘Improved French’ and ‘Muir Beauty’. Acta Hortic. 874, 163–168 http://dx.doi.org/10.17660/ActaHortic.2010.874.22. Gü r, 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 Appl. Sci. J. 4, 195–198. Kastori, R. (2005). Fiziologija Biljaka (Novi Sad, Srbija: Nauč ni Institut za Ratarstvo i Povrtarstvo), pp.527. 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. Liu, L., Huang, L., and Li, Y. (2013). Influence of boric acid and sucrose on the germination and growth of areca pollen. Am. J. Plant Sci. 04 (08), 1669–1674 http://dx.doi.org/10.4236/ajps.2013.48202. Milatović , D., and Nikolić , D. (2014). The effect of temperature on pollen germination and pollen tube growth of sour cherry cultivars. J. Agric. Sci. (Belgrade) 59, 45–52 10.2298/jas1401045m.
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