Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(3): 63-73, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-258 Citation: Y. Sharafi (2019) Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy. Caryolo- gia 72(3): 63-73. doi: 10.13128/cayolo- gia-258 Published: December 13, 2019 Copyright: © 2019 Y. Sharafi. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy Yavar Sharafi Department of Horticultural sciences, Faculty of Agriculture, Shahed University, Tehran, Iran E-mail: y.sharafi@shahed.ac.ir Abstract. Zinc is classically the second abundant moveable metal in plants after iron and represented in all enzyme classes. Zinc generally contributes in the biosynthesis of IAA and GA3 phytohormones which play the major role in fertilization and fruit set. Zinc deficiency leads to reduction in leaf and shoot size, photosynthesis and finally decreases fruit set. Foliar Zinc spray was shown to be efficient and fast for improving Zinc deficiency in fruit trees. In this research the effects of Zinc solution by (0, 3000 and 5000 mg. L-1) were studied on pollen penetration to the pistil and ovary in the four apple cultivars crosses which included “Golden Delicious”, “Red Delicious”, “Gala” and “Fuji”. Spraying was done on the shoots two weeks before blooming in the spring. Pollen penetration was studied using fluorescent microscopy technique 72 and 120 hours after field pollination. Results revealed that the effects of Zinc, crosses and their interaction were significant on pollen germination on the stigma and tube penetration into the primary, middle and beginning of the ovaries and the highest pollen germina- tion on the stigma (43.5%) was observed in the cross (♀Golden Delicious × Gala♂), in 3000 mg. L-1 of Zinc 120 hours after pollination and highest pollen tube penetration into the ovary (12/88%) was observed in this cross, respectively. Finally, it was shown that fluorescence microscopy is an accurate technique for nutrition assay in pollination and fruit set. The foliar application of Zinc increased pollen germination and pollen tube growth in all of the crosses. Keywords. Fruit set, micronutrient, ovary, pollination, pollen tube growth. INTRODUCTION Pollen can be used advantageously by breeders, geneticists, physiologists, germplasm supervisors and growers (Dafni and Frimage 2000; Sharafi et al. 2017; Sedgley 1990). In higher plants, pollen grains carry the male gam- ete on the female part of a flower and play a vital role in breeding program and assist in successful fruit set (Dafni et al. 2012; Hisock and Allen 2008; Sedgley 1990). Generally high crop yield is dependent on viable pollen grains (Dafni et al. 2005). Pollen fertility and viability have dominant prominence in natural and artificial hybridization programs (Sedgley 1990). 64 Yavar Sharafi Zinc (Zn) is an essential micronutrient in plants and has a vital role in cell division, nucleic acid metab- olism, protein synthesis, photosynthesis, carbohydrate metabolism, and phytohormones regulation (Broadley et al. 2007; Chen et al. 2008). Zn directly contributes in the biological synthesis of auxin (IAA) and gibberel- lin (GA3) which have utmost roles in the plant growth, pollination, fertilization and fruit set in fruit trees (Broadley et al. 2007). Also, Zinc plays a major role as a cofactor in the structure and function of more than 300 enzymes in plants, such as Cu/Zn superoxide (Cu/Zn- SOD), carbonic anhydrase (CA), and sorbitol dehydroge- nase (SDH). Zinc toxicity in plants is far less widespread than Zn deficiency (Andreini and Bertini 2012; Moghad- am et al. 2013; Nosarszewski et al. 2004; Weinthal et al. 2010). It was reported that about 30% of the agricultural soils in the world show Zn deficiency and Zinc is the most common micronutrient deficient, mostly in high- pH soils (Alloway 2008). Fruit trees which grown in such soils encounter Zn deficiency and show both poor growth and yield quantity and quality. On the other hand, apple (Malus domestica L.) commercial cultivars are self- incompatible and therefore, need to be planted along with cross-compatible pollinizer that generates sufficient favorable pollen (Hegedus et al. 2012; Losada and Herrero 2014; Sedgley 1990; Ramirez and Davenpor 2013). On the contrary, apple trees are highly susceptive to Zn deficiency (Alloway 2008). Zn deficiency decreas- es leaf and shoot size and reduces photosynthetic rates, and thus influences the apple yield and quality (Yan et al. 2010). Zinc deficiency in apple trees is observed as small leaves, late opening of flower and leaf buds, chlo- rosis between the lateral veins of the leaves, and terminal dieback (Marshner 2011; Macdonal 2000; Sedgley 1990;). In many fruit trees foliar applications of Zn have been effectively used to promote tree vigor, fruit set, and yield (Wojcik 2007). Some researchers (Golzer and Grant 2006; Qin 1996; Song et al. 2015; Song et al. 2016; Yadav et al. 2013; Zhang et al. 2014) have reported that in the most fruit trees; foliar applications of Zn on mature leaves is unsuccessful and does not provide significant Zn to new leaves produced after spray application or in the following spring. The best time for Zn foliar applica- tion is nearly after fruit harvest in the autumn or imme- diately after pistillate flower senescence followed by two weeks later. Zhang et al (2013 and 2016) reported that Zinc sul- fate spray before bud break increases the activity of car- bohydrate metabolic enzymes and regulates endogenous hormone levels in fruits of Fuji apple cultivar. Solar and Stampar (2001) reported that the yield of hazelnut trees was highest in the treatment with 2000 mg.kg–1 B + 2000 mg.L–1 Zn and lowest in the treatment with 1000 mg.L–1 B + 1000 mg.L–1 Zn. Hipps and Davies (2000) reported that foliar appli- cation of Zn after blooming could increase the Zn con- centration of apple fruit; and spraying Zn on leaves in autumn notably improves the flower Zn content in the coming year. Also, foliar Zn application promotes polli- nation and cell division. Moreover, foliar application of Zn was shown to be effective and fast for improving Zn decreasing symp- toms in many plants (Sanchez and Righetti 2002). Vari- ous studies in palm, citrus and apple showed that foliar application of Zn can significantly increase the Zn con- centration, fruit yield and quality (Karimi et al. 2017; Keshavarz et al. 2011; Rodríguez et al. 2005; Neilsen et al. 2004; Neilsen et al. 2005 Khayyat et al. 2007; Zhang et al. 2013). Boron, Iron and Zinc foliar applications have been observed to have a positive effect on chlorophyll contents in B, Fe and Zn deficient plants. Pollination is one of the most critical stages in the life cycle of a flowering plant, involving a complex series of cell-cell interactions that constitute the pollen-pistil interaction (Dafni et al. 2005; Dafni et al. 2012; Hisock and Allen 2008). In order for fertilization, pollen must first establish molecular compatibility with the stigma and then germinate to produce a pollen tube that pen- etrates the stigma and grows through the transmitting tissue of the style to locate on the ovule within the ovary (Radonic et al. 2017). Initiation and successful comple- tion of this sequence of events depends upon the stig- ma and style providing the exact requirements for pol- len germination and sustained growth and guidance of the pollen tube through the pistil and ovary. The pollen must therefore be programmed to respond appropriately at every step of this interaction. (Losada and Herrero 2014; Dafni et al. 1998; Nepi and Franchi 2000; Sedgley 1990; Shivanna 2003; Rodriguez-Riano and Dafni 2000). Zn deficiency can have a marked effect on pollination by affecting pollen production, pollen physiology, floral anatomy, and fruit set (Usenik and Stampar 2002). It has been demonstrated that the first action of stig- ma is to hook the pollen grains on its surface. For this mechanism receptive stigma must have an adhesive sur- face. Pollen-stigma interaction is instituted after adhe- sion of pollen grains on the stigmatic head and multifold incidents occur. The first step is the hydration of the pol- len grain and release of wall proteins that bind to recep- tors on the stigma surface (Radunic et al. 2017; Yellof and Hunt 2005). Also, f lorescence microscopy technique accom- plished to study pollen tube growth after field and lab- oratory-controlled pollination and used to identify the 65Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy self- and cross-(in) compatibility of cultivars, effective pollination period (EPP), and effects of pollen types on fruit set (Altagic et al. 2012; Kubitscheck 2017). Further- more, it has not been used for the investigation of the effects of macro and micro nutrients on the pollen ger- mination and tube grow especially in the apple cultivars. In spite of numerous researches on the response of deficient fruit trees to Zn foliar application, there is no enough information directly appropriate to apple trees. It could be said that because of the five partitions of the apple flower stigma and style; pollen penetration assays in the style have not been used for the screening of nutritional elements on the flower buds (Mularczyk- Oliwa et al. 2017; Sheffield et al. 2005). The objective of this study was to assess the effects of foliar application of Zn on the apple trees two weeks before bud break in the spring in some crosses by flores- cence microscopy technique. MATERIALS AND METHODS Plant materials, Zn treatments, crosses and pollination This research was carried out on four apple cul- tivars which included “Golden Delicious”, “Red Deli- cious”, “Gala” and “Fuji”. All of the trees were 12-year- old on EM126 rootstocks and foliar sprayed by ZnSO4 as the Zn source (0, 3000 and 5000 mg. L-1) two weeks before bud break in the spring. In the beginning volume of each treatment was calculated based on the fruit trees number and then ZnSO4 was dissolved in distilled water and so sprayed with sprayer on the shoots. Spraying was done in the in the morning (7-8 O clock), when the sky was cloudy and the weather moisture was 70%. Crosses among the cultivars (six crosses) were programmed as ♀Red delicious × Golden delicious♂, ♀Gala× Fuji♂, ♀Red delicious × Fuji♂, ♀Red delicious × Gala♂, ♀Golden delicious × Fuji♂ and ♀Golden deli- cious × Gala♂. For each cross four repeats in all direc- tion of the trees were considered and, in each repeat, at least 2 branches with 60 – 100 flower buds were labeled in winter. Selected female cultivar’s flower buds were bagged at ‘Balloon’ stage to prevent the entrance of for- eign pollens on the closed pistils. Pollens were collected from the male cultivar flower buds and maintained in freezer until usage in the field pollination time. Pollen germination was tested in an in vitro medium before field application on the pistils. Selected female cultivar’s flowers were pollinated with selected male parent pollen when the pistils were acceptable for pollens and repeated after 24 hours to increase the pollination accuracy. Florescence microscopy assessment Based on the apple trees EPP; 72-120 h is enough for the pollen tubes to reach the ovary hence, pollinated flow- ers were collected at 72 and 120 h after pollination, sliced and fixed in acid FAA solution (5 % v/v Formaldehyde (40%), 90 % v/v Alcohol ethylic 70% and 5 % Acetic acid glacial 96 %). After rinsing with water two to three times, pistils were cleared in 16% NaOH at room temperature for three days. They were then rinsed in water and stained with 0.1% aniline blue in 0.1% K2HPO4. Each part of the pistil was placed on a microscope slide with 10% glycerol and squashed under a glass cover slip. The number of pol- len tubes and the rate of pollen tube growth in the differ- ent parts of the style were measured using fluorescence microscopy (Olympus AX70). In each pistil the number of germinated pollen grains on the stigma, the number of pollen tubes in the upper and middle parts of the style and in the beginning of the ovary were determined by a fluorescent microscope (Fig 1). Pollen germination per- centage was determined by dividing the number of ger- minated pollen grains by the total number of pollen on the stigma and expressed as a percentage and normalized by angular transformation. The mean of the pollen tube number was calculated as the average number of pollen tubes in different parts of 10 pistils at least. Due to the five partitions of the apple flower stigma and style; the mean of the 5 parts was evaluated for each of them. Experimental design and data analysis The experiment was carried out as a factorial based on completely randomized design with three factors Fig 1. Florescence microscopy photographed from pollens of Red delicious germinated on the Golden delicious stigma and pollen tubes penetrated to the upper part of the style. 66 Yavar Sharafi including Zn in three levels (0, 3000 and 5000 mg. L-1), time (72 and 120 hrs.’ after pollination) and six crosses in five replications (at least ten styles per cross). The data was analyzed using SPSS (24) software. Mean values were analyzed by Duncan’s multiple range test. RESULTS The analysis of variance showed that the crosses of four cultivars “Golden Delicious”, “Red Delicious”, “Gala” and “Fuji” had a significant effect on pollen ger- mination percentage on the stigma and pollen tube number which penetrated into the upper and middle parts of the of style and also in the beginning of the ovary at p≤0.01 respectively. Also, Zn concentration and time independently affected the pollen germination per- centage on the stigma, the number of pollen tubes which penetrated to the upper and the middle parts of the style and the primary part of the ovary significantly at p≤0.01 (Table 1). The interaction among Zn concentra- tion × crosses was significant on the pollen germination on the stigma, penetration of pollen tubes in the upper and middle parts of the style and so the beginning of the ovaries at p≤0.01 (Table 1), but the interaction of crosses × time was not significant on the studied traits (Table 1). However, three ways interaction among the time × crosses × Zn concentration was not significant on the pollen germination percentage on the stigma, the num- ber of pollen tubes that penetrated into the middle and the end of the style and the number of pollen tubes in the beginning of the ovary (Table 1). Based on our findings, the foliar application of Zn on the apple trees enhanced the growth of pollen tubes toward the ovary. In addition, the use of Zn increased the pollen germination on the stigma. About 85 to 90% of pollen germination on the stigma occurred 48 hours after pollination in pollen which was treated by Zn (data not shown). The highest pollen germination percentage on the stigma (43.5%) was observed in the cross (♀Golden Delicious × Gala♂), in 3000 mg. L-1 of Zn 120 hours after pollination and the highest pollen tube penetration into the ovary (12/88%) was observed in this cross, respectively. These results may be related to the Zn positive effects on the cell division in pollen tube followed by elongation lead to arrival to the ova- ries (Fig. 2). In comparison with 3000 mg. L-1 and 5000 mg. L-1 concentration of the Zn, pollen germination rate decreased significantly and showed a toxic effect on pol- len. In the crosses which both of pollen parents style parents and were treated by 5000 mg. L-1 Zn, pollen germination on the stigma was decreased. This could be related to the toxic effect of Zn. The concentration of 3000 mg l-1 of Zn has a positive effect on germina- tion percentage and penetration of pollen tubes, and has been effective in maintaining and integrating the mem- brane of pollen cells. In this research pollen tubes which penetrated into the style and ovary was also affected. It appeared that the use of Zn on both the male and female parents led to increase in the pollen germination on the stigma in the in-vivo condition. However, in trees treated with 5000 mg. L-1 Zn, the number of pollen tubes and the swelling of the tip of the tubes were significantly reduced; this was in accordance with some researchers reports regarding the negative effects of Zn on the vital phenomenon in high concen- tration (Marschner, 2011; Sedgley, 1990; Sharafi et al., 2017; Zhang et al., 2013; Zhang et al., 2016) Based on the results of Figure. 1, the highest pollen germination percentage was observed in the cross ♀ Red delicious × Gala ♂ with 43.51% and the highest pollen tube penetration in the cross ♀Red delicious × Golden delicious with 19.27% respectively (Fig. 1). Table 1. Analysis of variance of the effect of time, crosses and Zn on the pollen germination on the stigma and tube penetration to upper and middle part of the style and so beginning of the ovary. Beginning of ovary Middle of style Upper of style Stigma Df Sources of Variation 9.01 ns 12.10 ns **55.07 **149.64 5 Cross **149.03 **207.02 **239.06 **4120.07 2 Zn concentration **765.05 **601.04 **974.05 **2245.44 1 Time 7.01 ns 15.00 ns 10.01 ns 55.97ns 5 Cross × time **32.04 **02.48 96.50** 83.02 ** 10 Cross × Zn *35.07 *52.06 38.07 * **327.71 2 Zn ×Time 14.01 ns 38.07 ns 19.01 ns 29.01ns 10 Cross × Zn ×Time 9.04 14.01 13.04 27.03 ns 144 Error 179 Total ns= Non Significant, * = Significant at p ≤0.05, ** = Significant at p ≤0.01. 67Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy Results were showed that pollens of Golden delicious on the Red delicious lead to increase the ovule fertiliza- tion and finally fruit set among all of the studied crosses (data not shown). The results of Figure 3 showed that the interaction of Zn and crosses significantly affected the pol- len tube penetration to the beginning of the styles in all of the six crosses. The highest (20.09%) and lowest (15.2%) pollen tube penetration to the beginning of the styles was observed in the cross ♀ Red delicious × Golden delicious ♂ in the 3000 mg. L-1 Zn and not treated crosses (control) respectively and thus, pollen tub penetration to the begin- ning of the styles was decreased in 5000 mg. L-1 Zn while it was higher than the controls in all of the crosses. It may be connected with the positive effects of Zn on the apple pollen. The positive effects of Zn in high concentration were reported in the most of the plants. The highest (18.08%) and lowest (13%) pollen tube penetration to the middle part of the styles was observed fed f fe f f fe abc a ae be abcd ab bf af bf bf cf bf 0 5 10 15 20 25 30 35 40 45 50 ♀ Red × G alla ♂ ♀ Red × Gol den ♂ ♀ Gol den × Fuj i ♂ ♀ Gal la × fuji ♂ ♀ Red × F uji ♂ ♀ Gol den × Gal la ♂ po lle n ge rm in at io n % Crosses control 3000 Mg.L-1 Zinc 5000 Mg.L -1Zinc dcb dcb d d dc abcd abc a cbd abcd abc ab bcd a abc abc bcd ab 0 5 10 15 20 25 ♀ Red × G alla ♂ ♀ Red × Gol den ♂ ♀ Gol den × Fuj i ♂ ♀ Gal la × fuji ♂ ♀ Red × F uji ♂ ♀ Gol den × Gal la ♂ po lle n tu be n um be r % crosses control 3000 Mg.L-1 Zinc 5000 Mg.L -1Zinc Fig 2. The interaction between Zn concentration and crosses on the pollen germination on the stigma in different crosses. Means in each column, followed by similar letter (s) are not significantly different at 1% probability level. Fig 3. The interactions between Zn concentration and crosses on the pollen tube penetration into the beginning of the style in different crosses. Means in each column, followed by similar letter (s) are not significantly different at 1% probability level. 68 Yavar Sharafi in the cross ♀ Red delicious × Fuji ♂ in the 3000 mg. L-1 Zn and not treated crosses (control) respectively and thus, pollen tube penetration to the middle part of the styles was decreased in 5000 mg. L-1 Zn while it was higher than the controls in all of the crosses (Figure 4). Also, the results of Figure 5 showed that the highest (12.88%) and lowest (10.28%) pollen tube penetration to the middle part of the styles was observed in the cross ♀ Red delicious × Golden delicious ♂ in the 3000 mg. L-1 Zn and not treated crosses (control) ♀Golden delicious × Gala ♂ respectively and thus, pollen tub penetration to the middle part of the styles was decreased in 5000 mg. L-1 Zn while it was higher than the controls in all of the crosses. Based on the results shown in Figures 6, 7, 8 and 9 the interaction of Zn concentration and the time after pollination significantly affected the pollen germination on the stigma, tube number in the upper and middle parts of the style and also in the beginning of the ovary in all of the crosses. Maximum (34.28%) and minimum cd d cd d d bcdbcd abc cd bcd a abcd bcd abc abcd ab bcd abcd 0 5 10 15 20 25 ♀ Red × G alla ♂ ♀ Red × Gol den ♂ ♀ Gol den × Fuj i ♂ ♀ Gal la × fuji ♂ ♀ Red × F uji ♂ ♀ Gol den × Gal la ♂ po lle n tu be n um be r % ه Crosses control 3000 Mg.L-1 Zinc 5000 Mg.L -1Zinc c c bc bc c bc abc ab bc bc abc abcbc ab abc a bc bc 0 2 4 6 8 10 12 14 16 ♀ Red × G alla ♂ ♀ Red × G old en ♂ ♀ Go lde n × Fuj i ♂ ♀ Gal la × fuji ♂ ♀ Red × F uji ♂ ♀ Go lde n × Gal la � po lle n tu be n um be r % Crosses control 3000 Mg.L-1 Zinc 5000 Mg.L -1Zinc Fig. 4. The interactions between Zn concentration and crosses on the pollen tube penetration into the middle part of the style in different crosses. Means in each column, followed by similar letter (s) are not significantly different at 1% probability level. Fig. 5. The interactions between Zn concentration and crosses on the pollen tube penetration into the beginning of the ovary in different crosses. Means in each column, followed by similar letter (s) are not significantly different at 1% probability level. 69Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy (14.2%) pollen germination on the stigma was observed in the 3000 mg. L-1 Zn and not treated crosses (control) respectively and thus, pollen germination on the stigma was decreased in 5000 mg. L-1 Zn while it was higher than the controls in all of the crosses (Figure 6). Ma ximum (24.73%) and minimum (9.9%) pol- len tube penetration to the beginning of the style was observed in the 3000 mg. L-1 Zn and not treated crosses (control) respectively and thus, tube penetration to the beginning of the style was decreased in 5000 mg. L-1 Zn while it was higher than the controls in all of the crosses (Figure 7). Maximum (26.48%) and minimum (8.63%) pollen tube penetration to the middle of the style was observed in the 3000 mg. L-1 Zn and not treated crosses (control) respectively and thus, tube penetration to the middle of c b c ab a ab 0 5 10 15 20 25 30 35 40 control 3000 mg.L 5000 mg.L P ol le n g er m in at io n % Zn concentration 72 hour 120 hour c b c b a b 0 5 10 15 20 25 30 control 3000 mg.L 5000 mg.L P ol le n t u b e n u m b er % Zn concentration 72 hour 120 hour d b b c a a 0 5 10 15 20 25 30 35 control 3000 mg .L 5000 mg .L po lle n tu be n um be r % Zn concentration 72 hour 120 hour d b bc a a 0 5 10 15 20 25 control 3000 mg.L 5000 mg. L P ol le n t u b e n u m b er % Zn concentration 72 hour 120 hour Fig. 9. The interactions between time and Zn concentration on the pollen tube penetration percentage on the beginning of the ovary. Means in each column, followed by similar letter (s) are not signifi- cantly different at 1% probability level. Fig. 6. The interactions between time and Zn concentration on the pollen germination percentage on the stigma. Means in each col- umn, followed by similar letter (s) are not significantly different at 1% probability level. Fig. 7. The interactions between time and Zn concentration on the pollen tube number in the beginning of style. Means in each col- umn, followed by similar letter (s) are not significantly different at 1% probability level. Fig. 8. The interactions between time and Zn concentration on the pollen tube penetration percentage on the middle part of style. Means in each column, followed by similar letter (s) are not signifi- cantly different at 1% probability level. 70 Yavar Sharafi the style was decreased in 5000 mg. L-1 Zn while it was higher than the controls in all of the crosses (Figure 8). However, maximum (19.8%) and minimum (5.47%) pollen tube penetration to the beginning of the ovary was observed in the 3000 mg. L-1 Zn and not treated crosses (control) respectively and thus, tube penetration to the beginning of the ovary was decreased in 5000 mg. L-1 Zn while it was higher than the controls in all of the crosses (Figure 9). In all of the crosses the highest pollen tube number in the beginning of the style were observed 120 hr after pollination. This phenomenon demonstrated that pollen germination and tube growth were increased followed by the time which may be related to the nutrition case in the style. However, there was a significant difference between the interaction of time and Zn concentration on the pollen tube number in the middle part of the style and in the beginning of the ovary respectively (Figures 8 and 9). Maximum pollen tube numbers in the middle part of the style and in the beginning of the ovary were observed 120 hr after pollination. Correllation between Zn and pollen germination and penetration to different parts of the style and ova- ry is showen in Table 2. There was a significant positive correlation between Zn concentrations and germination percentage of pollen on the stigma and the tube number which penetrated to the upper and middle parts of the style and also to the beginning of the ovary respectively (Table 2).The correlation between pollen tubes penetra- tion to the upper (./68) and middle of the style (./85) and the beginning of the ovaries was positive (Table 2). DISCUSSION In this research the pollen tube penetration per- centage into the styles and ovaries increased by Zn application two weeks before bud break especially in 3000 mg. L-1. In this study a large number of pollen grains ger- minated on the stigma exudate and formed callose, indicating good growth of pollen tubes by Zn treat- ment in apple crosses. However, few of the pollen tubes were observed to penetrate to the style. The aver- age number of pollen tubes was only slightly higher in some crosses. In addition, 120 h after pollination, the average pollen tube length and growth rate were slightly higher in all of the crosses. Previous studies have suggested that self-pollen tubes can grow slow- er or have higher rates of abrasion than cross-pollen tubes (Golzer and Grant 2006; Qin 1996; Song et al. 2015; Song et al. 2016; Yadav et al. 2013; Zhang et al. 2014; Sedgley 1990). In accordance with our results Pandey et al, (2006); observed that Zinc is critically required for pollen func- tion and fertilization in lentil. Also, Neilsen et al (2005) observsd that postbloom humic-and fulvic-based zinc sprays can improve apple zinc nutrition also Neilsen et al (2004) reported positive effects of zinc and boron in fertigated high density apple orchards. Keshavarz et al (2011); reported that foliar applica- tion of Zinc and Boron improves walnut vegetative and reproductive growth. They demonstrated the first report of the benefit of foliar B and Zn on pollen germination in walnut trees. There was clear positive effect of B and Zn applied as individual foliar applications and a syn- ergistic effect when applied in combination on walnut yield and quality parameters. Fei et al (2016); studied enzyme activities, and expression of Zn/Iron-regulated transporter-like pro- tein (ZIP) family genes in the mild, moderate, and severe Zn deficiency in (Citrus sinensis L. Osbeck). They reported that the expression of the ZIP family genes, ZIP1, ZIP3, and ZIP4, was promoted by Zn deficiencies. However, chlorophyll contents and net photosynthetic rate decreased with reduction in Zn contents reduction. Also, comparison of severe Zn-deficient and normal leaves revealed increased significant activities of peroxi- dase (POD) and catalase (CAT, but significantly reduced Zn-containing enzymes such as Cu/Zn superoxide dis- mutase (Cu/Zn-SOD). In plants, about half of the ZIP genes could be induced under Zn deficiency, while ZIP1-4 genes seem to be involved in plant Zn transport. Zinc/iron-regu- lated transporter-like proteins (ZIPs) play a key role for Zn uptake in plants and currently, over 100 ZIP fam- ily members have been recognized in diverse plant spe- cies (Andreini and Bertini 2012; Moghadam et al. 2013; Nosarszewski et al. 2004; Weinthal et al. 2010). Table 2. Pearson correlation coefficients for the effect of the Zn on the pollen germination percentage on the stigma and pollen tube penetration to the upper and middle parts of the style and so the beginning of the ovary. Correlation Stigma level Upper of style Middle of style Beginning of ovary Stigma level 1 Upper of style 0.59ns 1 Middle of style 0.45ns **0.79 1 Beginning of ovary **0.57 **0.68 0.85** 1 ns= Non Significant, * = Significant at p ≤0.05, ** = Significant at p ≤0.01 71Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy Furthermore, Zn is involved in various cellular pro- cesses, including photosynthesis, nucleic acid and lipid metabolism, protein synthesis, detoxification of reactive oxygen species (ROS), and membrane stability (Broad- ley et al. 2007). The main role of Zn is involvement in important procedures of cell division and gene expres- sion associated with nucleic acid and protein metabo- lism. Zinc play an essential role in processes leading to DNA synthesis, and DNA polymerase contains firmly bound zinc. These roles help to explain the nature of some of the symptoms characteristic of Zn deficiency in fruit trees, for example, ‘rosetting’ and shoot tip dieback (Broadley et al. 2007; Andreini and Bertini 2012; Mogh- adam et al. 2013; Nosarszewski et al. 2004; Weinthal et al. 2010). Zinc deficiency leads to malfunctioning of some enzymes, for example, alkaline phosphatase carbonic anhydrase. It could lead to decreased starch formation, accumulation of amino acids, and synthesis of Auxin via impaired tryptophan synthesis. It decreases carbonate dehydrogenase activity which has affected the balance of carbon dioxide and carbon acid and thus has indirectly influenced the rate of photosynthesis. Zinc deficiency has also been found to promote formation of abscisic acid causing premature abscission of leaves and flower buds (Andreini and Bertini 2012; Moghadam et al. 2013; Nosarszewski et al. 2004; Weinthal et al. 2010). Hence, Zinc is important in DNA and RNA metab- olism and protein synthesis and thus, maintains the structural integrity of biomembranes. More than 1,200 protein molecules (Zn metalloprotein) have been identi- fied including a large number of ‘zinc-finger’- containing proteins and transcription factors, oxidoreductases and hydrolytic enzymes such as metalloproteases. Further- more, Zn is a mechanical factor of ribosomes and thus vital for their structural integrity. It plays a major role in carbohydrate metabolism by regulating key enzymes, fructose 1,6-bisphosphatase and aldolase. Synthesis of auxin, and indole acetic acid, is particularly impaired under Zn deficiency (Broadley et al. 2007; Andreini and Bertini 2012; Moghadam et al. 2013; Nosarszewski et al. 2004; Weinthal et al. 2010). Finally by comparing the effects of the contols with 3000 and 5000 mg. L-1 Zn in Figures 1, 2, 3, 4, 5, 6 ,7 and 8 it was demomonstrated that pollen germination and tube penetration to the style and ovary increased until 3000 mg. L-1 Zn but, in 5000 mg. L-1 mentioned traits were decreased respectively. This could be inter- preted that by increasing the Zn concentration it may show toxic effects of pollen tube cell growth. However, in this study, 5000 mg. L-1 did not show toxic effect because it showed positive effects on pollen germination and tube penetration to the style and ovary in compari- son with the crosses which were not treated with Zn. CONCLUSION “It was concluded that f luorescence microscopy technique is a very accurate method for assay of nutrient effects on pollen germination and tube penetration to the pistils in fruit trees in compared with fruit set per- centage studies. In this research the mentioned method was used for assay of Zn effects in four apple cultivars crosses which included “Golden Delicious”, “Red Deli- cious”, “Gala” and “Fuji”. Results showed that the high- est pollen germination percentage on the stigma (43.5%) and penetration of pollen tube to the ovary (12/88%) were observed in the cross (♀Golden Delicious × Gala♂), in 3000 mg. L-1 of Zn 120 hours after pollination respec- tively. However, the foliar application of Zn by 1000 mg. L-1 on apple cultivars two weeks before bud break posi- tively affects pollen germination and tube penetration to the ovary in all of the crosses”. ACKNOWLEDGMENT We would like to acknowledge the research team of Shahed University. REFERENCES Alloway BJ. 2004. Zinc in soils and crop nutrition: Inter- national Zinc Association Brussels. Andreini C, Bertini I. 2012. A bioinformatics view of zinc enzymes. J. inorganic biochemistry 111: 150-156. Atlagić J, Terzić S. Marjanović-Jeromela A. 2012. Staining and fluorescent microscopy methods for pollen viabil- ity determination in sunflower and other plant spe- cies. J. Industrial crops and products 35 (1): 88-91. Broadley MR, White PJ, Hammond, JP, Zelko I, Lux A. 2007. Zinc in plants. J. New Phytologist 173 (4): 677- 702. Chen W, Yang X, He Z, Feng Y, Hu F. 2008. Differential changes in photosynthetic capacity, 77 K chlorophyll fluorescence and chloroplast ultrastructure between Zn‐efficient and Zn‐inefficient rice genotypes (Oryza sativa) under low zinc stress. J. Physiologia Plantar- um 132 (1): 89-101. Clément C, Pacini E, Audran JC. 2012. Anther and pol- len: from biology to biotechnology: Springer Science & Business Media. 72 Yavar Sharafi Dafni A, Firmage D. 2000. Pollen viability and longevity: practical, ecological and evolutionary implications. J. Plant Systematics and Evolution 222 (1): 113-132. Dafni A, Kevan PG, Husband BC. 2005. Practical pollina- tion biology. Practical pollination biology. Fei, X, Fu X.-z, Wang N-q, XI Jl, Huang Y, Wei Z, Ling L, Peng Lz. 2016. Physiological changes and expression characteristics of ZIP family genes under zinc defi- ciency in navel orange (Citrus sinensis). J. Integrative Agriculture 15 (4): 803-811. Glozer K, Grant JA. 2006. (101) Effects of Fall Applica- tions of Urea and Zinc Sulfate toBing’Sweet Cherry Spring Budbreak. HortScience 41 (4): 1030-1031. Hegedűs A, Lénárt J, Halász J. 2012. Sexual incompatibil- ity in Rosaceae fruit tree species: molecular interac- tions and evolutionary dynamics. Biologia plantarum 56 (2): 201-209. Hipps N, Davies M. 2000. Effects of foliar zinc applica- tions at different times in the growing season on tis- sue zinc concentrations, fruit set, yield and grade out of culinary apple trees. Paper read at IV International Symposium on Mineral Nutrition of Deciduous Fruit Crops 564. Hiscock SJ, Allen AM. 2008. Diverse cell signalling path- ways regulate pollen‐stigma interactions: the search for consensus. J. New Phytologist 179 (2): 286-317. Karimi HR, Mohammadi N, Estaji A, Esmaeilizadeh M. 2017. Effect of supplementary pollination using enriched pollen suspension with Zn on fruit set and fruit quality of pistachio. J. Scientia Horticulturae 216: 272-277. Kubitscheck U. 2017. Fluorescence microscopy: from principles to biological applications: John Wiley & Sons. Merkusheva MG. 2009. Agrokhimicheskoe mineral’noe syr’e : P, K, S i mikroėlementy. Ulan-Udė: Izd-vo Bur- iatskogo nauch. tsentra SO RAN. Moghadam HR, Zahedi TH, Ashkiani A. 2013. Effect of zinc foliar application on auxin and gibberellin hormones and catalase and superoxide dismutase enzyme activity of corn (Zea mays L) under water stress. J. Maydica 58 (2013): 218-223. Mularczyk-Oliwa, M., A. Bombalska, M. Kaliszewski, M. Włodarski, K. Kopczyński, M. Kwaśny, Szpakowska M, Trafny EA. 2012. Comparison of fluorescence spectroscopy and FTIR in differentiation of plant pollens. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97: 246-254. Neilsen G, Hogue E, Neilsen D, Bowen P. 2005. Post- bloom humic-and fulvic-based zinc sprays can improve apple zinc nutrition. J. HortScience 40 (1): 205-208. Neilsen G, Neilsen D, Hogue E, Herbert L. 2004. Zinc and boron nutrition management in fertigated high density apple orchards. Canadian J. plant science 84 (3): 823-828. Nepi M, Franchi G. 2000. Cytochemistry of mature angi- osperm pollen. J. Plant Systematics and Evolution 222 (1-4): 45-62. Nosarszewski M, Clements AM, Downie AB, Archbold DD. 2004. Sorbitol dehydrogenase expression and activity during apple fruit set and early development. J. Physiologia Plantarum 121 (3): 391-398. Pandey N, Pathak GC, Sharma CP. 2006. Zinc is critically required for pollen function and fertilisation in lentil. J. Trace Elements in Medicine and Biology 20 (2): 89-96. Pereira MR, Ribeiro H, Cunha M, Abreu I. 2018. Com- parison of pollen quality in Vitis vinifera L. cultivars. J. Scientia Horticulturae 227: 112-116. Phuntsho, Shon SHK, Hong S, Lee S, Vigneswaran S. 2011. A novel low energy fertilizer driven forward osmosis desalination for direct fertigation: evaluating the performance of fertilizer draw solutions. J. Mem- brane Science 375 (1): 172-181. Qin X. 1996. Leaf spraying with boron, zinc and magne- sium and their effects on the fruit production and quality of Jingchen oranges (Citrus sinensis Osbeck). J. Southwest Agricultural University 18 (1): 40-45. Radunić M, Jazbec A, Ercisli S, Čmelik Z, Ban SG. 2017. Pollen-pistil interaction influence on the fruit set of sweet cherry. J. Scientia Horticulturae 224: 358-366. Ramírez, F., and T. L. Davenport. 2013. Apple pollination: a review. J. Scientia Horticulturae 162: 188-203. Rawashdeh HM, Florin S. 2015. Foliar application with iron as A vital factor of wheat crop growth, yield quantity and quality: A Review. International J. Agri- cultural Policy and Research 3 (9): 368-376. Rodríguez VA,. Mazza SM,. Martínez GC,. Ferrero AR. 2005. Zn and K influence in fruit sizes of Valencia orange. J. Revista Brasileira de Fruticultura 27 (1): 132-135. Sanchez E, Righetti T. 2001. Misleading zinc deficiency diagnoses in pome fruit and inappropriate use of foli- ar zinc sprays. Paper read at International Symposi- um on Foliar Nutrition of Perennial Fruit Plants 594. Sedgley M. 1990. Flowering of deciduous perennial fruit crops. Horticultural Reviews, Volume 12: 223-264. Sharafi Y, Talebi SF, Talei D. 2017. Effects of heavy metals on male gametes of sweet cherry. J. Caryologia: 1-8. Sharpley AN. 2000. Agriculture and phosphorus manage- ment : the Chesapeake Bay. Boca Raton, Fla.: Lewis Publishers. Sheffield CS, Smith RF, Kevan PG. 2005. Perfect syncarpy in apple (Malus× domestica ‘Summerland McIntosh’) 73Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy and its implications for pollination, seed distribu- tion and fruit production (Rosaceae: Maloideae). J. Annals of Botany 95 (4): 583-591. Shivanna K. 2003. Pollen Biology and Biotechnology Sci- ence Publishers. Enfield, NH, USA. Solar A, Stampar F. 2000. Influence of boron and zinc application on flowering and nut set in’Tonda di Giffoni’hazelnut. Paper read at V International Con- gress on Hazelnut 556. Song C-Z, Liu M-Y, Meng J-F, Shi P-B, Xi Z-M, Zhang ZW. 2015. Promoting effect of foliage sprayed zinc sulfate on accumulation of sugar and phenolics in berries of Vitis vinifera cv. Merlot growing on zinc deficient soil. J. Molecules 20 (2): 2536-2554. Song C-Z, Liu M-Y, Meng J-F, Shi P-B, Zhang ZW, Xi Z-M. 2016. Influence of foliage-sprayed zinc sulfate on grape quality and wine aroma characteristics of Merlot. J. European Food Research and Technology 242 (4): 609-623. Upadhya JC. 1999. Analysis of sulphur, phosphorus and silica in metals, alloys, inorganic compounds and sol- vents. Mumbai: Bhabha Atomic Research Centre. Usenik V, Stampar F. 2001. Effect of foliar application of zinc plus boron on sweet cherry fruit set and yield. Paper read at International Symposium on Foliar Nutrition of Perennial Fruit Plants 594. Weinthal D, Tovkach A, Zeevi V, Tzfira T. 2010. Genome editing in plant cells by zinc finger nucleases. J. Trends in plant science 15 (6): 308-321. Wojcik P. 2007. Vegetative and reproductive responses of apple trees to zinc fertilization under conditions of acid coarse-textured soil. J. plant nutrition 30 (11): 1791-1802. Yadav V, Singh P, Yadav P. 2013. Effect of foliar fertili- zation of boron, zinc and iron on fruit growth and yield of low-chill peach cv. Sharbati. International J. Scientific and Research Publications: 693. Yan Z, Zhang Y, Zhang Y, Wang Y, F. Zhang, Shu H. 2010. Effects of different zinc application levels on the dynamic accumulation and distribution of dry matter and zinc in apple trees. J. Plant Nutrition and Fertilizer Science 16 (6): 1402-1409. Yeloff D, Hunt C. 2005. Fluorescence microscopy of pol- len and spores: a tool for investigating environmental change. Review of Palaeobotany and Palynology 133 (3): 203-219. Zhang Y, Fu C, Yan Y, Fan X,. Wang Ya, Li M. 2014. Foli- ar Application of Sugar Alcohol Zinc Increases Sug- ar Content in Apple Fruit and Promotes Activity of Metabolic Enzymes. J. HortScience 49 (8): 1067-1070. Zhang Y, Fu C, Yan Y, Wang Y. a, Li M, Chen M, Qian J, Yang X, Cheng S. 2013. Zinc sulfate and sugar alco- hol zinc sprays at critical stages to improve apple fruit quality. J. HortTechnology 23 (4): 490-497. Zhang Y, Yan Y, Fu. Li CM,. Wang Ya. 2016. Zinc sulfate spray increases activity of carbohydrate metabolic enzymes and regulates endogenous hormone levels in apple fruit. J. Scientia Horticulturae 211: 363-368. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 72, Issue 3 - 2019 Firenze University Press Role of humic acid against salt-induced cytotoxicity in Hordeum vulgare L. Selma Tabur*, Merve Dündar Yurtlu, Serkan Özmen Characterization of intraspecific hybrid in Clitoria ternatea (L.) using morpho-physiological, cytogenetic, metabolic and molecular markers Aparupa Naik1, Amiya K. Patel1, Sujit K. 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