Incompatible pollen tubes in the quince style and Incompatible pollen tubes in the quince style and Incompatible pollen tubes in the quince style and Incompatible pollen tubes in the quince style and theirtheirtheirtheir impact on fertilization success impact on fertilization success impact on fertilization success impact on fertilization success

Self-incompatibility presents one of the challenges in modern fruit production. It can be correlated with a lower yield of self-incompatible cultivars which also demand the planting of the pollinisers. The aim of this work was to investigate the phenomenon of incompatibility and its influence on fertilization success in quince ( Cydonia oblonga Mill.), using the cultivars ‘Leskovacka’, ‘Vranjska’, ‘Morava’, ‘Pazardzijska’, ‘Hemus’, ‘Asenica’, ‘Portugal’ and ‘Triumph’. Incompatible pollen tubes were determined by using fluorescence microscopy. In two types of pollination (self-pollination and open pollination) pollen tubes showed signs of incompatibility, mostly in the upper third of the style. The most common sign of incompatibility is the formation of swelling at the tip of a pollen tube. Also, sometimes twisted, bifurcated, and short and thickened pollen tubes along their entire length are formed. The incompatibility was significantly more pronounced in the self-pollination than in the open pollination variant in all tested cultivars. The highest number of incompatible pollen tubes in both pollination types was present in the cultivar ‘Pazardzijska’, while the lowest number was present in the cultivars ‘Leskovacka’ and ‘Vranjska’. The appearance of incompatibility affected the degree of fertilization in quince. The results showed that only ‘Leskovacka’ and ‘Vranjska’ are self-compatible, while other studied cultivars (‘Morava’, ‘Pazardzijska’, ‘Hemus’, ‘Asenica’, ‘Portugal’ and ‘Triumph’) are self-incompatible. The results provide a good background for the future research of reproductive biology and also for adequate management of the quince orchards.


Introduction Introduction Introduction Introduction
Self-incompatibility in flowering plants is a phenomenon of great evolutionary significance, which prevents self-fertilization and promotes cross-fertilization (Nettancourt, 2001;Frankling-Tong and Franklin, 2003). Gametophytic incompatibility is present in many fruit tree species, including quince. It is controlled by one gene with a large number of alleles called incompatibility alleles or S alleles. Fertilization occurs only in cases of compatible pollination when the pollen grain contains different S alleles than those in the pistil (Newbigin et al., 1993).
The wall of the pollen tube consists of two main layers of polysaccharides: the inner "callose" wall, which contains predominantly β-1,3-glucan, and the outer wall, which contains predominantly arabinan. The inner glucan layer fluoresces intensely when stained with fluorochrome dyes, such as aniline blue. This reveals transverse callose walls in pollen tubes containing sperm cells and other cytoplasmic elements of the pollen grain (Evans and Hoyne, 1982;Rae et al., 1985). Greater accumulation of callose in the tips of pollen tubes inhibits their growth through the style, which may show a marked difference between incompatible and compatible pollen tubes (Nettancourt, 2001). The distribution and amount of callose in the walls of pollen tubes vary depending on the species and external factors. It is sometimes distributed along the entire length of the tube, while sometimes it is localized in densely distributed plugs. It was also found that the amount of callose is much higher in self-pollination compared to controlled pollination (Halász and Hegedûs, 2006).
The way of the growth of incompatible pollen tubes at the beginning is similar to that of pollen tubes in compatible pollination, but at some stage, the growth becomes irregular. The walls become thicker and the tip thickened, while the growth of compatible tubes remains unchanged along the entire length of the style, so it can reach the ovary (Newbigin et al., 1993). The most significant sign of incompatibility is the formation of characteristic swellings at the tips of pollen tubes during their growth through the style Milatović et al., 2010). In addition to the expanded tip, other signs of incompatibility can be observed, such as the formation of loops and bifurcation of the pollen tube (Milatović et al., 2013a).
Self-incompatibility is most common in fruit tree species of the genus Prunus, especially in cherries and almonds (Milatović and Nikolić, 2007). Worldwide, there is almost no research addressing with the problem of self-incompatibility in quince, especially with more reliable methods, which include, among others, the method of fluorescence microscopy. In contrast to quince, many researchers have studied self-incompatibility with this method in various other fruit tree species. Thus, using the fluorescent microscopy method, has been used to determine incompatibility in apples (Adachi et al., 2009), pears (Jacquemart et al., 2006;Sanzol and Herrero, 2007), Japanese quince (Kaufmane and Rumpunen, 2002), European plums , Japanese plums (Ontivero et al., 2006;Jia et al., 2008), apricots (Andrés and Durán, 1998;Milatović and Nikolić, 2007;Milatović et al., 2013a, b), cherries (Lansari and Iezzoni, 1990), almonds (Certal et al., 2002;Dicenta et al., 2002;Alonso and Sociasi Company, 2005a;Čolić et al., 2010). In fruit growing practice, self-incompatible cultivars are undesirable because they cannot be grown in a single cultivar orchard, so it is necessary to provide suitable pollinisers. Also, self-incompatible cultivars often give lower yields, because fruit setting in them depends on the amount of pollen transfer from other cultivars (Milatović and Nikolić, 2007). The aim of this work was to examine the occurrence and characteristics of incompatible pollen tubes in the style of quince in self-pollination and open-pollination variants, in order to examine its impact on fertilization success.

Study area
The research was conducted at the experimental field "Radmilovac" of the Faculty of Agriculture -University of Belgrade, Serbia (Figure 1). The distance (airline) from the centre of Belgrade (Serbia) is 11.5 3 km (southeast); 320 km southwest of Cluj-Napoca (Romania). The climate is temperate continental, with an average annual temperature of 11.7 °C, an average temperature during the growing season (April-October) of 17.5 °C and a total annual precipitation of 693.9 mm.  Pollination and pistil fixation Two variants of pollination were examined -self-pollination and open-pollination. In the variant of self-pollination, the perianths and anthers were removed just before the flowering time. During full flowering (2 days after removing the perianths and anthers), each cultivar was hand pollinated with its own pollen. Isolation bags were not used because the flowers were emasculated and consequently not visited by insects. In addition, the use of isolation bags can increase the temperature inside the bag and lead to low fertilization (Vuletin Selak et al., 2011). On the same day when self-pollination was performed, branches with flowers in anthesis in the open pollination variant were marked for sampling. The fixation of the pistils was done on the 2 nd , 4 th and 6 th day after pollination. The FAA fixative (70% ethanol: glacial acetic acid: formaldehyde = 90:5:5 v/v/v) was used for fixation and the fixed pistils were stored at +4 °C.  Staining and microscopic observation of pistils Sample preparation for microscopic observation was done by pistil softening in 8N NaOH for 12 hours. After that, the staining was done with 0.1% aniline blue dissolved in 0.1 N K3PO4 (Kho and Baër, 1971). Before microscopic observation, the style was separated from the ovary, which was cut with the razor blade along the suture. Each stigma and style of the flower were examined. The microscopic observation was done with a fluorescent microscope Leica DM LS (Leica Microsystems, Wetzlar, Germany) with filters A (wavelength 340-380 nm) and I3 (wavelength 450-490 nm). For both types of pollination, 30 pistils per variant and per cultivar were observed.
The number of incompatible pollen tubes was determined and expressed as a percentage of the total number of pollen tubes in the upper third of the style. The results are presented as the average number of the three fixation periods. In addition, the percentage of fertilization, i.e., the number of pistils with pollen tubes reaching the nucellus on the sixth day after pollination, was determined.

Results Results Results
When studying the growth of pollen tubes through the pistil of quince cultivars, the presence of pollen tubes with typical signs of incompatibility was found in both pollination types (self-pollination and open pollination). These incompatibilities occurred mostly in the upper third of the style, much less in the middle third, and least in the lower third of the style, where their growth ceased. The most characteristic sign of incompatibility was the formation of typical swellings at the tips of pollen tubes due to a higher accumulation of callose ( Figure 3). During the examination, other signs of incompatibility were also observed, such as the appearance of short and thickened pollen tubes along the entire length of the pistil ( Figure 4) and the formation of twisted and bifurcated pollen tubes ( Figures 5 and 6). The number of incompatible pollen tubes was significantly different among pollination variants (Table 1). It was more expressed in the self-pollination variant. In the self-pollination variant, the highest number of incompatible pollen tubes in the style (Table 2) was found in the cultivar 'Pazardzijska' (5.75%), and the lowest in the cultivars 'Leskovacka' (1.62%) and 'Vranjska' (2.01%).  Table 3. Table 3. Table 3. The highest number of incompatible pollen tubes in the self-pollination variant was found in the upper third of the style in all cultivars. The most characteristic sign of incompatibility in the upper third of the style in all tested cultivars was the appearance of swellings at the tips of pollen tubes, which were most pronounced in cultivar 'Pazardzijska' (5.54%), and least pronounced in cultivars 'Leskovacka' (1.21%) and 'Vranjska' (1.25%).
Compared to the upper third of the style, the number of incompatible pollen tubes with swelling at the tips was lower in the middle third of the style and ranging from 0.16% ('Pazardzijska') to 1.19% ('Portugal'). The decrease in the number of incompatible pollen tubes with swellings at the tips continued towards the base of the style, so that no signs of incompatibility were observed in the lower part of the style in the cultivars 'Pazardzijska' and 'Triumph'. The reason for this is that in these two cultivars, the growth of the pollen tubes stopped mainly in the upper third of the style.
The occurrence of twisted pollen tube growth was most pronounced in the upper third of the style, but was much less pronounced compared to the swellings at the tips. They occurred to the greatest extent in the cultivars 'Portugal' (0.28%) and 'Morava' (0.27%), and in the least (0.02%) in the cultivars 'Vranjska' and 'Triumph'. The occurrence of bifurcation of the pollen tube (0.01%) in the upper third of the style was also observed in the variety 'Asenica'. 8 Figure 3. Figure 3. Figure 3.     (Table 3) compared to self-pollination. The number of incompatible pollen tubes in the open pollination variant was highest in the cultivar 'Pazardzijska' (3.56%), and the least in the 'Leskovacka' cultivar (1.25%). The largest number of incompatible pollen tubes was found in the upper third of the style, as in the case of selfpollination, with swelling predominating at the top of the pollen tube. Also, in the upper third of the style, the highest number of pollen tubes with swelling at the tips was observed in the cultivar 'Pazardzijska' (3.35%) and the lowest in the cultivars 'Vranjska' (0.90%) and 'Leskovacka' (1.01%).
Going towards the middle and lower part of the style, the number of incompatible pollen tubes decreased in all cultivars. Thus, the highest number of incompatible pollen tubes with swellings at the tips was found in the cultivar 'Portugal' (0.42%) in the middle third and in 'Hemus' (0.26%) in the lower third of the style, and the lowest number (0.04%) was found in the cultivar 'Leskovacka' in both parts of the style.
The occurrence of twisted pollen tubes was most pronounced in the upper third of the style in all cultivars, but to the greatest degree in the 'Leskovacka' cultivar (0.10%), while no twisted pollen tubes were observed on the pistils of cultivars 'Pazardzijska', 'Asenica' and 'Portugal'. In cultivars 'Leskovacka', 'Morava' and 'Pazardzijska', the occurrence of bifurcation of the pollen tube in the upper third of the pistil was determined in 0.01% of the pollen tubes.
The percentage of fertilization, i.e., the number of pistils with pollen tubes reaching the nucellus on the 6th day after pollination, differed significantly between cultivars and pollination variants (Table 1). It was higher in all cultivars in the open pollination variant (Table 4). In the self-pollination variant, the highest percentage of fertilization was found in the 'Leskovacka' (40.09%) and 'Vranjska' (14.74%) cultivars. In the 'Morava' and 'Pazardzijska' cultivars, a very low percentage of fertilization was found (1.11% and 1.08%, respectively). However, no signs of fertilization were registered in the other cultivars. In the open pollination variant, the highest percentage of fertilization was also determined in the 'Leskovacka' cultivar (42.65%), and the lowest in the 'Asenica' cultivar (14.10%).

Discussion Discussion Discussion
An earlier study on the same cultivars included a detailed analysis of the number of pollens tubes in the pistil and style, but also of the dynamics of pollen tube growth by pistil regions. Based on these results, 'Leskovacka' and 'Vranjska' were found to be self-compatible, while other cultivars ('Morava', 'Pazardzijska', 'Hemus', 'Asenica', 'Portugal' and 'Triumph') were self-incompatible (Radović et al., 2020). The study of incompatible pollen tubes in quince is a continuation of previous studies with the aim of analysing in detail the phenomenon of incompatibility in quince in two pollination modes (self-pollination and open pollination).
In our study, the occurrence of incompatible pollen tubes was found in both pollination modes, but to a much higher extent in the self-pollination variant compared to the open pollination. Our results are in agreement with those of Đorđević et al. (2014). The authors studied the European plum cultivar 'Čačanska lepotica' in three pollination variants (self-pollination, cross-pollination and open pollination) and found the highest number of incompatible pollen tubes in the variant with self-pollination.
Incompatible pollen tubes occurred most frequently in the cultivar 'Pazardzijska' and least frequently in the cultivar 'Leskovacka'. The most characteristic sign of incompatibility in our work was the formation of swellings at the tips of pollen tubes due to a larger accumulation of callose. These phenomena were most pronounced in the upper third of the style, which was experimentally confirmed in pears (Sanzol and Herrero, 2007), plums  and sour cherry (Tobutt et al., 2004). During the growth of pollen tubes through the style, in addition to the formation of swellings at the tips, the formation of twisted tubes and bifurcation of the pollen tube was also noticed, but to a much lesser degree, which is in accordance with the results of Milatović et al. (2013a). This was also observed in plums , and almonds (Čolić et al., 2010).
In apricot, it was established that pollen tubes stop their growth most often in the lower half of the style, i.e., at about 3/4 of the length of the style (Andrés and Durán, 1998;Milatović and Nikolić, 2007;Milatović et al., 2013a). In contrast, Čolić et al. (2010) found that in almonds, pollen tubes stop their growth just below the stigma or in the upper quarter of the style.
The cultivar is considered self-compatible if the pollen tubes during self-pollination reach the ovary in at least 25-30% of the pistils (Lopez et al., 2001). Based on the above, the cultivars 'Vranjska' and 'Leskovacka' are classified as self-compatible, while the other cultivars are classified as self-incompatible (Radović et al., 2020). Milatović and Nikolić (2007) and Milatović et al. (2013b) consider as self-fertile those apricot cultivars in which at least one pollen tube reached the pistil in most of the analysed pistils. Similarly, Alonso and Sociasi Company (2005b) consider almond cultivars in which pollen tubes in more than 50% of the pistils have reached the base of the pistil as self-fertile, and those with less than 25% of the pistils to be nonself-fertile. However, the authors consider cultivars in which the pollen tubes reached the base of the style in 25-50% the pistils as uncertain in terms of self-fertility.
Many authors reported great differences between quince cultivars in terms of their self-fertility (Booth and Klimenko, 2001;Akbari and Qorbani, 2011). Similarly, quince cultivars in our study behaved differently with respect to the degree of self-fertility. The cultivars 'Leskovacka' and 'Vranjska' had the lowest number of incompatible pollen tubes and the highest percentage of self-fertilization.
Incompatibility is a phenomenon that greatly affects the success of fertilization. In practice, it creates problems for producers, because such cultivars cannot be grown in mono cultivar plantations. Therefore, appropriate pollinisers must be provided, which will enable abundant fruit set and high yields (Dicenta et al., 2002). It is considered that for high yields in quince, more than 20% of the fruit must be set (Szabó et al., 1999;Benedek et al., 2000).

Conclusions Conclusions Conclusions Conclusions
Analysis of the growth of pollen tubes in the pistil showed the occurrence of incompatibility in quince. It was expressed to different degrees depending on the genotype and pollination variant. In all tested cultivars, this phenomenon was significantly more pronounced in the self-pollination variant. The incompatibility was most pronounced in the upper third of the style. The most characteristic sign of incompatibility was the formation of a swelling at the tip of the pollen tube. The appearance of incompatibility affected the degree of fertilization in quince. In the self-pollination variant, the highest percentage of fertilization was observed in the cultivars with the least number of incompatible pollen tubes ('Leskovacka' and 'Vranjska'). On the other hand, the most unfavourable results in terms of fertilization were found in the cultivars with the most pronounced incompatibility. Based on these data, the cultivars 'Leskovacka' and 'Vranjska' can be considered as self-compatible, while the cultivars 'Morava', 'Pazardzijska', 'Hemus', 'Asenica', 'Portugal' and 'Triumph' are self-incompatible. Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.