Abilities of the Newly Introduced Apple Cultivars (Malus × domestica Borkh.) ‘Eden’ and ‘Fryd’ to Promote Pollen Tube Growth and Fruit Set with Different Combinations of Pollinations

Abstract

Apple production in Western Norway faces challenges due to climatic constraints and varying phenology. It is essential for cultivars to adapt to regional ecological factors, while suitable pollinators are necessary for successful cultivation. This study examined the reproductive biology of two newly introduced apple cultivars, ‘Eden’ (Wursixo) and ‘Fryd’ (Wuranda), over two years (2022–2023). Key qualitative and quantitative parameters of reproductive biology were analyzed, including in vitro pollen germination, pollen tube growth within the style and ovary locules, flowering overlap time, and fruit set. The study involved cross-pollination between the pollen recipient cultivars ‘Eden’ and ‘Fryd’, with various pollenizers: ‘Rubinstep’, ‘Red Aroma’, ‘Elstar’, ‘Asfari’ and ‘Professor Sprenger’, as well as self-pollination and open pollination. According to the results from the progamic phase of fertilization and fruit set, the cultivars ‘Rubinstep’, ‘Asfari’, and ‘Fryd’ were the best pollenizers for ‘Eden’. In contrast, ‘Rubinstep’, ‘Eden’, and ‘Elstar’ were the best pollenizers for ‘Fryd’. Looking only at the overlapping of the flowering time between pollen recipient and pollen donor, ‘Professor Sprenger’ and ‘Fryd’ were the best pollenizers for ‘Eden’, while ‘Professor Sprenger’ and ‘Eden’ were good pollenizers for ‘Fryd’.

1. Introduction

The most commonly produced fruit in Norway is the apple, which is primarily cultivated in the most suitable growing conditions in southern, eastern, and Western Norway. The apple-growing area increased from 1428 ha in 2010 to 1651 ha in 2023. During the same period, the annual production of apples increased from 11,491 tonnes to 20,577 tonnes, and the yield per hectare increased from 8.04 tonnes to 12.46 tonnes [1]. The apple yield level per area in Norway is much lower than further south due to a cool and short growing season.

In Norway, consumers prefer crispy, sour, and sweet red apple cultivars, with the cultivars ‘Discovery’, ‘Summerred’, ‘Red Gravenstein’, ‘Red Aroma’, and ‘Rubinstep’ being the most popular [2]. In order to meet the expected taste preferences of consumers, there is a constant demand on the market for new cultivars with improved taste and pleasant aroma with different harvesting times. Breeders must ensure that newly introduced cultivars are easy to grow, have large and tasty fruits, are resistant to diseases, and are capable of withstanding shipping and having good storage properties. The existence of apple production in Norway has economic and commercial value and is also a social, environmental, and traditional issue [3]. However, there are forecasts that apple production will increase in the coming years and decades in Norway if the markets allow, mainly due to climate change and temperature increase due to the planting of new orchards at higher altitudes and high market demand [4]. In 2020, two new club apple cultivars (licensed for growing and marketing), ‘Eden’ (‘Wursixo’) and ‘Fryd’ (‘Wuranda’), were introduced to Norwegian apple growers. The breeder of those cultivars is ‘Fresh Forward’, from the Netherlands. ‘Eden’ has striped red-yellow fruits with sweet-sour taste while ‘Fryd’ has bright red, refreshing fruit with a sweet taste, enormous juiciness, and crunchy texture due to the cross with ‘SQ159’ × ‘Honeycrisp’. Both cultivars are mid-season apples, ripening at the mid to end of September in Norway, and are resistant to scab [5].

Apples are one of the most widely cultivated fruits globally and are a Rosaceae family member. Most apple cultivars cannot self-fertilize due to a multi-allelic S-locus (S-RNase) mechanism that causes gametophytic self-incompatibility [6]. Diploid apple cultivars are mostly self-incompatible, with only a few cultivars identified as being either pseudo-compatible (‘Cox’s Orange Pippin’) or fully compatible (‘Megumi’, ‘Orin’), so they mostly require cross-pollination by insects to produce fruit and seeds [7]. The increase in self-compatibility in some plant species with self-incompatibility can be caused by different environmental factors, such as temperature and flower age [8]. However, self-incompatibility in apple is broken down by polyploidization, which is shown in tetraploid apples [9]. Moreover, some apple cultivars can generate unfertilized or apomictic seeds [10,11]. The yield stability of apple cultivars depends on various internal and external factors and genetically controlled mechanisms that ensure a successful reproductive process during flowering. Key factors include flower attraction [12], pollen viability [13], stigmatic receptivity [14], pollen tube growth [15], embryo sac development [16], ovule longevity [17], the effective pollination period [14], and fruit set [18]. An important factor in successful fertilization is the effective pollination period, which equals the longevity of the ovule minus the time required for pollen tube growth to the ovule. The duration for this period in apple can vary between 2 and 9 days [14].

To ensure uniform cropping in an orchard, allocating approximately 10% of the area to pollenizer cultivars, which serve as pollen donors, is advisable. These cultivars can be another compatible apple variety or a specialized crabapple pollenizer cultivar [19,20]. Another aspect of successful pollination is determined by the ratio between available pollen grains and the ovules to be fertilized [21]. Pollen limitation occurs when the ratio between pollen grains and the ovules is unbalanced, with more ovules than pollen which can lead to a reduction in pollination-dependent crop productivity [22,23]. On the other hand, low fruit and seed set have a negative effect on the quality of apple fruit which can lead to obtain malformed fruits with low calcium content, which shortens their shelf life [24]. In addition, pollination performance accounts for ~65% of market production per hectare, as it affects both the quality and quantity of apples, as illustrated by Garratt et al. [25]. In Norway, the main limitations are the amount and locations of pollinizers, overlapping in flowering time and pollinating insects being available. In cool and rainy weather, pollination insects will not operate. The climatic environment, including temperature, heat and chill requirements, global warming, nutrient deficiencies, and water stress, plays a crucial role in plant growth and can determine how species evolve based on their adaptability to specific ecologic factors. In response to these environmental influences, plants modify their phenological phases, which impact their life cycle events concerning seasonal temperature changes. Flowers’ male and female reproductive organs are susceptible to temperature changes throughout their development, including before, during, and after pollination [26,27]. This sensitivity results in a gradual shift in the distribution of cultivars, favoring those better adapted to specific temperatures regarding their reproductive behavior [28]. This is especially evident in Western Norway, where extreme weather conditions can adversely impact apple trees’ flowering, pollination, and fruit setting. Earlier studies on pears and plums have shown that pollen germination in vitro and the efficiency of pollen tube growth within the pistil and fruit set depend on temperature in these often-unfavorable climate conditions [29,30]. Also, apple trees are susceptible to water stress during the growing season, reducing fruit set and quality, mainly due to a reduction in fruit size and weight at harvest time and/or due to premature June and August fruit fall [31]. In cool apple production areas, like Norway, the alternate bearing of apple trees is very common. Chemical thinning is carried out during flowering or when fruitlets are 10–15 mm in diameter and corrected by hand thinning later [32]. The aim of this study was to evaluate the progamic phase of fertilization in two newly introduced apple cultivars ‘Eden’ and ‘Fryd’. The objective was to determine the ability of newly introduced cultivars to support the pollen tube growth and to select the best pollenizer when pollinated with ‘Rubinstep’, ‘Red Aroma’, ‘Elstar’, ‘Asfari’, ‘Professor Sprenger’, ‘Eden’, and ‘Fryd’, including self-pollination and open pollination under ecological conditions in Western Norway. It was hypothesized that the longest flowering overlap of the pollenizers with the main cultivars, the longest flowering time, the highest pollen quality, the highest number of pollen tubes in the style and ovary locules, a fast growth dynamic from the style to the ovule locules and the highest fruit set after artificial pollination indicate the best pollenizer for these two new apple cultivars. By recommending the best pollenizers for these two cultivars, producers will be ensured to have high and consistent yields of high fruit quality.

2. Materials and Methods

2.1. Plant Material

The apple cultivars ‘Eden’ (Wursixo) and ‘Fryd’ (Wuranda) were used as pollen recipients, in addition to their self- and open pollination. The cultivars ‘Rubinstep’, ‘Red Aroma’, ‘Elstar’, ‘Asfari’, and ‘Professor Sprenger’ (crab apple) were used as pollen donors, and the pollen from ‘Fryd’ and ‘Eden’ was used for mutual pollination. Each cultivar was represented with at least 20 trees, and all were planted in orchards belonging to the Norwegian Institute for Bioeconomy, Lofthus, Ullensvang.

The studies were conducted in an experimental apple orchard at Lofthus, NIBIO Ullensvang, Western Norway, at latitude 60°19′8.03″ N, longitude 6°39′14.31″ E, over two consecutive years (2022–2023). The trees were planted in 2020 as two-year-old feathered trees, with all cultivars grafted on the rootstock M9 with ‘Santana’ as an interstock. The trees were spaced 4 × 1 m apart and trained as slender spindle trees. Trees selected for the experiment consistently flowered and represented the average bloom intensity and tree size of the orchard. Having good management, the trees gave a small yield the year after planting and significant the third and fourth year. No thinning was conducted. The crossings were carried out on two- and three-year-old branches. The orchard management included grass between the rows and a 1-m-wide vegetation-free strip within the rows. During the season, the trees were daily fertigated from drip lines along the tree rows and based on evaporation.

2.2. Climate Conditions and Flowering Time

Western Norway is characterized by cool summers and mild winters, with an average temperature of 7.6 °C and 1705 mm of rainfall. Weather fronts usually come from the Atlantic Ocean, and thus clouds, rain, and wind dominate throughout the year. Regardless of whether the Gulf Stream is moderating its climate, cold temperatures can occur during spring. In the Hardanger region, most of the rainfall occurs during the winter. Summers are dryer, and water has to be supplied to the fruit trees. The average temperature during the growing season (May–September) is 14.4 °C, and the amount of rainfall is 459 mm. Climatic data were collected from the meteorological stations in Lofthus place. Daily mean, maximum, and minimum temperatures, as well as precipitation in millimeters, were recorded during the flowering period of the tested apple varieties over two years. Graphs depicting the climate parameters by month for both years were obtained with permission from the NIBIO and Norwegian Meteorological Institute from the website https://lmt.nibio.no/ (accessed on 1 March 2025).

The phenophase of flowering of each tested cultivar was recorded according to the BBCH system to code the phenological growth stages of plants, history, and publications (BBCH scale) [33]. The start of flowering (BBCH stage 61) was observed when roughly 10% of the flowers had opened. Full bloom (BBCH stage 65) occurred when 50% of the flowers were open, and the end of flowering was recorded when most of the petals had fallen (BBCH stage 67).

2.3. Pollen Germination In Vitro

The anthers from at least 100 flowers originated from five different trees from each cultivar were collected at the late balloon stage (BBCH stage 60) and allowed to dehisce for 24 to 48 h at room temperature (22 °C). To evaluate in vitro pollen germination for all tested pollinizers, the pollen was placed in Petri dishes containing an artificial medium of 1% agar and 14% sucrose and then kept at 22 °C for 24 h. The number of germinated pollen grains was counted in three microscopic observation fields using a Leica DM LS light microscope (magnification 100×). Pollen grains with tubes exceeding their radius were considered as germinating [34].

2.4. Pollination Procedure

During the balloon stage, branches with flowers from 2–3 trees/pollination combination of pollen recipient cultivars ‘Eden’ and ‘Fryd’ were selected and marked. Open flowers were removed, and the remaining ones were emasculated. Approximately 250 to 300 flowers were prepared (emasculated and marked) for each combination of crosses, self-pollination, and open pollination. For hand pollination, ‘balloon’ flowers were collected, and the anthers were removed in Petri dishes, leaving them open at room temperature to dry and release pollen grains. Once the pollen was shed, the dishes were closed and refrigerated at +4 °C. Pollination was performed 24 to 72 h after emasculation, during the early hours when the mother trees were in full bloom (when nearby branches had wide-open flowers and the anthers were changing color), and when stigma secretion was visible. Before pollination, the closed dishes were shaken in all directions to create vibrations, which helped release more pollen grains. Hand pollination of emasculated flowers was carried out by dipping a finger into the Petri dish containing the pollen and then touching the exposed stigma twice. For this study, the following combinations were carried out: self-pollination and open pollination of ‘Eden’ and ‘Fryd’, as well as cross-pollination between these two cultivars and the following pollinators: ‘Rubinstep’, ‘Red Aroma’, ‘Elstar’, ‘Professor Sprenger’, and among each other.

2.5. Pollen Tube Growth In Vivo

Gathered pistils (three repetitions × 10 pistils) from every combination of crossing were picked on the third, sixth, and tenth days after pollination (DAP) and placed in the mixed FPA solution (90:5:5 ratio of 95% ethanol, propionic acid and formaline (40% formaldehyde), respectively). Fixed materials were kept at +4 °C until processing and staining with aniline blue according to the Preil [35] and Kho and Baër [36] methods. To make native (short term) preparations, each pistil was halved by removing the ovary from the styles. In each combination of pollination and each fixation day (third, sixth, and tenth DAP), pollen tubes were counted, and the growth dynamic from stigma toward ovary locule was followed. The dynamic of pollen tube growth through the upper, medium, and basal parts of the styles and ovary locule was given as the percentage of pistils where pollen tubes could be observed. Fluorescent microscopes Leica DM LS (Leica Microsystems, Wetzlar, Germany) with filters A (wavelength 340–380 nm) and I3 (wavelength 450–490 nm)] were used to study pollen tube growth in vivo (magnification 50×).

2.6. Fruit Set

A group of 250–300 open-pollinated flowers in both cultivars was chosen to test the fruit set. The percentage of the initial fruit set was counted around one month after full bloom (before the ‘June’ drop), while the final fruit set was recorded just before the harvest (BBCH 79). The harvesting time of cultivar ‘Fryd’ was on 12 October 2022 and 22 September 2023. Cultivar ‘Eden’ was harvested 23 September 2022 and 13 September 2023. The initial/final fruit set was calculated as a percentage of formed/harvested fruits per 100 flowers.

2.7. Statistical Analysis

Two factorial analyses of variance (ANOVA) were used to process the obtained data. Differences were compared using the Tukey test (significant differences at p < 0.05). Statistical analyses were performed using STATISTICA for Windows 6.0 (StatSoft Inc., Tulsa, OK, USA) software package.

3. Results

3.1. Time of Flowering and Climatic Conditions

The earliest onset of the flowering phenophase (10% open flowers, BBCH61) for the ‘Rubinstep’ cultivar was recorded on 11 May 2022, which was seven days earlier than in 2023, when both ‘Rubinstep’ and ‘Red Aroma’ cultivars began flowering on 18 May (Figure 1). In 2023, the latest start of this flowering phase was observed on 21 May for the ‘Fryd’ and ‘Eden’ cultivars, while the ‘Professor Sprenger’ cultivar began flowering on 22 May. In both years, the ‘Rubinstep’, ‘Elstar’, and ‘Asfari’ cultivars exhibited the shortest flowering window. In 2022, the ‘Elstar’ cultivar had the shortest flowering interval of 10 days, followed by ‘Rubinstep’ and ‘Asfari’, each with a 12-day interval. In contrast, in 2023, the ‘Asfari’ cultivar had the shortest interval of 12 days, followed by the ‘Rubinstep’ and ‘Elstar’ cultivars, each with a 13-day interval. The longest flowering periods were noted for ‘Professor Sprenger’ and ‘Eden’ in 2022, lasting 16 days. This duration was slightly shorter in 2023, totaling 15 days for both ‘Red Aroma’ and ‘Professor Sprenger’.

The examined flowering period ranged from 10% to 50% open, concluding at 80% fallen flowers, as outlined by the BBCH scale. In 2022, this period lasted 23 days, while in 2023, it was shorter, lasting only 16 days. In 2022, the phenophase at 50% open flowers (BBCH65) was observed in both the ‘Fryd’ and ‘Eden’ cultivars on 23 May, which closely coincided with the same phenophase in ‘Professor Sprenger’, recorded on 24 May. This phenophase began on 29 May for the ‘Eden’ and ‘Fryd’ the following year, aligning best with the ‘Asfari’ cultivar on 28 May and the ‘Professor Sprenger’ on 29 May. In 2022, this phenophase started 2 to 4 days earlier with four pollenizers: ‘Rubinstep’, ‘Red Aroma’, ‘Elstar’, and ‘Asfari’. In 2023, the start of the flowering was even earlier, 4 to 6 days ahead of the ‘Rubinstep’, ‘Red Aroma’, and ‘Elstar’ cultivars.

3.2. Air Temperature and Precipitation

The temperature and precipitation figures for 2022 and 2023 are presented in Figure 2 and Figure 3, respectively. In 2022, temperatures varied from a low of −7.7 °C on 14 December to a high of 29.6 °C on 15 June. The month with the highest average temperature was August, recording an average of 15.3 °C (Figure 2A). Deviations from the average temperature ranged from 0.1 °C to ±2.8 °C in certain months. That year also had a total of 154 days with precipitation, amounting to 1604.8 mm, less than the average long term value of 1708 mm (Figure 2B). The average temperature during the growing months from May through September was 13.5 °C, and the amount of precipitation was 381 mm. This is almost 1 °C lower than the 30-year average.

In 2023, temperatures ranged from a low of −9.1 °C on 10 March to a high of 28.8 °C on 24 June. The highest average temperature recorded was 16.1 °C in July (Figure 3A). Temperature deviations from the average long term values varied between −2.1 °C and 3.7 °C in certain months. During this year, there were 154 days of precipitation, totaling 1513.9 mm, which is lower than the average for 2022 (Figure 3B). Notwithstanding, in the months of March, April, May, and June, there was drought, with 65 mm, 60 mm, 20 mm, and 50 mm, respectively, which is less rainfall than the average. This year, the average temperature from May to September was 14.4 °C, which is similar to the average temperature. The amount of precipitation was 509 mm.

During the flowering period of 28 days, from the opening of the earliest cultivar’s flowers to the petal fall of the latest cultivar, the average daily air temperature in 2022 was 11 °C (Figure 4). That same year, the mean maximum temperature reached 15.6 °C, while the minimum was 7.9 °C. There was precipitation on 21 days during this period, totaling 112.1 mm. For the duration of the progamic phase of fertilization, from 20 May to 30 May, the average temperature was 11.6 °C, with an average minimum of 8.6 °C and a maximum of 16.3 °C.

The following year, the average daily air temperature increased slightly by one degree during the flowering period, reaching 12 °C. The flowering period for the examined apple varieties was shorter by nine days compared to 2022, lasting 19 days. The average maximum temperature during this period was 16.6 °C, while the average minimum was 8.2 °C. Precipitation occurred on only seven days, totaling 43.3 mm.

During the progamic phase of fertilization, from 24 May to 3 June 2023, the average daily temperature was 11.1 °C. The average minimum temperature was 7.9 °C, and the maximum was 15 °C, which was the same as or slightly lower than the previous year. During that period, there were six days of precipitation, totaling 21.9 mm, which is slightly less than the amount recorded in 2022.

3.3. In Vitro Pollen Germination

The assessment of pollen germination in vitro serves as the primary indicator of pollen functional viability. Statistical analyses of the germination data revealed significant differences between cultivars (Figure 5). In 2022, the average germination rate for all tested cultivars was slightly higher than in 2023, with 70.5% ± 3.8 and 68.3% ± 3.4, respectively, but not statistically different. Also, interaction genotype × year did not show any statistically significant influence on pollen germination.

The ‘Red Aroma’ exhibited the highest pollen germination rates in 2022 (89.9% ± 5.5) while cultivar ‘Fryd’ showed the highest rate in 2023 (83.6% ± 3.4). Cultivar ‘Asfari’ recorded the lowest average percentages of germinated pollen tubes in 2022 (49.2% ± 2.2) and in 2023 (51.5% ± 2.1).

3.4. Pollen Tube Growth in the Pistil

In our pollination studies, it was common to observe an overload of pollen grains on the surfaces of all five stigmas in all crossing combinations (Figure 6A). Pollen tubes then penetrated the ‘transmitting tissue’ of the five styles in a progressive way, ‘bound’ in an intercellular manner, moving through the tissue and the individual obturators to reach the locules of the ovary. Throughout this growth process, the number of pollen tubes decreased significantly, especially as they traveled through different sections of the styles—upper, medium, and lower (Figure 6B–D). The pentacarpelar apple gynoecium consists of five ovary locules, each containing two anatropous ovules. Pollen tubes were observed moving toward the ovule locules. After traveling through the funiculus and placental tissue, the pollen tubes make a 180° turn inside each locule (Figure 6E). This maneuver allows the pollen tube to penetrate over the obturator to the micropyle of the ovule. In cases of self-pollination, the growth of incompatible pollen tubes was stopped in the upper part of the styles (Figure 6F).

The upper parts of the styles and ovary locules are crucial for assessing the effectiveness of pollen tube growth. The average number of pollen tubes in the pistil parts varied across different crossing combinations and years of study (Figure 7).

In 2022, the average number of pollen tubes observed in the upper part of the style was higher in ‘Fryd’ (60.6 ± 4.1) compared to ‘Eden’ (30.2 ± 2.8). However, in the following year, this difference decreased, with ‘Fryd’ averaging 42.9 ± 2.8 and ‘Eden’ averaging 34.9 ± 2.2. Significant differences were particularly noted in specific pollination combinations during certain years. In the same year, ‘Eden’ displayed the highest average number of pollen tubes in the upper parts of its styles when pollinated with ‘Professor Sprenger’ (50.1 ± 3.5) and ‘Rubinstep’ (50.9 ± 7.6). In contrast, the lowest average number of pollen tubes was observed during self-pollination (8.5 ± 0.8) and when pollinated with ‘Asfari’ (19.9 ± 1.5). The same year, ‘Fryd’ achieved the highest average number of pollen tubes in the upper third of the styles during open pollination, reaching an average of 75.6 ± 5.1. It also recorded a high average pollen tube number with ‘Elstar’ (70 ± 4.9) and ‘Red Aroma’ (70.3 ± 4.4). In contrast, the lowest average number of pollen tubes in the upper parts of the styles for ‘Fryd’ was observed when pollinated with ‘Eden’ (40.3 ± 3.4).

The following year, cultivar ‘Eden’ exhibited the highest average number of pollen tubes in the upper third of the styles when pollinated with ‘Professor Sprenger’ (50.1 ± 3.3) and ‘Rubinstep’ (30.9 ± 2.5). In contrast, it had the lowest count when pollinated with ‘Red Aroma’ (21.7 ± 1.5). For the ‘Fryd’, the highest average number of pollen tubes in the upper third of the styles was observed during open pollination (58 ± 3.0) and when pollinated with ‘Eden’ (56 ± 3.1) and ‘Professor Sprenger’ (46.7 ± 2.9). This cultivar recorded the lowest average in self-pollination (23.2 ± 2.2) and with ‘Red Aroma’ (28.5 ± 2.3).

The average number of pollen tubes calculated for all pollination combinations observed in the ovary locules for ‘Fryd’ was 6.5 ± 0.5 in 2022. This is significantly higher than the average of 3.7 ± −0.3 pollen tubes compared to ‘Eden’ during the same year. In the following year, ‘Eden’ demonstrated a slight improvement, averaging 5.1 ± 0.4 pollen tubes in the ovary locules, compared to ‘Fryd’, which averaged 4.8 ± 0.4 pollen tubes. It is important to note that in self-pollination, neither ‘Eden’ nor ‘Fryd’ showed any penetration of pollen tubes into their ovary locules.

In 2022, the ‘Eden’ exhibited the highest average number of pollen tubes in its ovary locules when pollinated with ‘Rubinstep’ (5.7 ± 0.3) and ‘Red Aroma’ (5.3 ± 0.3). The lowest average was recorded with ‘Professor Sprenger’ (1.5). The following year, open pollination proved the most effective method, resulting in an average of 6.7 ± 0.5 pollen tubes in ‘Eden’. This was closely followed by the combination of ‘Asfari’ (6.1 ± 0.4) and ‘Professor Sprenger’ (5.0 ± 0.4).

For the ‘Fryd’ as pollen recipient, the highest average number of pollen tubes across all pollination combinations was recorded with ‘Elstar’ (9.6 ± 0.6) and ‘Red Aroma’ (8.1 ± 0.6). In the subsequent year, ‘Eden’ (5.8 ± 0.5) and ‘Professor Sprenger’ (5.1 ± 0.4) produced the highest average number of pollen tubes in the ovule locules of ‘Fryd’.

Figure 8 shows the dynamic of pollen tube growth in all pollination combinations of ‘Eden’ and ‘Fryd’ in 2022. The highest rates of pistils (100%) containing pollen tubes in ovary was determined in crossing combination ‘Eden’ × ‘Professor Sprenger’, followed by crosses ‘Eden’ × ‘Rubinstep’ and ‘Eden’ × ‘Asfari’ (both achieving a success rate of 94.1% during the tenth day after pollination). Conversely, the lowest percentage of pistils (15%) with pollen tubes reaching the ovule locules was found in the open pollination of ‘Eden’. In self-pollination of ‘Eden’, 60% of the pistils exhibited pollen tubes that extended only to the lower parts of the styles.

In the ‘Fryd’ cultivar, 100% of the pistils showed successful pollen tube penetration, with the tubes reaching the ovule locules when pollinating with ‘Rubinstep’, ‘Red Aroma’, ‘Professor Sprenger’, and ‘Eden’. In contrast, during open pollination, only 37.5% of the ‘Fryd’ pistils had pollen tubes that reached the ovule locules, which was the lowest percentage observed. Additionally, in the self-pollination of ‘Fryd’, pollen tubes penetrated only up to the lower parts of the styles in 20% of the examined pistils.

The following year, the highest percentage of pistils (93%) with pollen tubes that penetrated the locule of the ovary were recorded in combinations of pollination ‘Eden’ × ‘Fryd’ on the tenth day after pollination (Figure 9). The pollination of ‘Eden’ with ‘Red Aroma’ and ‘Rubinstep’ resulted in 80% of the pistils having pollen tubes that penetrated the ovary locules simultaneously. The lowest percentage of pistils with pollen tubes that penetrated ovary locules was found in ‘Eden’ during both open pollination (53.8%) and self-pollination (20%).

In the cultivar ‘Fryd’ the highest percentage of pistils with pollen tubes penetrating the ovary locules was observed in two specific pollination combinations. The first one is ‘Fryd’ × ‘Eden’, which achieved an 80% success rate, while the second combination, ‘Fryd’ × ‘Professor Sprenger’, demonstrated a 70% success rate ten days after pollination. In contrast, the self-pollination of ‘Fryd’ resulted in the lowest percentage, with only 80% of the pistils having pollen tubes that reached the lower parts of the styles by the tenth day after pollination.

In both years of the study, signs of incompatibility and arrested pollen tubes were frequently observed through the styles in the mother cultivars ‘Eden’ and ‘Fryd’ during self-pollination. Specifically, for cultivar ‘Eden’, 10% of the pistils showed pollen penetration into the ovary locules during self-pollination.

3.5. Fruit Set

The fruit set values for the mother cultivars ‘Eden’ and ‘Fryd’ across all pollination combinations, including self-pollination and open pollination, are illustrated in Figure 10 for both 2022 and 2023. All factors examined—genotype, year of study, and their interactions—significantly impacted the fruit set. When averaging results from all types of pollination, the final fruit set in 2022 was slightly higher at 24.3 ± 1.7% compared to 21.7 ± 1.6% in 2023. In cultivar ‘Eden’, the average fruit set across all pollination combinations was 34.1% ± 2.1 in 2022, but decreased to 13.4% ± 0.9 in 2023. Cultivar ‘Fryd’ also declined, with a fruit set of 28.5% ± 1.8 in 2022 to 14.8% ± 1.1 in 2023. In specific years, pollination combinations significantly affected the final fruit set rate in both ‘Eden’ and ‘Fryd’.

In 2022, the cultivar ‘Eden’ achieved the highest average final fruit set with various pollination combinations, reaching 46.2% ± 2.2 when pollinated with ‘Rubinstep’ and 57.0% ± 3.5 with ‘Red Aroma’. However, it had the lowest yields with self-pollination and open pollination, producing only 1.4% ± 0.1 and 13% ± 0.9, respectively. The following year, these percentages significantly decreased. Regarding cultivars, the highest average final fruit set was recorded in pollination combination with ‘Professor Sprenger’ (21.7% ± 1.2) and ‘Asfari’ (17.5% ± 1.1).

The cultivar ‘Fryd’ demonstrated the highest average final fruit set in 2022 when pollinated with ‘Rubinstep’ (46.6% ± 1.6) and ‘Elstar’ (40.5% ± 1.2). In contrast, the lowest fruit set percentages were observed with ‘Red Aroma’ (18.8% ± 0.9) and during self-pollination (0%). Similar trends were noted the following year with ‘Eden’ and other pollenizers, showing a decline with all pollination combinations. Notably, the highest fruit set for ‘Fryd’ was achieved in pollination combinations with ‘Eden’ (33.7% ± 1.3) and ‘Rubinstep’ (16.7% ± 1.1). Otherwise, previous studies indicated that the cultivars ‘Eden’ and ‘Fryd’ are each other’s best pollenizers due to their complete cross-compatibility and similar flowering dates [38]. Conversely, ‘Fryd’s’ lowest fruit set percentages were again observed in the self-pollination combination (0%) and with ‘Red Aroma’ (10%). In the cultivar ‘Eden’, the final fruit set from self-pollination ranged from 1.4% in 2022 to 2.3% in 2023. In contrast, the pollen recipient cultivar ‘Fryd’ exhibited a final fruit set percentage of 0% in both years.

4. Discussion

4.1. In Vitro Pollen Germination

Several elements affect pollen germination, including the cultivar, flower age, the time of collection, the season, the conditions under which the pollen is stored and tests used for germination assessment [39]. The viability and germination percentages of pollen among various diploid apple cultivars ranged from 56% to 95.5%, depending on geographical regions and climatic conditions [7,40,41]. In our research, the average germination rate for all cultivars in both years was 69.5 ± 3.6%. The lowest individual pollen germination rate recorded was 50.3% ± 3.1 (‘Asfari’), while the highest was 90% ± 5.4 (‘Red Aroma’). Although not statistically significant between the years, the variations in pollen germination between the cultivars are influenced by other external and internal factors affecting the development of pollen grains. The variation in pollen germination percentage can be due to the difference in the pollen protein and carbohydrate content, which results from a genetic variation among cultivars studied [42]. Also, climatic conditions, mostly extreme temperature and drought, and lack of some micronutrients or excess of heavy metals can make pollen germination very low [43,44,45].

4.2. Pollen Tube Growth In Vivo

The growth of the pollen tube into the pistil begins when pollen grains germinate on the funnel-shaped surface of the stigma. The stigma is the specialized receptive part of the style that captures pollen and is a natural medium for pollen hydration and germination and other functions in the pollen–stigma interaction [46]. In the apple, no matter whether the stigmas are multicarpelar, all show receptivity at the same time [47]. The stigma of the apple is characterized by a wet surface, papillate receptive cells, and moderate to slight secretions that flood interstices. The liquid sticky exudate is rich in carbohydrates, amino acids, proteins, lipids, and S-RNases [48,49]. When the period of receptivity ends, the stigma dries out and turns brown. In apple corymb, king flowers have an intense but short stigmatic receptivity, which is at its maximum at anthesis, whereas lateral flowers have a more discrete but much longer stigmatic receptivity, with a maximum of three days after anthesis [47].

The quality of pollen, stigma receptivity, activation and guidance of pollen tubes, among other factors, play a crucial role during the progamic phase of fertilization [50,51]. Additionally, less than 8% of the progamic phase is dedicated to pollen germination [52]. Meanwhile, the remaining time is associated with the growth of the pollen tube from the stigma to the ovule [53]. Pollen tube growth in all crossing combinations of pollination begins with the germination of pollen grains on the stigma surface. The number of pollen tubes gradually decreases from the stigma to the locules of the ovary. These changes are accompanied by the emergence of intercellular substances that contribute to various processes, such as nourishing the growing pollen tubes and guiding them through different female structures in the final stages of the progamic phase of fertilization [54,55,56]. Some results suggest that pollen tube growth is further modified by the temperature under which pollen tube growth occurs [15]. These findings may be more closely related to the maternal cultivar than the paternal one, which indicates that maternal genotype can influence pollen tube growth and fertilization [57].

Our study found that when self- and open pollination were excluded, the average number of pollen tubes for all pollenizers in the upper styles of ‘Eden’ was 31.4 ± 2.1 in 2022 and 33.3 ± 2.2 in 2023. In the ovule locules, these average numbers were 4.4 ± 0.3 in 2022 and 4.8 ± 0.4 in 2023. In the case of the ‘Fryd’ cultivar, there were more noticeable variations over the years. The average number of pollen tubes for all pollenizers in the upper styles was 59.2 ± 3.4 in 2022 and 43.9 ± 3.0 in 2023, while in the ovule locules, the average numbers were 6.9 ± 0.4 in 2022 and 4.9 ± 0.3 in 2023. Although the differences in the average number of pollen tubes in the upper styles and ovary locules between the years were not statistically significant, it is noteworthy that ‘Fryd’ had higher counts in 2022 compared to 2023. This discrepancy might be due to the average daily temperature during the first six days of the progamic phase of fertilization, which was 2.7 °C higher in 2022 (12.9 °C) than in 2023 (10.2 °C). During this period, the penetration of pollen tubes into the ovule locules is noticeable. Pollen growth rates are considered more efficient at higher temperatures in ‘Fryd’ than in ‘Eden’. In 2022, ‘Fryd’ exhibited the most efficient growth of pollen tubes, with 100% of the pistils showing pollen penetration to the lower part of the style when pollinated with ‘Rubinstep’, ‘Red Aroma’, ‘Elstar’, and ‘Eden’. Pollen tube penetration into the lower part of the styles was observed at a rate of 100% only in the pollination combination of ‘Eden’ with ‘Professor Sprenger’. The following year, the pairing of ‘Eden’ with ‘Fryd’ was the only combination that achieved 100% pistil penetration by pollen tubes in the ovary locules.

Additionally, these results indicated that each genotype responded differently to temperature changes [26,58,59,60]. This conclusion also applies to individual pollenizers, since for ‘Eden’, the pollenizer ‘Professor Sprenger’, demonstrated the highest number of pollen tubes in the upper part of the style during both years. In contrast, the pollenizers ‘Rubinstep’ in 2022 and ‘Asfari’ in 2023 recorded the highest average number of pollen tubes that penetrated the ovule locules of ‘Eden’. Observations for ‘Fryd’ in 2022 revealed that ‘Elstar’ was the most effective pollinizer, producing the highest average number of pollen tubes in the upper parts of the styles and ovary locules. However, in 2023, ‘Eden’ became the best pollinizer for the ‘Fryd’. These findings reinforce earlier conclusions that climate factors, including temperature and humidity, significantly impact pre- and post-fertilization processes [26].

Regarding self-pollination, the penetration of pollen tubes was observed in the lower part of ‘Fryd’ in both years and in ‘Eden’ only in 2023. In 2022, pollen tube penetration into the ovary locules was confirmed in 10% of the pistils of ‘Eden’. However, in most cases, pollen tubes showed characteristic looping, forkening, and thickening as signs of self-incompatibility which indicate that both cultivars could be considered as self-incompatible.

4.3. Fertilization and Fruit Set

Pollination is critical in the fruit set, growth, quality, and seed production of temperate fruits, mostly apple cultivars. Also, successful fertilization, where the ovary transforms into a rapidly growing young fruit, relies on the efficiency of the progamic phase of fertilization, which includes transferring pollen to the stigma, growing pollen tubes through the pistil, and fertilizing the ovules [51]. Several factors, including weather conditions, pollinator activity, compatibility between cultivars, and the timing of flowering, can influence the limit of fruit sets, lack of nutrients, and early flower drop [61,62]. Water stress can sometimes affect fruit drop in June, July, and August. Otherwise, in the climatic conditions in Western Norway, variations in fruit set vary between the years, which have already been observed in certain varieties of pear and plum [29,30]. Moreover, biennial bearing in apples, which is a persistent problem, can influence fruit set in booth ‘on’ and ‘off’ year. Biennial bearing apple cultivars can bear fruit on more than 90% of terminal buds in ‘on’ year, versus annual bearing cultivars where the number of terminal buds carrying fruit is usually around 20% [63,64].

The results of the progamic phase of fertilization and fruit set for all pollination combinations except self- and open pollination in both ‘Eden’ and ‘Fryd’ cultivars showed differences over the years. Eden’s average fruit set was from 46.2 ± 2.2% in 2022 to 16.1% ± 0.8 in 2023, while ‘Fryd’ decreased from 38.8% ± 2.1 to 17.2% ± 1.8. Throughout both years, ‘Eden’ consistently showed a higher average number of pollen tubes than ‘Fryd’, observed in the upper styles, ovary locules, and pollen tube growth dynamics (Figure 7, Figure 8 and Figure 9). This study revealed that the different pollinizers influenced the average number of pollen tubes in the upper part of the style and ovary and the percentage of pistils with pollen tubes that penetrated the ovary’s locule. It should be noted that ovule viability is an important factor in fruit set in addition to the progamic phase of fertilization [65].

Both ‘Eden’ and ‘Fryd’ originate from the Netherlands, where higher temperatures during flowering occur. Although the quantitative parameters of the progamic phase of ‘Fryd’ are better than those of ‘Eden’, the fruit set of ‘Fryd’ is somewhat lower than that of ‘Eden’.

Data collected over two years show that the average fruit set from open pollination for the ‘Fryd’ was 19.2% ± 1.1, higher than ‘Eden’ (12.6% ± 1.1). Both cultivars showed slightly higher fruit sets in 2022 compared to 2023. The fertilization efficiency of the ‘Eden’ and ‘Fryd’ was similar, with average pollen tube numbers in the style and ovary locules often exceeding control crossings. This suggests that in open pollination, both cultivars have comparable pollen receptivity during full bloom and receive similar amounts and qualities of pollen, impacting their fruit set values over the years.

Besides depending on temperature, the pollenizer’s success can depend on the influence of the pollen recipient cultivar, which can affect fruit set values, as observed in some other fruit species [28,29]. In the self-pollination variant, ‘Eden’ produced 1.4% ± 0.1 in 2022 and 2.3% ± 0.2 in 2023 of fruit set. In contrast, ‘Fryd’ had no fruit set in either year. These results of in vivo pollen tube growth and fruit set indicate that both cultivars are self-incompatible. Generally, most apple cultivars are primarily self-incompatible, with only a few that can self-pollinate [66].

During the two-year study, both cultivars had a much higher fruit set in 2022 than in 2023. The fruit set average of both cultivars was ~2.5 times higher in 2022 (41% ± 2.1) compared to 2023 (16.7% ± 1.1). This difference can be attributed primarily to the impact of the appearance of water stress in May and June 2023. May 2023 had ~1.7-fold lower precipitation than in 2022, and June 2023 had ~2.7-fold lower rainfall than the same month in 2022 (Figure 2B and Figure 3B). This discrepancy could have affected the severe June drop in 2023 and the much lower fruit set. Similar findings were discovered by Racsko et al. [67], who argued that the effect of preceding temperature and water deficit are important factors responsible for the June drop in apples. The June drop mainly occurs 5–6 weeks after full bloom, when cell division and cell expansion of the fruitlets are energy and sugar dependent, so they become a strong sink organ. When fruitlets start ‘fighting’ with young growing shoots for translocated photoassimilates, extreme competition can lead to fruit abscission [68]. Moreover, the June drop is related to protein synthesis, mineral nutrition, the number of fertilized ovules per fruit, and embryo abortion [69].

Another scenario that can explain the much lower fruit set in 2023 compared to 2022 is preharvest fruit drop (PFD), which begins approximately four weeks before harvest and is mainly caused by a surge of endogenous ethylene. This physiological phenomenon can vary significantly between orchards due to differences in climate and agricultural practices, even within the same year. Factors such as wind, temperature, nutrient availability, water supply, sunlight, and disease pressure contribute to this physiological behavior. Under extreme conditions, plant resource deprivation can lead to growth changes and premature fruit drop in response to stress [70,71].

In 2022, ‘Red Aroma’, ‘Rubinstep’ and ‘Asfari’ were the best pollenizers for Eden (giving 57.0% ± 3.5, 46.2% ± 2.2, and 45.3% ± 2.5 fruit set, respectively), while in the second year those were ‘Professor Sprenger’, ‘Asfari’ and ‘Red Aroma’ (21.7% ± 1.2, 17.5% ± 1.1, and 17.5% ± 0.5 fruit set, respectively). For ‘Fryd’ the top pollinators in terms of fruit set in 2022 were ‘Rubinstep’ (46.6% ± 1.6) and ‘Elstar’ (40.5% ± 1.2). In 2023, the effectiveness shifted to ‘Eden’ (33.7% ± 1.3) and ‘Rubinstep’ (16.7% ± 1.1). Our results coincide with those of Bessho et al. [72] and Das et al. [73], who evaluated crabapples for their suitability as pollinizers for commercial apple cultivars in terms of bloom time, pollen compatibility, seed number, and productivity and found that crabapple was an effective pollinizer in terms of fruit set.

Even though in vitro and in vivo pollen tube growth among the tested pollinators does not correspond with their fruit settings, these parameters strongly impact the final fruit set.

4.4. Overlapping in Flowering Time

Flowering overlap is crucial to ensure cross-pollination in apple. Since apples are self-incompatible, one or more compatible pollenizers are required to fruit set and produce a commercial yield successfully. Cultivars with a long flowering season, e.g., those that flower both on one-year-old, long shoots and on spurs, and those that bloom annually, rather than biennially, should be useful as cross pollinizers [74,75]. According to Kron et al. [76], the greater the flowering overlaps between apple cultivars, the higher is the siring success of the pollenizer. According to Delaplane and Mayer [77], it is necessary to plant both early- and late-blooming pollinizers so that the main apple cultivar blooms in between in order to have optimal yields.

In 2022, the period between the start of flowering of the earliest pollenizer, ‘Rubinstep’, and the end of the flowering for the latest ‘Eden’ was 23 days. Considering the flowering overlap, ‘Eden’ and ‘Fryd’ had the most extended overlap with the pollenizer ‘Professor Sprenger’ (8–9 days) and the least with ‘Rubinstep’ (4 days). In addition, the pollenizers ‘Asfari’, ‘Elstar’, ‘Red Aroma’, and ‘Rubinstep’ had full bloom 2 to 4 days earlier than ‘Eden’ and ‘Fried’. The following year, ‘Professor Sprenger’ had the most extended overlapping flowering with ‘Eden’ and ‘Fryd’ (5 days).

In the following year, the time between the beginning of the flowering of the earliest cultivar (‘Rubinstep’) and the end of the latest flowering cultivar (‘Professor Sprenger’) was shorter than 2023, lasting only 16 days. That year, the pollenizer ‘Professor Sprenger’ had the most extended flowering time, so its full bloom overlapped for 5 days with the full bloom of ‘Eden’ and ‘Fryd’. The full bloom of other pollenizers overlapped just with the beginning of flowering of ‘Eden’ and ‘Fryd’, and this lasted from 6 days (‘Asfari’) to 10 days (‘Red Aroma’). The best overlap occurred between ‘Eden’ and ‘Fryd’ as mutual pollenizers, with 9 days in 2022 and 6 days in 2023. Apart from the coincidence in flowering dates, the duration of the bloom period is especially relevant since a long-lasting flowering period can lead to a more stable year-to-year fruit set [78]. The cultivar ‘Professor Sprenger’ exhibited a long blooming period, which overlaps with many other cultivars and potentially can be used to pollinate cultivars from different flowering groups. Previously it was reported that ‘Prof. Sprenger’ had very high potential for use as pollinizer to supplement pollination of Gala and Fuji cultivars [79].

5. Conclusions

Introducing new apple varieties—in this case, ‘Eden’ and ‘Fryd’—requires carefully selecting pollenizers because these cultivars are self-incompatible. This is particularly important in Western Norway, where apple cultivation faces climatic challenges, varying flowering times, and a shortage of well-adapted cultivars and suitable pollenizers for those that can be successfully grown in this region.

The studied reproductive parameters in vitro pollen germination, progamic phase of fertilization, flowering time overlap, and fruit set reveal varying adaptability levels among pollen recipient and pollenizer cultivars in Western Norway’s environmental conditions. Based on the aforementioned reproductive parameters, the ‘Eden’ cultivar had a higher fruit set in both years than the ‘Fryd’. Otherwise, in 2023, specific climatic conditions in Western Norway caused a significant shortage of rainfall during May and June. This stress led to substantially reduced fruit sets in all pollination combinations of the mother cultivars with the tested pollenizers. In addition, it was also confirmed that ‘Eden’ and ‘Fryd’ belong to a group of self-incompatible apple cultivars.

Based on evaluating certain parameters of the progamic phase of fertilization and fruit set, cultivars ‘Rubinstep’, ‘Asfari’, and ‘Red Aroma’ proved to be the best pollenizers for ‘Eden’. In contrast, ‘Rubinstep’, ‘Eden’, and ‘Elstar’ were the best pollenizers for ‘Fryd’. When considering the parameters that indicate a longer overlap between flowering and high fruit set, ‘Professor Sprenger’ and ‘Fryd’ were identified as the best pollenizers for the cultivar ‘Eden’. In addition, ‘Professor Sprenger’ and ‘Eden’ were the best pollenizers for ‘Fryd’. This overlap in flowering significantly boosts the chances of successful pollination, leading to higher yields of quality in the apple fruit.

From the producer’s point of view, combining ‘Eden’ with ‘Red Aroma’ and ‘Asfari’ and ‘Fryd’ with ‘Rubinstep’ and ‘Eden’ seems to represent a good solution. If growing together in the same orchard, cultivars ‘Rubinstep’ (as one of the best pollenizers for both cultivars) and ‘Professor Sprenger’ (the longest overlap in flowering, and a source of high-quality pollen for bees [80]) would be a win-win combination for everybody.