2022 Volume 91 Issue 3 Pages 276-285
Low temperature negatively affects pollen germination in many species, but artificial pollination can help alleviate these negative effects. With ongoing environmental changes, it is important to select pollinizers with pollen that has superior germination properties at lower temperatures. To select candidate cultivars for a new Pyrus pollinizer for pollen with superior germination properties at low temperatures, in vitro pollen germination was studied in 129 Pyrus plants. The highest germination percentage for most Pyrus plants was observed at 25.0°C and was < 20% at 10.0°C. However, ‘Narayoshinokoboku’, ‘Imamuranatsu’, and ‘Tosanashi’ showed a higher germination rate than the other Pyrus plants at 10.0°C, and the germination percentage rates of three plants were > 30%. The fruit setting percentage of the cultivars ‘Kosui’, ‘Hosui’, and ‘Gold-Nijisseiki’ were > 85% by pollination using these three selected cultivars in an open field. In addition, S-genotyping in selected cultivars was determined as follows: ‘Tosanashi’ (S1S7), ‘Imamuranatsu’ (S1S12), and ‘Narayoshinokoboku’ (S1S9). From these results, we concluded that all three cultivars showed cross-compatibility in most pear cultivars of the four major Japanese pear cultivars. The percentage of flower bud formation in ‘Tosanashi’ was > 50%, and the amount of pollen from ‘Narayoshinokoboku’ was more than double the amount of ‘Chojuro’. By assessing the chilling requirement in selected cultivars, a safe area for growth for each cultivar was evaluated as all areas with the exception of the Kyushu and Shikoku regions, based on the ongoing progress of global warming (average daily temperature increase of 3.7°C). In conclusion, we selected ‘Narayoshinokoboku’ and ‘Tosanashi’ as new Pyrus pollinizers because they combine the advantages of low-temperature germinability and high pollen load.
In Japanese pear cultivars, artificial pollination using pollen from other cultivars is necessary because the Japanese pear has S-RNase-based self-incompatibility, and does not have the parthenocarpy found in the European pear (Nyéki et al., 1998). Extremely low pollen germination rates may cause fruit-setting failure owing to ovule degradation before the pollen tube reaches the ovary (Vasilakakis and Porlingis, 1985). Therefore, pollen germination during pollination is important for stable fruit set and production of high-quality fruit. Pollen germination is affected by multiple biotic and abiotic stresses, including low temperature (Ohnishi et al., 2010; Thakur et al., 2010). Low temperature generally has negative effects on pollen germination and tube growth in many species (Cohen et al., 1989; Rosell et al., 1999). The optimum temperature for pollen germination varies among species and cultivars of the same species, as demonstrated previously in several woody species, such as avocados (Sedgley and Annells, 1981), almonds and peaches (Weinbaum et al., 1984), walnuts (Luza et al., 1987), pistachios (Polito et al., 1988), apricots (Egea et al., 1992), and mangos (Sukhvibul et al., 2000). In addition, the optimum temperature for pollen germination in Pyrus plants was 20°C in many cultivars (Kuroki et al., 2017). However, considerably accelerated flowering time due to global warming in recent years has been reported for Japanese pear cultivation in Japan (Sugiura et al., 2007). Fruiting failure in Japanese pears has occurred as pollen germination has been inhibited by early flowering owing to frequent low temperatures (Kuroki et al., 2017). Therefore, with ongoing global environmental changes, when using artificial pollination it is important to select pollinizers that have pollen with superior germination properties at lower temperatures.
In previous research, pollen of Pyrus communis L., and the ‘La France’ European pear combined the advantages of higher germinability and higher monocarpy under low temperature conditions (10°C) (Kuroki et al., 2017). However, the amount of pollen per 100 flowers in these cultivars was low (Kuroki et al., 2017). Therefore, to select a pollinizer requires selection from other pear cultivars with a higher percentage of flower bud formation and amounts of pollen. Additionally, during selection, estimating suitable crop areas for cultivars selected as pollinizers is important. Global climate change resulting from a predicted absolute increase in temperature, frequency, and amplitude of temperature variations could jeopardize plant cultivation in some areas (Intergovernmental Panel on Climate Change (IPCC), 2001). The consequences of global climate change are already visibly shifting species distributions and flowering times (Hedhly et al., 2005). In pear plants, a lack of winter chilling to break endodormancy has caused problems in the promotion of bud break during spring due to global warming in low-latitude areas (Kingston et al., 1990; Klinac and Geddes, 1995; Petri and Herter, 2002; Petri et al., 2002). The most recent report by the IPCC indicated that the temperature in 2081–2100 may have risen by 3.7°C (likely range between 2.6°C and 4.8°C), compared with preindustrial levels in the case of the RCP8.5 scenario (IPCC, 2013). To grow pear pollinizer cultivars as perennials, suitable crop areas with simulated future climates are needed.
In the present study, we comparatively assessed pollen germination in plants of the main Japanese pear cultivar, and selected cultivars with pollen having higher germination properties under low temperature conditions (10°C). We then investigated the effect of fruit set and fruit quality in the main Japanese pear cultivar by pollination treatment using pollen from selected pear cultivars in an open field. Additionally, we investigated the percentage of flower bud formation, and amount of pollen in the selected cultivars. Finally, to assess suitable crop areas for the future considering ongoing global warming, we evaluated suitable crop areas in selected pear cultivars using the Agro-Meteorological Grid Square Data.
We used pollen from 129 Pyrus plants grown in the orchards of Tottori University (35.5° N, 133.7° E) collected from each plant in April 2017. Pollen was collected from ‘Narayoshinokoboku’, ‘Imamuranatsu’, and ‘Tosanashi’, which were selected as cultivars as they had pollen with superior germination properties at low temperatures, and ‘Chojuro’ as the control in 2018 (‘Imamuranatsu’ was not included in 2019). Pollen was extracted from the anthers of flowers at the balloon stage for germination assays. These anthers were dried on a piece of paper for 24 h at 25°C and separated from pure pollen using acetone. The pollen grains were then immediately germinated in a polystyrene preparation at four temperatures (10.0, 15.0, 20.0, and 25.0°C). The pollen was scattered into the in vitro system, which had already been injected with a solidified germination medium consisting of 10.0% (w/v) sucrose, 1.0 mM boric acid, and 1.0% (w/v) agar. Pollen germination was arrested after 5 h. The percentage of pollen germination was then determined using a microscope (Optiphot; Nikon, Japan).
Pollen germination was defined as when the length of the pollen tube exceeded the diameter of the pollen grain (Kuroki et al., 2017). For each treatment, the percentage of pollen germination was determined three times, and fields in which the pollen had reached at least 100 were selected. Statistical analyses were performed using Tukey-Kramer’s HSD tests. P < 0.05 was considered significant.
Effect of fruit set and fruit quality by pollination treatment using pollen from selected pear cultivars in an open fieldThe experiment was conducted using three 31-year-old Japanese pear cultivars: ‘Kosui’, ‘Hosui’, and ‘Gold-Nijisseiki’, which were grafted onto P. betulaefolia seedlings that were planted in a field at Tottori University, Tottori, Japan. ‘Kosui’, ‘Hosui’, and ‘Gold-Nijisseiki’ were hand pollinated in April 2018 and ‘Kosui’ in April 2019 using pollen from the three selected cultivars and ‘Chojuro’ collected in 2017. The percentage of fruit set for all pollinated flowers was assessed four weeks after pollination and calculated from the number of fruits set among all pollinated flowers. Statistical analyses were performed using the chi-squared test. P < 0.05 was considered significant.
An experiment was performed to evaluate the effects of pollination on fruit quality (n = 30) in ‘Kosui’ in 2018. Fruit quality and the number of seeds per fruit (seed yield) were evaluated on the days of fruit harvest. We determined fruit weight, length and diameter, and flesh firmness (as a parameter of fruit quality) using electronic scales, calipers, and a hardness tester, respectively. Total soluble solids and pH of the fruit juice were determined using ATAGO PAL-1 (Atago, Japan) and a LAQUAtwin-pH-11 pH Meter (HORIBA, Japan), respectively. We evaluated the number of seeds per fruit; good seeds were defined as fully inflated seeds with a diameter of 4 mm or more. Statistical analyses were performed using Tukey-Kramer’s HSD test. P < 0.05 was considered significant.
Percentage of flower bud formation and amount of pollen in selected cultivarsWe used ‘Narayoshinokoboku’, ‘Imamuranatsu’, and ‘Tosanashi’, which were selected as cultivars because they had pollen with superior germination properties at low temperatures, and ‘Chojuro’ as the control. We evaluated the formation of flower buds on branches of 1 m or more in February 2018 by visual observation. The percentage of flower bud formation was determined from the ratio of axillary flower buds to the total number of buds. The amount of pollen was evaluated as the weight of pure pollen using acetone per 100 flowers in April 2018.
Analysis of S-genotyping of selected pear cultivarsDNA was extracted from young leaves of the three selected cultivars using a modified version of the cetyltrimethylammonium bromide (CTAB) protocol. PCR amplification was conducted using four S-RNase-specific primer combinations: FTQQYQ and anti-(I/T)IWPNV (Takasaki et al., 2004). PCR conditions were as described previously (Takasaki et al., 2004). Reproducible amplified target fragments were cloned into the pGEM-T Easy Vector (Promega, Australia), transformed into Escherichia coli, identified by colony PCR, and then bidirectionally sequenced using an ABI PRISM 3100 Genetic Analyzer (Thermo Fisher Scientific, USA).
Assessment of chilling requirement and suitable crop area for selected pear cultivarsTo estimate the chilling requirement to break endodormancy in the selected pear cultivars, the one-year-old shoots of each cultivar were periodically collected from November 2019 to February 2020. The shoots were divided into five-node cuttings consisting of five lateral leaf buds. Leaf bud break was defined as the percentage of green-tip buds that reached more than 70% of the total, and the chilling requirement (CR) was calculated for each cultivar as described by Takemura et al. (2013). Based on these results, we evaluated a suitable crop area by the date of endodormancy termination in selected cultivars using the Agro-Meteorological Grid Square Data, NARO (Ohno et al., 2016; Kominami et al., 2019). The date of endodormancy termination was calculated as described below, taking into consideration the addition of a negative factor when the temperature rose above 15°C. The starting date of the computation was November 1 (Takemura et al., 2021).
Chill Unit (CU) (/day) = −7·10−7·t6 + 4·10−5·t5 − 0.0003·t4 − 0.0085·t3 − 0.0206·t2 + 0.041·t + 24.016 (t = °C (/hour))
The RCP8.5 scenario in the IPCC estimate a global mean surface air temperature increase of about 3.7°C (a likely range between 2.6°C and 4.8°C), by 2081–2100, compared to the preindustrial level (ICPP, 2013). The estimated date of endodormancy break (the reaching date) was assessed as the normal value of the daily average temperature and increased to 3.7°C above the normal value.
Table 1 shows the extent of pollen germination for each Pyrus plant at different incubation temperatures in 2017 in descending order of percentages at 10.0°C. The highest germination percentage for most plants was observed at 25.0°C. Among the 129 plants that were tested, the germination percentage of plants was < 20% at 10.0°C. However, ‘Narayoshinokoboku’, ‘Imamuranatsu’, and ‘Tosanashi’ showed a higher germination rate than the other plants at 10.0°C, and the germination percentage rates of the three cultivars were > 30%. The percentages of pollen germination in ‘Imamuranatsu’ and ‘Chojuro’ as controls in 2018 were < 10% at 10.0°C, despite having a percentage of > 60% at 25.0°C (Table 2). That of ‘Narayoshinokoboku’ and ‘Tosanashi’ was significantly higher than that of ‘Imamuranatsu’ and ‘Chojuro’ in the 10.0°C treatment in 2018. Furthermore, the germination percentage of ‘Narayoshinokoboku’ and ‘Tosanashi’ was > 20% under the same conditions in 2019.
Germination percentages of the pollen of Pyrus plants at different temperatures (2017).
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Germination percentages of the pollen of selected cultivars at different temperatures (2018 and 2019).
The fruit setting percentages of the cultivars ‘Kosui’, ‘Hosui’, and ‘Gold-Nijisseiki’ were > 85% by pollination using the three selected cultivars (‘Narayoshinokoboku’, ‘Imamuranatsu’, and ‘Tosanashi’) in an open field (Table 3). Furthermore, the fruit setting percentage of ‘Hosui’ using pollen of ‘Narayoshinokoboku’ and ‘Imamuranatsu’ was significantly higher than that of ‘Chojuro’.
Effect of fruit set by pollination treatment using the pollen of selected cultivars.
Next, an experiment was performed to evaluate the effects of pollination on fruit quality in ‘Kosui’. The fruit weight, diameter, and pH of fruit juice in ‘Kosui’ by pollination treatment using the pollen of ‘Imamuranatsu’ were higher in the ‘Chojuro’ treatment (Table 4). The flesh firmness and pH of fruit juice by pollination treatment using the pollen of ‘Tosanashi’ were higher and lower in the ‘Chojuro’ treatment, respectively. The number of seeds per fruit (good and dead seeds) did not significantly differ between the cultivars for the different types of pollination.
Effect of fruit quality by pollination treatment using the pollen of selected cultivars.
The percentages of flower bud formation in ‘Tosanashi’ and ‘Chojuro’ as the controls were > 50% (Table S1) and those in ‘Narayoshinokoboku’ and ‘Imamuranatsu’ were 33.3% and 10.9%, respectively. In addition, the amount of pollen per 100 flowers in ‘Tosanashi’ was similar to that in ‘Chojuro’. However, the amounts of pollen in ‘Narayoshinokoboku’ and ‘Imamuranatsu’ were more than double and 1.4 times the amount of ‘Chojuro’, respectively.
Analysis of S-genotyping of selected pear cultivarsPCR with a set of primers resulted in PCR products with different lengths from the three cultivars: ~350 bp from ‘Tosanashi’, ~500 and 350 bp from ‘Imamuranatsu’, and ~1,400 and 350 bp from ‘Narayoshinokoboku’, respectively (Fig. 1). These PCR products were sequenced, and S-genotyping in each cultivar was determined as follows: ‘Tosanashi’ (S1S7), ‘Imamuranatsu’ (S1S12), and ‘Narayoshinokoboku’ (S1S9).
PCR analyses of S-genotypes in selected cultivars. S-RNase fragments of the three cultivars were amplified by PCR using FTQQYQ and anti-(I/T)IWPNV primers. The lanes in each gel are from left to right; 1: ‘Tosanashi’ (S1S7), 2: ‘Imamuranatsu’ (S1S12), and 3: ‘Narayoshinokoboku’ (S1S9).
The percentage of leaf bud break in the three selected cultivars was < 50% for CU 800 and 1,000 (Table 5). Thereafter, the bud break percentage in ‘Narayoshinokoboku’ increased by > 70% at CU 1,400. That of ‘Imamuranatsu’ and ‘Tosanashi’ increased by > 70% at CU 1,600.
Seasonal changes in the percentage (%) of leaf bud break after 28 days of forcing at 23°C on selected cultivars.
The analysis using a 1 km × 1 km grid map indicated that the reaching date at CU 1,400 based on normal values in most areas was from mid-January to mid-March (Fig. 2A). The reaching date at CU 1,600 based on normal values in a wide range of areas in the Kyushu region was from mid-March to late-March (Fig. 2B). When the temperature increased by 3.7°C above the normal value, areas where endodormancy broke (the reaching area) at CU 1,400 after late March was most common in the Kyushu area, followed by the coastal area of Chugoku, Shikoku, Kansai, and Tokai regions (Fig. 2C). At CU 1,600, the temperature increased by 3.7°C above the normal value, and the reaching area after late March spread to the rest of the mountains in wide areas of Kyushu and Tokai regions (Fig. 2D).
Assessment of estimated endodormancy breaking date in selected cultivars based on a temperature increase of 3.7°C compared with the daily average temperature. (A) Estimated breaking date at the normal value in ‘Narayoshinokoboku’. (B) Estimated breaking date at the normal value in ‘Imamuranatsu’ and ‘Tosanashi’. (C) Estimated breaking date at the normal value + 3.7°C in ‘Narayoshinokoboku’. (D) Estimated breaking date at the normal value + 3.7°C in ‘Imamuranatsu’ and ‘Tosanashi’.
The percentage of pollen germination and pollen tube development are important for fertilization. Excessively low rates may cause failure of fruit setting due to ovule degradation before the pollen tube reaches the ovary (Mellenthin et al., 1972; Therios et al., 1985). Accordingly, the present study was performed to select an optimal pollinizer with a stabilizing influence on the fruit setting of Japanese pear cultivars at a low temperature.
Regarding the extent of pollen germination at different temperatures in Pyrus plants centered on the Japanese pear, most varieties did not germinate at 10.0°C (Table 1). However, ‘Narayoshinokoboku’, ‘Imamuranatsu’, and ‘Tosanashi’ showed a higher germination rate than other Pyrus plants. Pollen germination varies every year due to many environmental factors (Kuroki et al., 2017). At 10.0°C in 2018 and 2019, the germination percentage of these three cultivars was < 30%, but those of ‘Narayoshinokoboku’ and ‘Tosanashi’ were significantly higher than that of ‘Chojuro’, which was used because it is one of the most common pollinizers in Japan. From these results, we selected candidate pollinizer cultivars with pollen that had high germination properties at low temperatures. Previous studies have demonstrated that viability, germination, and tube growth of pollen vary significantly according to the species and cultivar (Hedhly et al., 2004; Du et al., 2006). Additionally, it has been established that in vitro germination tests of Pyrus may provide an index for the fertilizing capacity of pollen because germinability in vitro and fertilizing capacity in the field are correlated (Kuroki et al., 2017). The fruit setting percentage of ‘Hosui’ by the pollen of ‘Narayoshinokoboku’ and ‘Imamuranatsu’ was significantly higher than that of ‘Chojuro’ in an open field (Table 3). Conversely, fruit setting percentages of ‘Kosui’ and ‘Gold-Nijisseiki’ were not affected by the pollen selected. In addition, pollination treatment had a relatively lower effect on fruit quality in ‘Kosui’ using the pollen from selected cultivars. The effect of the pollen source on fruit characteristics is known as xenia (Focke, 1881), and it has been reported in some fruit trees (Denney, 1992). However, in this study, we did not perform a fruiting test assuming low temperature conditions; therefore, further investigation is required.
In addition, S-genotyping in selected cultivars was determined as follows: ‘Tosanashi’ (S1S7), ‘Imamuranatsu’ (S1S12), and ‘Narayoshinokoboku’ (S1S9). From these results, we concluded that all three cultivars showed cross-compatibility with the four major cultivars of Japanese pear (‘Kosui’(S4S5), ‘Hosui’(S3S5), ‘Nitaka’(S3S9), and ‘Nijisseiki’(S2S4)). Additionally, we assume that the three cultivars show cross-compatibility with most pear cultivars other than the following combinations: S1S7, S4S7, S1S12, S4S12, S1S9, and S4S9. Although the cultivation record of ‘Tosanashi’ and ‘Imamuranatsu’ was described in the late 1800s (Kajiura and Sato, 1990), the parents of each cultivar were unknown. Interestingly, all three cultivars had the S1 gene in common, but pollen germination in other cultivars (‘Ichiharawase’, ‘Suisei’, ‘Doitsu’, ‘Hayatama’, ‘Yakumo’, and ‘Akaho’) with the same S1 gene was < 10% at 10°C in 2017.
It is also important when selecting a pollinizer to collect pollen to estimate the percentage of flower bud formation and amount of pollen in selected cultivars. We found that the percentage of flower bud formation in ‘Tosanashi’ was > 50%, and the amount of pollen in ‘Narayoshinokoboku’ was more than double than that in ‘Chojuro’ (Table S1). In previous studies, the amount of pollen in P. communis L. and ‘La France’ (selected as a cultivar because of pollen with high germination rates in low temperature conditions) were the same and about one-third of that in ‘Chojuro’, respectively (Kuroki et al., 2017). The germination percentages of pollen in P. communis L. and ‘La France’ (European pear) were > 50% and < 10%, respectively, at 10°C (Kuroki et al., 2017). The germination percentages of the three selected cultivars were not more than 50% under the same temperature conditions in both 2018 and 2019; therefore, it was considered that the percentages were lower than those in P. communis L. and ‘La France’. However, we suggest that ‘Narayoshinokoboku’ and ‘Tosanashi’ show cross-compatibility with most pear cultivars and can be used as pollinizers for efficient and stable pollen harvesting. In addition, the flowering start date in all three selected varieties in Tottori in 2020 was approximately one week earlier than that of the control ‘Chojuro’ (Table S1).
We estimated the CR and suitable crop areas for these cultivars. A previous study showed that the CR of Japanese pears ranged widely from 800 to 1,800 CU, and suitable crop areas with high CR cultivars were limited due to global warming (Takemura et al., 2013). In previous studies we reported that the CRs in P. communis L. (selected as a cultivar because of pollen with high germination rates in low temperature conditions) and ‘Chojuro’ (the most common pollenizer) were CU 1,800 and CU 1,000, respectively (Takemura et al., 2013). From the percentage of leaf bud break in the three selected cultivars, we determined the CR of ‘Narayoshinokoboku’ as CU 1,400, and that of ‘Imamuranatsu’ and ‘Tosanashi’ as CU 1,600. However, these three cultivars had high CR, and it is important to estimate suitable crop areas to adjust for global warming in the future. For apples and satsuma mandarins, to estimate the effect of global warming on the cultivation environment, analysis was performed in units of about 10 km × 10 km using climate change mesh data (Sugiura and Yokozawa, 2004). Furthermore, Sugiura et al. (2019) predicted the distribution of grapes suitable for cultivation due to climate change based on the frequency of occurrence in years when the color of the grape skin was poor using the grape skin color as an index. In the present study, based on an increase of 3.7°C (likely ranging between 2.6°C and 4.8°C) in the 2081–2100 RCP8.5 scenario by the IPCC (2013), we evaluated the suitable crop area by the date of endodormancy termination at CU 1,400 and 1,600. Areas in which the date of endodormancy termination reached late-March were assessed as unsuitable crop areas because of a risk of poor flowering due to lack of chilling. From our results, we considered that it is possible to grow cultivars with CU 1,400 and 1,600 in the plains of all regions except those of southern Kyushu. However, when the temperature increased to 3.7°C above the normal value, the reaching area at both CUs after late March was most in the Kyushu areas, followed by the coastal area of Chugoku, Shikoku, Kansai, and Tokai regions. Therefore, we suggest that safe areas for the growth of selected cultivars include all areas in the Tohoku region and Chubu region, except areas on the Pacific side and coastal areas of the Chugoku, Shikoku, Kansai, and Tokai regions, based on ongoing global warming. Additionally, we suggest that in the future, it will be necessary to select and breed cultivars that can produce pollen with high germinability at lower temperatures and with a low chilling requirement to achieve endodormancy breaking.
In conclusion, we selected ‘Narayoshinokoboku’ and ‘Tosanashi’ as new Pyrus pollinizers because they combine the advantages of low-temperature germinability and a high pollen load.
