2024 Volume 93 Issue 1 Pages 68-75
Strawberries, the most economically well-known berry crop, are known for their taste, nutritional value, and antioxidant compounds. Various spatiotemporal or seasonal factors are known to affect the strawberry pollen germination rate; however, determinating which factor most influences the variation in the pollen germination rate remains challenging. This study aimed to determine the optimal pollen germination media and pollen short-term storage methods in the Japanese strawberry cultivars ‘Shindai SUS-1’ and ‘Shindai BS8-9’. Each strawberry pollen was incubated for 3 h in the dark on a solid medium with 1.5% agar and different sucrose and boric acid concentrations. The pollen germination rate was then investigated. The optimal pollen germination medium for both cultivars was 1.5% agar, 10% sucrose, and 0.1% boric acid. The small amount of strawberry pollen used in this study was collected immediately after flowering in the morning. Therefore, we also investigated a method for collecting a large amount of pollen while maintaining pollen activity. For both cultivars, the pollen germination rate for dry pollen, incubated for 24 h with silica gel after the petals and calyx were removed, was the same as that of the fresh pollen collected immediately after flower collection. In addition, dry pollen was more efficient in terms of short-term storage than fresh pollen. For ‘Shindai BS8-9’, the germination rate for dry pollen was also higher than that of the fresh pollen when stored in vacuum at −25, 4, 15, 20, and 25°C for 3 and 7 days.
Strawberries (Fragaria × ananassa Duch.) are the most economically important berry crop in the world. They have adapted to various environments from tropical areas to sub-arctic climates, in fresh and processed forms (Hytönen and Kurokura, 2020; Kim et al., 2021; Simpson, 2018). Strawberries are popularly known for their taste and are a pseudo fruit well-known for their nutritional value and antioxidant compounds, which can aid in prevention of numerous diseases (Ariza et al., 2016; Moreira et al., 2022). The global production of strawberries is ~9.2 million t, with a harvested area of ~395,844 ha (FAO, 2019). In Japan, field cultivation was common when strawberry cultivation began, with harvest time between May and June. Subsequently, the cultivation method changed from tunnel cultivation in fields to greenhouse cultivation. The breeding of summer strawberries also progressed, and strawberries became available in stores and supermarkets throughout the year.
Currently, there are over 300 strawberry cultivars in Japan, and the number is increasing annually (Tochigi prefecture, 2020). Plant breeders have always been interested in crossing varieties and species to produce new and improved strawberries that are better suited to the requirements of farmers and customers. However, many of these attempts have failed due to spatiotemporal or seasonal differences in the flowering of the selected parents or the pollen grains failing to germinate on the stigma, especially under high temperatures (Gill, 2014). Ledesma and Sugiyama (2005) reported that pollen germination and pollen tube elongation for ‘Toyonoka’ were suppressed under high-temperature conditions at 30°C, resulting in unfertilized ovules. In contrast to ‘Toyonoka’, most of the ‘Nyoho’ pollen germinated on the stamen, elongated through the style, and reached the ovule, regardless of the temperature treatment. The strawberry cultivars ‘Toyonoka’ and ‘Nyoho’ exhibit different resistances to high temperatures. Therefore, it is important to determine the optimal temperature range and characteristics of each cultivar. Failure of crossing due to spatiotemporal or seasonal differences can be overcome by preserving viable pollen from the pollen parent until the female parent has reached the optimal maturation state.
Pollen is normally sensitive to temperature and can easily lose viability under natural conditions; therefore, its preservation can be a challenge. Different storage temperatures are known to be suitable for different species and varieties; that is, the optimal pollen storage and germination temperatures depend on the species and vary between cultivars (Du et al., 2018; Loupassaki et al., 1997). Strawberry pollen has been stored successfully in tubes and Petri dishes at room temperature, 4, −4, and −18°C for over 1 year. However, this approach depends on the cultivar (Aslantas and Pirlak, 2002; Zebrowska, 1995). Given that these methods take up a large amount of storage space, it is important to develop a method to store pollen in a smaller space. For example, when watermelon pollen was stored at −25°C for 1 year under nitrogen or vacuum conditions, the fruit set rates were 86.4% and 66.7%, respectively (Akutsu and Sugiyama, 2008). For watermelon pollen, the fruit set rate was lower when stored in a vacuum than under nitrogen conditions. However, watermelon pollen can be stored in a smaller space and under dry vacuum conditions.
The pollen germination rate is closely related to pollen tube elongation and fertilization. Plant pollen fertility is evaluated by crossing and investigating the rate of fruit set. However, it is possible to confirm the pollen activity prior to species crossing by examining the pollen germination rate on an artificial medium. The strawberry pollen germination rates on a solid medium containing 8% sucrose and 0.6% agar were evaluated (Hortynski and Zebrowska, 1991; Zebrowska, 1995). In addition, a liquid medium containing 10 or 20% sucrose (Aslantas and Piulak, 2002; Voyiatzsis and Paraskevopoulou-Paroussi, 2005) and 10–20% sucrose and 100–350 ppm boric acid were tested (Ariza et al., 2006). Further, the importance of boric acid during in vitro and in vivo pollen germination has been highlighted (Akutsu and Sugiyama, 2008; Horsley et al., 2007; Imani et al., 2011; Kosel et al., 2018; Naik et al., 2016). Addition of boric acid to the pollen germination medium is effective for pollen tube elongation in the Japanese strawberry cultivar ‘Nyoho’ (Yoshida and Tanimoto, 1999). However, confirming whether variations in the pollen germination rate are due to the strawberry pollen tested, the composition of the germination medium, characteristics of the strawberry varieties, or culture conditions such as temperature or humidity remains difficult.
This study investigated the optimal pollen germination medium on a solid medium, for two strawberry cultivars, ‘Shindai BS8-9’ and ‘Shindai SUS-1’; the agar concentration in the solid medium was fixed at 1.5% and various sucrose and boric acid concentrations were used. However, the strawberry cultivars used in this study had low amounts of pollen per flower, rendering is difficult to collect enough pollen for storage. Thus, in a second experiment, we investigated a method for collecting large amounts of pollen while maintaining pollen activity. Furthermore, we investigated whether the large amount of pollen collected in the second experiment could withstand pollen storage and remain viable for germination.
The strawberry cultivars ‘Shindai BS8-9’ and ‘Shindai SUS-1’ were cultivated in a greenhouse at the Alpine Field Center at Shinshu University. All seedlings for both cultivars were grown by collecting runners from parental stock grown in the previous year. They were then transferred to a 17-m elevated bed system filled with a mixed medium of 40% bark compost, 24% rice husk, 18% mountain sand, and 18% bedding soil, with 25 cm between plants and two staggered rows. The temperature in the greenhouse was set to automatically turn on the heating below 8°C with the side windows open at 25°C. Liquid fertilizer composed of a mixture of Tank Mix B and Tank Mix F (OAT-Agrio Co., Ltd., Japan) diluted 500–600 times was used. All pollination types, except hand-pollination, were performed by black bumble bees (Bombus ignitus).
Optimization of the culture medium for in vitro germination of strawberry pollen (Experiment 1)The flowers from ‘Shindai BS8-9’ and ‘Shindai SUS-1’ were harvested in the morning (8:00 to 10:00 am) for pollen collection using a horsehair brush. Next, the pollen was assayed to optimize the culture medium for in vitro germination and germination ability on various media. For ‘Shindai SUS-1’, the media contained 0, 5, 10, 15, 20, 25, or 30% sucrose, 0, 0.1, or 0.5% boric acid, and 1.5% agar (WAKO Ltd., Osaka, Japan). For ‘Shindai BS8-9’, the media contained 5, 10, 15, or 20% sucrose, 0 or 0.1% boric acid, and 1.5% agar. A total of 1 mL of each medium was dispensed onto a glass slide, the pollen was placed on the slide with the medium in a plastic container lined with Kim towels (Nippon Paper Crecia, Co., Ltd., Japan) that were dampened with distilled water. Next, the germination percentage was determined after incubation in the dark for 3 h at 25°C by counting random samples of more than 100 pollen grains from each medium using an optical microscope. Pollen with pollen tubes longer than the pollen diameter was considered as germinated.
Optimization of strawberry pollen collection methods (Experiment 2)The flowers from both cultivars were harvested in the morning (8:00 to 10:00 am) and then the petals and sepals of each flower were removed and placed onto a filter paper laid on silica gel in a glass Petri dish or onto filter paper in a glass Petri dish without silica gel. They were sealed with Parafilm (Bemis Flexible Packaging, Ltd., IL, USA) and incubated overnight, ~24 h, at 25°C in the dark. The germination rates were compared by recording the fresh pollen collected immediately after flowering, after being incubated for ~24 h with silica gel (dry pollen), and after being incubated for ~24 h without silica gel (wet pollen).
Optimal temperature for short-term pollen storage and pollen collection procedure (Experiment 3)The fresh and dry strawberry pollen from ‘Shindai BS8-9’ were packaged in paraffin paper after assaying the pollen germination rate to determine the optimal temperature for short-term pollen storage. They were each packaged in a plastic bag (Hiryu BX20; Asahi Kasei Co., Ltd., Japan), using a vacuum packaging machine for foods (SQ-205S; Sharp Co., Ltd., Japan). They were then stored at either −25, 4, 15, 20, or 25°C in a vacuum for 1 week to observe the best short-time storage temperature for strawberry pollen. In addition, the pollen germination rate was investigated after storage for 0, 3, and 7 days.
The germination percentage under the experimental treatments was determined before and after incubation for 3 h at 25°C in the dark, by counting random samples of more than 100 pollen grains for each optical medium, as detailed in Experiment 1. The pollen germination rates in all experiments were calculated using the following formula: pollen germination rate (%) = the number of germinated pollen grains/the total number of pollen grains × 100. Experiments mentioned in Experiment 1 and 2 had ten replicates, and those in Experiment 3 had seven replicates for each treatment and medium. The significance of differences was tested by Tukey’s multiple-range test (P < 0.01 or 0.05) using statistical software (Statcel Oms-publishing, Japan). Pollen was collected from the greenhouse at Shinshu University in Nagano, Japan, from April 9, 2021 to February 25, 2022. Pollen was collected from March 9–12, 2022 and April 27, 2021 to November 18, 2021 for ‘Shindai SUS-1’ and from for ‘Shindai BS8-9’, respectively.
For ‘Shindai SUS-1’, pollen was germinated on the medium without boric acid and with 0.1% boric acid, and almost no pollen was germinated on the medium with 0.5% boric acid. The highest pollen germination rate was observed on media without boric acid, but with 15 and 20% sucrose, at 28.4 and 28.1%, respectively. There was no significant difference in germination rates on media with 10 and 25% sucrose addition. The highest pollen germination rate was on the media with 0.1% boric acid and 10 and 15% sucrose, at 73.0 and 70.4%, respectively. Significant differences were observed in germination rates on the media with 5 and 20% sucrose addition (Fig. 1).
Differences in the fresh pollen germination rate based on the concentration of boric acid and sucrose for ‘Shindai SUS-1’ (n = 10). * All pollen used in this experiment was fresh pollen (8:00–10:00) from the day of flowering. Bars represent the standard error. Different letters denote significant differences on Tukey’s multiple-range test based on the boric acid concentration. Significance was set at P = 0.01. There was no significant difference with the 0.5% boric acid addition.
For ‘Shindai BS8-9’, the germination rate was examined on media supplemented without boric acid or with 0.1% boric acid, and 5, 10, 15, or 20% sucrose because no pollen germination was observed on the medium with 0.5% boric acid. Media without boric acid, but with 10 and 15% sucrose, exhibited the highest pollen germination rate of 56.2 and 55.2%, respectively. The highest pollen germination rate on media with 0.1% boric acid and 10 and 15% sucrose was 68.3 and 66.5%, respectively. Significant differences were observed in germination rates for both boric acid concentrations with the 5 and 20% sucrose concentrations (Fig. 2).
Differences in the pollen germination rate based on the concentration of boric acid and sucrose for ‘Shindai BS8-9’ (n = 10). * All pollen used in this experiment was fresh pollen (8:00–10:00) from the day of flowering. Bars represent the standard error. Different letters denote significant differences on Tukey’s multiple-range test. Significance was set at P = 0.05.
Addition of 0.1% boric acid significantly increased the germination of both strawberry cultivars compared to the 0% boric acid control. The germination of both types of strawberry pollen peaked with 0.1% boric acid and 10% sucrose and then decreased. There was a significant difference in the germination rates between the 0 and 0.1% boric acid concentrations (Figs. 1 and 2).
Optimization of strawberry pollen collection methodsIn this experiment, the ‘Shindai BS8-9’ showed almost no visible pollen in the morning. However, pollen was visible around noon, suggesting that collecting pollen during the early afternoon may be suitable. Therefore, as a preliminary experiment, we examined the pollen germination rate on the dame flower in the morning (9:00 am) and at noon. As a result, the pollen germination rate for ‘Shindai BS8-9’ was the highest in the morning, with a small amount of pollen. The amount of pollen increased at noon, but the pollen germination rate decreased. The germination rates for pollen collected at 9:00 am and 12:00 pm were 71.4 and 46.8%, respectively, with a significant difference of P < 0.01.
There was a significant difference in the pollen germination depending on the time of pollen collection between 9:00 am and 12:00 pm (Fig. 3). Therefore, this experiment was conducted to develop a method for collection of more pollen while maintaining a constantly high germination rate.
Differences in the pollen germination rate based on the time (at 9:00 am and 12:00 pm) of pollen collection ‘Shindai BS8-9’ (n = 10). * Pollen was collected from flowers immediately. Bars represent the standard error. Different letters denote significant differences on Tukey’s multiple-range test. Significance was set at P = 0.01.
For ‘Shindai BS8-9’, the germination rates of fresh pollen (pollen germinated immediately after collection), dry pollen (pollen incubated for one night, for ~24 h with silica gel), and wet pollen (pollen incubated for ~24 h without silica gel) were 67.8, 72.7, and 56.6%, respectively. There was no significant difference between the germination rate of fresh and dry pollen. However, significant differences in germination rate were observed for wet pollen (P < 0.01; Fig. 4A). For ‘Shindai SUS-1’, the germination rate for fresh, dry pollen, and wet pollen was 61.6, 55.1, and 59.3%, respectively, with no significant differences (Fig. 4B).
Differences in the pollen germination rate for types of pollen based on the collection methods for ‘Shindai BS8-9’ (A) and ‘Shindai SUS-1’ (B). * Fresh pollen was collected from flowers immediately (8:00–10:00), dry pollen was collected after being incubated for ~24 h with silica gel, and wet pollen was collected after being incubated for ~24 h without silica gel. Bars represent the standard error. Different letters denote significant differences on Tukey’s multiple-range test. Significance was set at P = 0.01.
Three days after the storage of fresh and dry pollen at 25, 20, 15, 4, and −25°C, the pollen germination rates of the dry pollen were 2.9-, 4.7-, 4.2-, 3.2-, and 2.2-fold higher than that of the fresh pollen, respectively. Seven days after the storage of the fresh and dry pollen at 25, 20, 15, 4, and −25°C, the pollen germination rates for dry pollen were 2.2-, 6.8-, 4.3-, 2.1-, and 2.1-fold higher than that of the fresh pollen, respectively. Significant differences were observed for each storage period at 3 and 7 days (P < 0.01; Fig. 5).
Differences in the fresh and dry pollen germination rates based on the storage temperature and period (days) for ‘Shindai BS8-9’ (n = 7). Storage temperatures were 25°C (A), 20°C (B), 15°C (C), 4°C (D), and −25°C (E). * Fresh pollen was collected from flowers immediately (8:00–10:00), dry pollen was collected after being incubated for ~24 h with silica gel. Bars represent the standard error. Different letters denote significant differences on Tukey’s multiple-range test. Significance was set at P = 0.01.
Boric acid is an essential microelement that enhances pollen germination and pollen tube growth in vitro and in vivo. It may play a role in the control of secretion activities in pollen tubes by maintaining the cell wall integrity of the growing pollen tubes (Brown et al., 2002) and by serving as a chemotactic agent for pollen tube growth (Eshghi et al., 2010; Vaknin et al., 2008). In strawberry cultivars, addition of boric acid to a liquid medium increased pollen germination (Ariza et al., 2006). Therefore, here, the effects of the pollen germination medium were examined by adding various concentrations of sucrose and boric acid to strawberry cultivars ‘Shindai BS8-9’ and ‘Shindai SUS-1’. The medium comprising 1.5% agar, 10% sucrose, and 0.1% boric acid was found to be optimal for pollen germination in both cultivars. For ‘Shindai SUS-1’, the pollen germination rate significantly increased on the medium with 0.1% boric acid compared to that on the medium with 0% boric acid, except when adding 25 or 30% sucrose. In ‘Shindai BS8-9’, the pollen germination rate was slightly higher on the medium containing 0.1% boric acid compared to that on the control medium (0% boric acid), although the difference was not significant when adding 10 or 15% sucrose (Figs. 1 and 2). Furthermore, addition of boric acid was effective in the solid pollen germination medium.
In the preliminary experiment, ‘Shindai BS8-9’ produced significantly less pollen when pollen collection was performed immediately after flowering at ~9:00 am (morning), at 1 mg of pollen for an average of 23.3 ± 1.1 flowers (n = 10); however, at ~12:00 pm (noon), more than 1 mg of pollen per flower was collected from the same flowers. The pollen germination rates were 73% and 40% for flowers at 9:00 am and 12:00 pm, respectively, showing a significant decrease in the pollen germination rate from morning to noon. Temperature has been reported to affect pollen (Patel and Mankad, 2014), and high-temperature stress negatively influences the early stages of anther and pollen development (Afif, 2011; Harsant et al., 2013). In this experiment, the temperature in the greenhouse-cultivated strawberries was 23.6°C at ~9:00 am, but was at 36.8°C at ~12:00 pm, even with ventilation. Therefore, it is possible that the collected pollen may have died or lost its germination ability due to high temperatures.
Strawberry pollen can maintain a 10% or more pollen germination rate for approximately 4 months when stored at 22 ± 2°C (Aslantas and Pirlak, 2002). Therefore, we attempted to promote the dehiscence of anthers collected from ‘Shindai BS8-9’ or ‘Shindai SUS-1’ with the petals and sepals removed while maintaining the pollen germination rate for ~24 h in an incubator controlled at 25°C. We also investigated whether flowers with petals and sepals, removed from both cultivars, could be collected in drier conditions using silica gel while maintaining pollen viability.
For ‘Shindai BS8-9’, the dry pollen (pollen incubated for ~24 h with silica gel) had a higher pollen germination rate than the fresh pollen (immediately after collection). Compared to the dry and fresh pollen grains, the wet pollen (pollen incubated for ~24 h without silica gel) exhibited a significantly decreased pollen germination rate. For ‘Shindai SUS-1’, the pollen germination rates for fresh, dry, and wet pollen were almost the same, with no noticeable difference. However, in both cultivars, the dry pollen could be collected in larger quantities than the fresh and wet pollen (data not shown). The difference in the pollen quantity depended on the pollen collection time (9:00 am or 12:00 pm). Relative humidity during pollen collection and storage affects the pollen’s moisture content and its viability (Sidhu, 2019). Therefore, it was important to examine whether the pollen used in the experiment is resistant to desiccation or is weak depending on the plant species used. This is because some pollen, such as that of Cucurbita pepo, is resistant to desiccation, affecting pollen viability, while other pollen, such as that of Petunia hybrida, is sensitive to desiccation (Nepi et al., 2010). The pollen of the two strawberry cultivars used in these experiments is likely resistant to desiccation because the dry pollen maintained the same pollen germination rate as that of the fresh pollen. Pollen from ‘Shindai BS8-9’ may be more resistant to dehydration than ‘Shindai SUS-1’ because dry pollen from ‘Shindai BS8-9’ maintained a statistically significantly higher pollen germination rate than the wet pollen. These results suggest that strawberry pollen may be tolerant to drying, although this tolerance varies among strawberry cultivars. Furthermore, it is suggested that drying strawberry pollen to a certain degree may suppress the decrease in the pollen germination rate and increase anther opening, which may in turn increase the amount of pollen collected.
Crossing between different strawberry cultivars in conventional breeding uses all the initial material. The production of interparietal hybrids can be difficult due to asynchronous flowering when using stored viable pollen that can facilitate crossing. For the Fragaria genous, long-term viable stored pollen could also be used as a plant resource (Zebrowska, 1995), which would be useful for breeding new strawberry varieties. This experiment examined whether dry pollen could maintain pollen germination when stored at various temperatures for 1 week after vacuum treatment.
The fresh pollen from ‘Shindai BS8-9’ had a pollen germination rate of less than 20% under all the temperature treatments, except when stored at −25°C for 3 days. In contrast, the pollen germination rate of dry pollen stored at −25°C for 3 days was ~60% for the different temperature treatments. The pollen germination rate for dry pollen stored under different temperatures for 7 days remained ~40%, while the pollen germination rate for fresh pollen stored at 25, 20, and 15°C was 10% or less (Fig. 5). Therefore, dry pollen from ‘Shindai BS8-9’ may be more suitable for pollen storage than fresh pollen because it had a significantly higher pollen germination rate than fresh pollen stored for 3 days and 7 days under the different treatment temperatures. Zebrowska (1995) reported that the strawberry pollen of the cultivars ‘Dukat’, ‘Senga Sengana’, and the clone ‘B-302’, which was placed in a Petri dish and dehydrated with silica gel, still had sufficient capacity for fruit set after being stored at −18°C for 4 months. Aslantas and Pirlak (2002) reported that the pollen from ‘Aliso’, ‘Brio’, and ‘Cruz’, when kept in tubes with the tops covered with one-line CaCl2, maintained a pollen germination rate of ~20% after being stored −18°C for 1 year. This was mainly due to the pollen absorbing moisture within the tubes. Therefore, pollen can be stored for over a year while maintaining a constant high pollen germination rate and fruit set ability. The dry pollen germination rate for ‘Shindai BS8-9’ at the start of storage was two-fold higher than that of the strawberry cultivars tested by Zebrowska (1995). However, it was almost the same as that from ‘Brio’ tested by Aslantas and Pirlak (2002). In conclusion, for the two Japanese strawberry cultivars, ‘Shindai BS8-9’ and ‘Shindai SUS-1’, evaluated in this study, dry pollen collected after incubation for ~24 h with silica gel after removal of the sepals and petals more efficiently maintained the same germination rate as fresh pollen collected from flowers immediately. In addition, the pollen germination rate was significantly higher at 9:00 am than at 12:00 pm, so collecting pollen in the morning was considered to be better. Furthermore, dry pollen retained a significantly higher pollen germination rate than fresh pollen during a short-term storage period. We plan to evaluate quality factors from ‘Shindai BS8-9’ and ‘Shindai SUS-1’, including pollen germination and viability rates, after storage for 1 month, 6 months, or 1 year. Additionally we plan to estimate the strawberry quality after cultivar crossing. Furthermore, the anther opening under silica gel conditions, which was found to be useful in this study, could be evaluated in other plant varieties.
The authors thank Professor Shigemitsu Kasuga, Mr. Atsushi Nakamura and the other technical staff of AFC at Shinshu University. They also thank Mr. Taisei Watanabe and Mrs. Riho Kuroda at the Laboratory of Horticulture.
