Effect of freezing conservation time on loquat (Eriobotrya japonica) pollen germination

Aim of study: Several studies point out that storage at -20 oC is a suitable method for preserving pollen of many species in the long term. Part of those studies indicate the total storage time at which these conditions are optimal. However, we have found a lack of information about the freezing time conditions and incubation temperature of loquat pollen. The main objective of this study was to evaluate the effect of the -20 oC conservation temperature on loquat (Eriobotrya japonica (Thunb.) Lindl.) pollen. Area of study: The study was conducted in Montserrat (Valencia, Spain). Material and methods: Loquat flowers were collected in November 2017 and stored at -20 oC for three time periods: 4 (T1), 6 (T2) and 8 (T3) months. Subsequently, pollen grains were incubated at different temperatures for 72 h. We analyzed (i) the effect of freezing conservation time; (ii) the effect of incubation temperature on germination; (iii) the interaction between these two factors. Main results: T1 showed higher germination percentage and tube length values (mean and maximum) than T2 and T3. The highest germination percentage (52.77%) was detected for T1 at an incubation temperature of 25 oC. The interaction between freezing time and incubation temperature showed more consistent results for T1 than for T2 and T3. Research highlights: This suggests that storing at -20 oC for more than 4 months affects pollen grain and reduces germination and pollen growth. Therefore, -20 oC loquat pollen storage should not exceed 4 months. Additional key words: pollen germination rate; pollen tube length; fruit tree; Rosaceae; pollen conservation. Authors’ contributions: Conceived, designed and performed the experiments: RB, NC, AG and HM. Analyzed the data: RB, GA and HM. Contributed reagents/materials/analysis tools: AG and CZ. Wrote the paper: RB. All authors read and approved the final manuscript Citation: Beltrán, R; Cebrián, N; Zornoza, C; Garmendia, A; Merle, H (2020). Effect of freezing conservation time on loquat (Eriobotrya japonica) pollen germination. Spanish Journal of Agricultural Research, Volume 18, Issue 3, e0804. https://doi.org/10.5424/sjar/202018316626 Received: 02 Mar 2020. Accepted: 01 Oct 2020. Copyright © 2020 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Roberto Beltrán: robelmar@upvnet.upv.es Spanish Journal of Agricultural Research 18 (3), e0804, 10 pages (2020) eISSN: 2171-9292 https://doi.org/10.5424/sjar/2020183-16626 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) OPEN ACCESS RESEARCH ARTICLE Funding agencies/institutions Project / Grant Asociación Club de Variedades Vegetales Protegidas, as a part of a project undertaken with the Universitat Politècnica de València UPV-20190822


Introduction
Loquat (Eriobotrya japonica (Thunb.) Lindl.) is a subtropical evergreen tree that originates from China (Blasco et al., 2016). This species was introduced in Europe in 1784 when several plants were acquired by the National Garden, Paris (Sharpe, 2010). It commenced to be cultivated at the beginning of 19th century. Throughout that century, loquat orchards extended to several European countries and the United States (Agustí, 2010). Nowadays, it is cultivated mainly in subtropical climate countries. The world's main loquat producers are China, Japan and Spain (Caballero & Fernández, 2002). E. japonica belongs to the Maloideae subfamily of Rosaceae. One of the main characteristics of this species is that the flowering season takes place in autumn in Mediterranean countries, unlike other Rosaceae species (Agustí, 2010). Loquat trees can withstand low temperatures of up to -12 ºC (Freihat et al., 2008). The optimum temperature for its development ranges from 20 ºC to 30 ºC, depending on variety. Likewise, it has been observed that temperatures above 35 ºC can be unfavorable for its plant growth (Freihat et al., 2008).
The pollination and fertilization processes of E. japonica are similar to other Rosaceae species. Some studies cite loquat as a self-compatible species (Cuevas et al., 2003;Freihat et al., 2008), but the study performed by Sharafi et al. (2011) reported most loquat cultivars with gametophytic self-incompatibility. Several studies consider cross-pollination in loquat to be an essential factor for high yields (Freihat et al., 2008;Sharafi et al., 2011). It has been specifically proven that fruit set and fruit size depend on reproduction and pollination processes (Yang et al., 2012). After fecundation, several seeds degenerate and only 3 or 4 seeds reach the mature state (Qin et al., 2008). Cuevas et al. (2003) indicated the importance of pollinators activity on loquat cross-pollination. However, pollination is also associated with the formation of more seeds, which results in reduced fruit quality (Yang et al., 2012). Loquat pollination studies remain fundamental to improve the main crop variables. Sharafi et al. (2011) conducted an in vitro germination study of loquat pollen, where pollen was incubated only at 22 ºC for 1 day. Instead, pollen germination studies at several temperatures in other Rosaceae species can be frequently found. Among them, the studies of Vasilakakis & Porlingis (1984) in Pyrus communis L., Egea et al. (1992) in Prunus armeniaca L., Hedhly et al. (2004) in Prunus avium (L.) L., Hedhly et al. (2005) in Prunus persica (L.) Batsch, and Sorkheh et al. (2011) in Prunus dulcis (Mill.) D.A.Webb, stand out. Recently, the effect of temperature on the pollen germination of several Rosaceae species has been published (Beltrán et al., 2019). In these studies, the maximum pollen germination and maximum pollen tube length values were achieved by incubating pollen grains at 20 ºC.
Pollen germination studies are also available at different temperatures in species from other families, such as those published by Kakani et al. (2002) in groundnut (Arachis hypogaea L.), Reddy & Kakani (2007) in Capsicum spp., and Acar & Kakani (2010) in Pistacia spp. In these cases, the optimum temperature for pollen tube growth ranged between 20 ºC and 30 ºC.
Other loquat pollen germination studies have focused on stigma receptivity duration and the time at which pollen tubes reach the ovule but have left aside the temperature at which these processes occur (Qin et al., 2008). In most studies conducted with loquat pollen or anthers, pollen was either used fresh or stored in a refrigerator at 0-4 ºC for a short period of time.
Fresh pollen has been used to study the relation between loquat pollen tube length and genomic characterization (Carrera et al., 2009), the effect of rain on loquat pollen adhesion to stigma (Yang et al., 2011), and induced parthenogenesis on loquat (Blasco et al., 2016). Qin et al. (2008) stored loquat pollen at -20 ºC in their in situ pollen study, and then used that pollen for the cross-pollination of loquat trees.
Several studies report pollen germination capability after a certain period at low temperature. Weinbaum et al. (1984) stored pollen of Prunus dulcis and P. persica at -20 ºC until further use and did not observe loss of germination rates. Some studies conducted on pollen grains of Solanum melongena L. pointed out that storage at temperatures below -20 ºC for 48 weeks provided better germination results than those obtained with fresh pollen (Khan & Perveen, 2006). Similar results are reported for the same authors in Citrullus lanatus L. (Khan & Perveen, 2010), Lagenaria siceraria (Molina) Standley (Khan & Perveen, 2011) and five citrus species (Khan & Perveen, 2014).
Although the results obtained in other species indicate that long-term freezing is a suitable pollen conservation method, information about long-term loquat pollen preservation by freezing and loquat pollen germination at different temperatures is scarce.
Hence the aim of this study was to: (i) evaluate the germination rates of loquat pollen stored at -20 ºC depending on freezing times; (ii) analyze these germination rates depending on incubation temperatures; and (iii) check the interaction between both these factors.

Materials and methods
Loquat flowers were collected in November 2017 in an orchard located in the municipal district of Montserrat (Province of Valencia, Spain; Latitude: N 39.359629, Longitude: E -0.547494, Altitude: 153 m). The site's climate is cold semiarid, BSk in the Köppen & Geiger (1936) classification, with an average temperature of 16.8 ºC and an average rainfall of 432 mm. The employed loquat variety was ˈAlgerieˈ and trees were 10 years old. Fifty flowers per tree from five trees were taken at anthesis. All the samples were kept in bags and stored in a freezer at -20 ºC for 4 (T1 or treatment 1), 6 (T2 or treatment 2) and 8 (T3 or treatment 3) months. Samples were placed inside a humid chamber at 4 ºC for 2 h before extracting pollen to achieve its pre-hydration (Mesejo et al., 2006). Three or four anthers were taken, and pollen grains were extracted using binocular lenses and placed in 5 mL of modified BK medium containing 100 g L -1 sucrose, 0.1 g L -1 H 3 BO 3 , 0.3 g L -1 Ca (NO 3 ) 2 , 0.1 g L -1 KNO 3 and 10 g L -1 agarose (Brewbaker & Kwak, 1963) to induce their germination on 90-mm Petri dishes. These dishes were incubated for 72 h in the dark at: 5, 10, 15, 20, 25 and 30 ºC (Hedhly et al., 2004;Beltrán et al., 2019). The pollen germination percentage, the average pollen tube length and the maximum pollen tube length were calculated for each treatment (number of freezing months) and temperature. Pollen was considered germinated when the pollen tube length exceeded the diameter of its pollen grain. Pollen tube length was measured as the ratio to pollen diameter. These variables were measured for the first 100 Effect of freezing time on loquat pollen pollen grains observed on each dish. If there were only a few grains, the variables were calculated for the total number of grains.
All the statistical analyses were done using R (R Core Team, 2017) and RStudio (RStudio Team, 2016). The ANOVAs, Kruskal-Wallis rank sum and Tukey post hoc tests were used to make comparisons between treatments (temperature and freezing times) using the "agricolae" package (Mendiburu, 2019). When significant differences were found, Levene's test and eta-squared statistics were calculated to assess the homogeneity of variances and the size effect in the ANOVA, respectively. Normality of residuals was tested by the Shapiro-Wilk test and by looking at the density curves.

Effect of freezing time on pollen germination
The highest values of pollen germination percentage, average pollen tube length and maximum pollen tube length were obtained at T1 (4-month frozen pollen; Fig.  1, Table 1). The average germination percentage for all the temperatures at T1 was 27.64%. The mean pollen tube length and the maximum pollen tube length were 1.83 and 2.47, respectively (ratio of tube length to pollen diameter).
The lowest values for these variables were obtained at T2 (6-month frozen pollen). At T2, the average pollen germination was 11.57%. The values observed for the longest freezing time (T3: 8-month frozen pollen) were slightly higher than those observed at T2. Significant differences were observed for the germination percentage between T1 and T2, but not at T3. All the differences observed for pollen tube length between T1 and the other two treatments were significant (see Table 1).

Effect of incubation temperature on pollen germination
The highest germination percentages and mean pollen tube length values were obtained at 25 ºC, while the minimum pollen germination (6.09%) was observed at 5 ºC. A gradual increase in the pollen germination percentage was noted between 10 ºC and 25 ºC, although differences were not significant, except for those between 5 ºC and 20 ºC (Fig. 2, Table 2). The highest mean pollen tube length was obtained at 20 ºC (1.62) while the longest pollen tube value was found at 10 ºC (2.55). However, no significant differences were observed between pollen tube length at different temperatures, which was probably due to the remarkably wide variability of pollen tube lengths.

Interaction between freezing time and incubation temperature
T1 showed the most consistent germination pattern. Significant differences between the two highest incubation temperatures and the lower temperatures were recorded only for T1 (Fig. 3). In this case, the highest germination percentage was observed at 25 ºC (52.77%), while the lowest one was noted at 5 ºC (4.4%). The germination percentage clearly increased with the rise in the incubation temperatures for T1. For T2 and T3, no significant differences in the germination percentage appeared among the incubation temperatures.
No significant differences were found among the temperatures at T1 and T2 for the mean pollen tube length, but significant differences were found between 10 ºC and the two highest temperatures obtained for T3 (Fig.  4). Finally, the maximum pollen tube length did not show any significant differences for T1, but several significant differences appeared among the incubation temperatures for T2 and T3 (Fig. 5). Germination failed for incubation temperatures 15 ºC and 20 ºC at T3.

Discussion
The highest pollen germination rates were reached between 20 ºC and 25 ºC for all the tested freezing times  Table 2). Several studies have also shown that the optimum germination temperature of loquat pollen fluctuates within this range. Qin et al. (2008) and Demirkeser et al. (2007) obtained their highest germination and tube length values at 20 ºC. The study conducted by Sharafi et al. (2011) in Iran with several loquat genotypes also obtained the highest pollen germination percentages at 22 ºC in some genotypes. Several studies carried out on other Rosaceae species have also indicated that the optimal temperature range for pollen germination oscillate between 20 ºC and 25 ºC. For example, Weinbaum et al. (1984) noticed that the maximal pollen germination percentage for P. persica was set at 23 ºC in a cold sensitivity study, while Hedhly et al. (2004) obtained the highest pollen germination rate in two P. avium varieties at 20 ºC.
These same authors indicated that the pollen germination rates for two P. persica cultivars were also optimal at 20 ºC (Hedhly et al., 2005). Sorkheh et al. (2018) demonstrated that the optimal temperature for pollen germination in several Iran-native almond genotypes fell within the 20-25 ºC range. In a recent study, Beltrán et al. (2019) pointed out that the highest pollen germination percentages for Cydonia oblonga Mill., P. avium, Prunus domestica L., P. dulcis, P. persica and P. communis were close to 20 ºC. No clear pattern was observed for pollen tube length in relation to the six tested incubation temperatures . This result could be related to the prolonged freezing time at -20 ºC. T1 showed longer tube length compared to T2 and T3. In any case, we should consider that  . In this study, significant differences were found in pollen tube length between genotypes, but the highest average pollen tube length value was given at 20 ºC. This result coincides with the pollen tube lengths observed in loquat pollen by Demirkeser et al. (2007). In other Rosaceae species, the results were similar. Sorkheh et al. (2018) obtained the longest pollen tube length values for several almond genotypes between 20 ºC and 30 ºC. In Prunus cerasus L., the maximal pollen tube length was detected between 15 ºC and 20 ºC (Cerović & Ružić, 1992), and an identical result was reported years later in several P. armeniaca cultivars (Pirlak, 2002). There is very little information available in the literature about the long-term viability of loquat pollen stored at low temperatures. Some studies have shown that using pollen stored between 0 and 4 ºC is another effective way to handle loquat pollen (Germanà et al., 2006;Sharafi et al., 2011). Likewise, other studies have worked with loquat pollen stored in freezers at -20 ºC. Qin et al. (2008) demonstrated that loquat pollen can be stored at this temperature for up to 3 years with no loss of its total germination capacity, and the longterm stored pollen was used to carry out the cross-pollination of loquat trees. However, our results revealed that germinability loss in these cases would be high. Similar studies are found on pollen germination capability in other species. In this sense, Khan & Perveen (2014) carried out a study with five species of the genus Citrus, where the germination of pollen stored at 4 ºC was compared to pollen frozen at different temperatures (-20, -30 and -60 ºC). Freezing at -60 ºC was found to be the best method to conserve pollen. Similar conclusions have been drawn by other studies previously conducted by the same authors for different species, like S. melongena (Khan & Perveen, 2006), C. lanatus (Khan & Perveen, 2010) and L. siceraria (Khan & Perveen, 2011). In all these species, freezing at -20 ºC or -30 ºC did not completely reduce germination. Therefore, the authors recommended freezing as a suitable method for longterm pollen preservation. Another study indicated that deep-frozen pollen (-196 ºC) gave better germination rates than fresh pollen in sweet orange (Citrus sinensis (L.) Osbeck), mandarin (Citrus reticulata Blanco) and other citrus fruits (Ahmed et al., 2017).
The higher mean pollen germination rates were close to 28% (Table 2). Sharafi et al. (2011) found a great variability in pollen germination rates among twenty loquat genotypes, recording the highest pollen germination rate close to 95% while the lowest one remained at 15%. Another study conducted by Reig et al. (2014) obtained 72% of pollen germination in loquat cv. 'Algerie' non treated (control) pollen. We found that pollen germination percentages went down below 15% at 5 ºC and 10 ºC. No other studies for E. japonica pollen germination at temperatures below 10 ºC have been reported. More studies conducted on pollen germination with other Rosaceae species under cold conditions indicate that these temperatures do not favor pollen grain germination. Some studies highlight that low temperatures lead to poor pollen growth in P. avium and P. communis (Sanzol & Herrero, 2001). Likewise, a study on several Citrus species reports no pollen growth at 10 ºC (Distefano et al., 2012).
The herein obtained pollen germination percentages were lower than 60% at all the tested temperatures. Sharafi (2011) indicated germination percentages between 30% and 40% for some stone fruit cultivars after incubating pollen at 24 ºC. One study has reported a germination percentage below 50% in P. armeniaca, P. avium and P. cerasus after running germination tests on sucrose media (Bolat & Pirlak, 1999). Germination percentages between 20% and 60% have been published for Gossypium pollen germination tests (Kakani et al., 2005). Towil (2010) indicated that pollen quality loss could occur after long-term storage between -10 ºC and -20 ºC in some species. Although many studies have emphasized that freezing can be a suitable method to preserve pollen, they have also observed how different freezing temperatures and times can modify germination patterns. In a study about Malus pumila L., the germination capability of pollen was lower at -20 ºC than at -60 ºC (Perveen & Khan, 2008). Seyrek et al. (2016) reported that pollen germination percentage and pollen tube length values in Actinidia eriantha Benth were clearly lower after 1 year of freezing at -20 ºC compared to 6-month freezing at the same temperature. Our results revealed loquat pollen quality loss after increasing freezing times as prolonging freezing times lowered the mean values of the three studied variables.
Freezing allows pollen to be preserved in the long term with no loss of total germination capability. In our study, loquat pollen germinated after 8 months of freezing, but with a lower germination percentage. For T1, the pollen germination pattern corresponded to that observed in previous studies conducted with loquat and other Rosaceae and fruit tree species. However, different and more erratic patterns appeared for pollen Different letters on KW mean significant differences for alpha = 0.05. SD: standard deviation. NA: not available. germination, tube length and maximum tube length at T2 and T3.
In conclusion, the results showed that storing loquat pollen at -20 ºC for 4 months preserves pollen and allows its posterior germination. Germination percentages above 50% were observed at 25 ºC and 30 ºC for incubations after 4 months of freezing (T1). The mean pollen tube length and maximum pollen tube length values showed no significant differences among the incubation temperatures for T1.
Instead no significant differences were observed among the incubation temperatures after 6 months of freezing (T2 and T3). The germination capability of the loquat pollen stored at -20 ºC for 8 months was the lowest, and no pollen grain germinated at 15 ºC and 20 ºC. After 4 months of freezing time at -20 ºC, the germination rates of loquat pollen did not follow a clear pattern. Accordingly, long-term freezing at -20 ºC can modify pollen germination. These results indicate that studies into the effect of temperature on pollen  germination or pollen tube growth should be carried out using fresh pollen or short-term freezing to avoid any possible adverse effects on pollen caused by prolonged storage at low temperatures.