1 Introduction

Based on the response to desiccation, seeds can be divided into three categories: orthodox, recalcitrant (Roberts 1973) and intermediate (Ellis et al. 1991). Orthodox seeds can tolerate desiccation to low moisture content < 5% on a fresh weight basis (water potential between − 35 and − 40 MPa) (Chmielarz 2010a; Roberts 1973), with no decrease in their germinability. Recalcitrant (desiccation–sensitive) seeds are killed by drying below a relatively high level of moisture content ≤ 20–50% (Pritchard 2004; Suszka and Tylkowski 1980). The range of tolerance in recalcitrant seeds is narrow and usually limited to approximately 20% moisture content (− 12 MPa); however, the threshold is species-specific. Intermediate seeds tolerate desiccation to moisture contents of ca. 7–12% but lose viability rapidly during storage at colder temperatures (Ellis et al. 1991; Chmielarz 2007; Hong et al. 1998).

Mature recalcitrant seeds have a relatively high water content and do not exhibit a long quiescent period (Berjak et al. 1999). These seeds remain metabolically active throughout the post-maturation period and up to the beginning of the germination process. Therefore, recalcitrant seeds are usually short-lived and impossible to store using conventional storage methods (Subbiah et al. 2019). The direct cause of recalcitrant seed-tissue death at temperatures below 0 °C is the formation of ice crystals (Chmielarz 1997).

Oak seeds usually do not tolerate a desiccation below 30–40% acorn moisture content (Gordon 1992; Chmielarz and Walters 2007). However, desiccation sensitivity depends on species-related physiology. Seeds of the white oaks—section Quercus (synonyms Lepidobalanus and Leucobalanus)—mature in 6 months and are very sensitive to desiccation and difficult to store successfully for more than 1–2 years. Seeds of the black oak group—section Lobatae (synonym Erythrobalanus)—mature in 18 months and are more desiccation tolerant than those of the white oak group (Schroeder and Walker 1987; Gosling 1989; Connor and Sowa, 2002, 2003; Chmielarz and Walters 2007). Earlier studies showed that the critical moisture content for pedunculate oak (Quercus robur, section Quercus) seeds was approximately 40%, below which viability declined rapidly (Suszka and Tylkowski 1980; Gosling 1989; Poulsen and Eriksen, 1992).

Snow cover is an important protection against abiotic stresses such as frost desiccation and cold air. Winter conditions are a major factor influencing seedling establishment (Renard et al. 2016). Effective protection of the ground vegetation from low temperatures (− 30 °C or lower) during winter depends on the duration and thickness of the insulating snow cover (Domisch et al. 2018). However, due to climate changes snowdays and snow cover duration decrease in temperate zones, which is especially apparent in mountain ecosystems (Kreyling and Henry 2011; Yeo et al. 2017). For example, under the RCP 4.5 and 8.5 emissions scenarios, a 49–95% reduction in snow cover in the northeastern USA is projected for 2100 (Reinmann et al. 2019). Without insulation, many seeds and seedlings can be affected by low winter temperatures and desiccation, which in result can limit their growth or in more severe cases lead to their death.

Recalcitrant seeds can be stored only under non-freezing temperatures and appropriate relative humidity levels for a short time, from several months to 1–2 years (Tompsett 1998; Suszka et al. 1996). During such storage, the seeds may either germinate or be affected by pathogens (in acorns, mainly by the fungus Ciboria batschiana (Zopf) N.F. Buchw.) Schröder 2002; Suszka 2002; Berjak and Pammenter, 2000). To date, the optimal storage conditions identified for pedunculate oak acorns are a moisture content of 40% and a temperature of − 3 °C in containers that are not tightly closed. However, over 2 years, pedunculate oak seeds rapidly lose their ability to germinate (Suszka et al. 1996). Because of the infrequent occurrence of mast years in Europe (usually 3–8 years), a longer period of safe acorn storage in controlled conditions is needed (Suszka et al. 1996). Suszka et al. (1996) reported that pedunculate oak acorns (at moisture content 42–45%) can be stored for 1.5–2 years without loss of seeds germinability and seedling emergence at temperature − 1° or − 3 °C.

Data available in the literature regarding seed frost resistance are usually related to several months of acorn storage. In our research, we attempted to answer the question of how acorn moisture content influences seed freezing sensitivity, for better understanding the survival of pedunculate oak seeds during cold, snowless winters and possibly increasing the safe seeds storage in controlled conditions. We hypothesized that a reduction in acorn moisture content can increase frost resistance of pedunculate oak seeds. We examined, for the first time, the sensitivity of seeds to freezing temperature on the basis of individual acorn germination, seedling emergence and their growth in correlation with their individual moisture content.

2 Materials and methods

2.1 Seed material

Acorns from two Polish provenances of Quercus robur L., Czmoń (later in the study referred to as seed lot A)—forest subdistrict (District Babki: 52°18′41.8″N 16°56′33.1″E)—and Siedlisko (later in the study referred to as seed lot B)—forest subdistrict (District Nowa Sól: 51°46′48.2″N 15°43′18.5″E), were collected in the middle of October in the western part of Poland, 100 km apart, in 2018 for seed lot A and in 2017 for seed lot B. After collection from the ground, acorns were transported to the Institute of Dendrology PAS in Kórnik, where they were preliminarily stored at 3 °C in containers that were not tightly closed. Acorns of seed lot A were stored in these conditions for 8 months until the experiment started. Acorns of seed lot B were preliminarily stored in similar conditions for 1 month. The mean moisture content of the acorns upon delivery was 37.7% of fresh weight for seed lot A and 40.8% for seed lot B. During preliminary storage, the acorns were not affected by Ciboria batschiana; thus, no thermotherapy was required before the experiment.

2.2 Acorn drying

Acorns of both seed lots were partially dried to obtain different moisture content levels at room temperature (approximately 20 °C) for up to 29 days. During drying, acorns were mixed regularly to reach uniform drying. Acorns from seed lot B collected in 2017 were dried in an open space in an air stream forced by a fan. This caused fast acorn desiccation, and consequently, some of the acorns showed a cracked pericarp. The final mean moisture content of these dried acorns after 29 days remained at 24.3%. Acorns collected in 2018 (seed lot A) were dried in a closed room with a higher air humidity reaching, after 29 days, a mean moisture content of 29.8%. The conditions used in the last method of acorn drying prevented the acorns from cracking (smaller spread in acorn moisture content at the final stage of drying of seed lot A, Fig. 1). Overall, − 3278 acorns were used for seed lot A, and 4400 acorns were used for seed lot B.

Fig. 1
figure 1

Effect of Quercus robur acorn drying time, up to 29 days, on the moisture content of the apex and distal parts. Czmoń (A) seed lot—acorns collected in 2018—and Siedlisko (B) seed lot—acorns collected in 2017. Cor—Spearman’s correlation coefficient

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2.3 Determination of the moisture content of individual acorns

After each drying period, 30–90 individual acorns (seeds and pericarp) from each seed lot were cut into apex and distal parts. The corresponding distal parts of the acorns were used to determine the moisture content after oven drying at 103±2 °C for 17 h. The results were used to create a standard curve, which allowed us to determine a total acorn moisture content (Appendix Fig. 4). Moisture content was calculated on a fresh weight (FW) basis and expressed in percentage.

The average moisture content of drying samples of acorns was determined for three replicates of 10 halves of acorns using a Sartorius MA37 weight dryer (120 °C for 20 min) calibrated according to a classical oven-drying method (103±2 °C for 17 h).

2.4 Acorn freezing

After desiccation, acorns were packed in polyethylene bags (300 acorns/bag) and frozen directly down to temperatures of − 3°, − 5°, − 7°, − 9°, − 11° or − 13 °C for seed lot A and down to − 3°, − 10° or − 18 °C for seed lot B. Frozen samples were kept at each temperature for 2 weeks. The control treatments for the two seed lots were stored at a temperature of 3 °C for two weeks.

2.5 Germination/seedling emergence test

An earlier described test of pedunculate oak was applied (Giertych and Suszka 2010; Giertych and Suszka, 2011). Before sowing acorns into the medium, the distal end (hilum end) of each acorn was cut off, and the remaining part (apex part) was planted with the radicle end upwards. Seeds with a radicle at least 5 mm long were considered germinated.

Individual acorns (their apex part) were sown vertically in a one 1-l container filled with a moist mixture (1:1, v/v) of quartz sand (< 1 mm fraction) and sieved peat (pH 5.5–6.5) and covered with a lid. In experiments with seed lot A, 90 acorns, and for seed lot B, 100 acorns, were sown in 3 containers for each treatment of the experiment. The thermal conditions for germination and seedling emergence within an environmental chamber were optimized at 20 °C and a 16/8 h photoperiod under a light intensity of 60 μmol m−2 s−1 provided by a fluorescent lamp (Fluora, OsramTM) (Suszka and Tylkowski 1980). Germinating seeds were left in the same containers for 4– weeks to monitor seedling emergence (Suszka and Tylkowski, 1980). When the shoot was at least 5 cm long, the seedling was classified as emerged. After 2–3 weeks of seedling growth the lids were removed to decrease the air humidity for growing shoots and to prevent fungal development on the small leaves.

2.6 Seedling biomass

After 3 months of further cultivation of seedlings under the same light and thermal conditions, the seedlings finished their growth, having 4–6 leaves and a terminal bud on the shoot top. Height, root collar diameter and dry mass (above- and belowground) were recorded on these three months old seedlings (Suszka et al. 1996). The dry mass was determined after drying at 105 °C for 17 h.

2.7 Statistical analysis

All statistical analyses were conducted in R software (R Core Team 2021), and because of differences in experimental design, seed lots were analysed separately. Dataset used for analysis can be found in Figshare repository (Wawrzyniak et al. 2021). The correlation between the moisture contents of the apex part and distal end of acorns was derived from linear regression analysis. Prior to analysis, databases were cleaned using the tidyverse package (Wickham et al. 2019). Outliers were identified using the interquartile range criterion and removed from the database to obtain a better model fit, expressed by the Akaike information criterion (AIC) value of the model. The effect of acorn drying time on germination and seedling emergence was evaluated using a generalized linear model (GZLM) with a binomial distribution (Ranal and de Santana 2006). In the case of overdispersion, the models were fit using a quasibinomial distribution (Crawley 2013). For the post hoc test, Tukey’s method was used with adjusted p values. Post hoc tests were performed using the emmeans package (Lenth 2022). A GZLM was also used to calculate the seedling emergence probability at a given moisture level under different temperatures. The GZLM was fit using a binomial distribution with a logit-link function. The model was chosen depending on the AIC. To examine the goodness of fit of the model, Mc Fadden’s R squared and the area under the curve (AUC) were analysed (Crawley 2013). For the linear regression models used in growth analysis, outliers were detected and removed using Cook’s distance estimates (Field et al. 2012). All data were log transformed to reach the normality of the distribution and stabilize variance homogeneity. Model assumptions were tested using Shapiro-Wilk’s test and Levene’s test. Additionally, adherence to the assumptions was checked using plot visualization. For all data visualization, the ggplot2 package was used (Wickham et al. 2016)

3 Results

3.1 Effect of acorn drying on the moisture content of the apex and distal parts

The average moisture content of the distal parts was lower (non-desiccated: mean moisture content 36%, dried 29 days: mean moisture content 28%) than that of the apex part (non-desiccated: mean moisture content 39%, dried 29 days: mean moisture content 32%) in both examined seed lots (Fig. 1). After a longer period of drying of acorn samples, divergence of extreme values of the individual moisture content of acorns was observed for both the distal and apex parts within the samples. This was particularly evident from day 15 for seed lot A and from day 23 for seed lot B. High variation of individual acorn moisture content shows how different can be mean moisture content after the drying period. Even after a month of desiccation, some acorns retained high moisture content (ca. 40%). Acorns drying was very uneven and restrained by pericarp.

3.2 Effect of acorn moisture content on freezing sensitivity

Generally, seeds desiccation resulted in decreased germination and emergence at almost all tested freezing temperatures in both seed lots (Table 1 and Appendix Table 4). However, an exception was observed for a slight drying of acorns. For example, undried seeds from seed lot A stored at − 7 °C germinated at a significantly lower frequency (40%) than seeds dried for 2 days (90%), with seedling emergence frequencies of 36% and 86%, respectively. Seeds frozen in acorns at − 9 °C that were not desiccated germinated at a frequency of 19% (emergence of 17%). Acorns desiccated for 2 days frozen at − 9 °C exhibited an increased germination frequency of 70% (emergence of 67%). Prolonged desiccation (more than 18 days) decreased germination significantly in all tested temperatures (Table 1). Protective effect of drying from subzero temperature was observed in − 11° and − 13 °C. Seeds of non-desiccated acorns frozen at − 13 °C showed a germination frequency of 2%. Temperature of − 18 °C has been proven to be lethal; frozen seeds did not germinate or emerge, regardless of whether the acorns were undried or desiccated.

Table 1 Germination and seedling emergence of Quercus robur seeds desiccated in acorns for up to 29 days (non-frozen)—control and seeds desiccated and then frozen for 2 weeks at − 3°, − 5°, − 7°, − 9°, − 11° or − 13 °C (seed lot A) and at − 3°, − 10° or − 18 °C (seed lot B). One-way ANOVA; means marked with the same letter are not significantly different at p < 0.05 according to Tukey’s test. Means±SEs.Acorns moisture content in % of fresh weight. Full model results can be found in Appendix Table 4
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The probability of seedling emergence of non-frozen seeds above 38% of moisture content was 100% (Fig. 2 and Table 2). Acorns with a moisture content of 30.7–30.9% cooled to − 3 or − 5 °C had a seedling emergence probability of 75% (in the 3rd quartile) (Table 3). The probability of seedling emergence from acorns desiccated before freezing at − 3°, − 5°, − 7°, or − 9 °C to a moisture content range of 25.4-26.4 was only 25% (1st quartile) (Table 3). The probability of obtaining seedlings from acorns frozen for two weeks at − 18 °C was 0% (seed lot B) (Fig. 2B).

Fig. 2
figure 2

Probability of Quercus robur seedling emergence depending on acorn moisture content and freezing temperature, i.e., − 3°, − 5°, − 7°, − 9°, − 11° or − 13 °C (seed lot A) and − 3°, − 10°, or − 18 °C (seed lot B), for 2 weeks. Non-frozen seeds—stored at 3 °C—were used as the control. Logistic regression analysis, model parameters can be found in Table 2

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Table 2 Model parameters for logistic regression. Seedling emergence of Quercus robur of different moisture content (MC) and interaction with tested temperature − 3°, − 5°, − 7°, − 9°, − 10°, − 11° − 13° or − 18 °C. (A) seed lot A; (B) seed lot B
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Table 3 Modelled quartiles of acorn seedling emergence probabilities as a function of acorn moisture content and storage temperature. See Table 2 for model parameters
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3.3 Effect of acorns freezing on the growth of the 3-month-old seedling

A significant effect of acorn drying on the dry mass of the 3-month-old seedlings was observed regardless of the freezing temperature used (p< 0.05, Fig. 3). The dry mass of seedlings decreased as acorns reduce their moisture content. There was no significant effect of temperature on plant dry mass, which in general was characterized by high variability (Fig. 3). However, the smallest seedlings with the lowest dry mass were obtained from seeds desiccated and frozen at the lowest temperature, − 11 °C or − 13 °C.

Fig. 3
figure 3

Effect of drying of Quercus robur acorns (non-frozen) on the dry weight of derived 3-month-old seedlings in relation to freezing temperature: − 3°, − 5°, − 7°, − 9°, − 11° or − 13 °C. Non-frozen acorns from seed lot A stored at 3 °C were used as the control. Partial R2 values are presented

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4 Discussion

4.1 Freezing sensitivity

Our results clearly demonstrated that relatively high levels of acorn moisture content (40%) previously described for stored seeds of pedunculate oak (Suszka et al. 1996), was the best moisture content for frozen acorns in our experiments. The safe cooling of seeds, down to − 7 °C, required a reduction in the initial acorn moisture content by drying between 2 and 14 days. However, Chmielarz (1997) did not find − 7 °C to be a destructive temperature during cooling of pedunculate oak acorns with higher moisture content levels of 42–43%, cooling of 1 °C/min down to − 15 °C and maintenance at this temperature for 15 min. Seeds cooled further to − 9 °C, − 11 °C and − 13 °C remained viable (seedling emergence of 42–72%) (Chmielarz 1997). The examined exotherms (ice crystallization and tissue death) in this earlier experiment appeared in different acorns at temperatures of − 8° and − 10 °C (a thermocouple was placed between cotyledons). In our experiment, due to summer drought, the collected acorns were relatively dry (initial moisture content of 37.7%). After being frozen for 2 weeks at − 11 °C or − 13 °C, they germinated and a gave rise to seedlings at a lower level at these temperatures in comparison with those of acorns in our previous experiment with a higher moisture content of 42–43% (Chmielarz 1997). This freezing sensitivity will probably change for seeds stored for longer periods. It was also noticed that unfavourable weather conditions during seed maturation limit their storability (Guerrant et al. 2004). Acorns with a lower moisture content during collection are more sensitive, and their germination decreases more rapidly after longer storage under classic conditions (− 3 °C) (personal communication).

This shows that the frost resistance of pedunculate oak seeds depends on different factors that affect seeds before storage. Comparing the results for freezing temperatures of − 9 °C, − 10 °C and − 11 °C in the present study with our earlier observations (Chmielarz 1997), as well as the findings of studies performed on pedunculate oaks of Polish provenances using DTA methods (crystallization exotherms) (Suszka 2002), we conclude that seeds from all investigated origins have a frost resistance threshold of approximately − 10 °C.

Systematic studies on the storage of acorns conducted by Suszka and Tylkowski (1980) showed seed resistance to temperatures from − 1 °C to − 3 °C even during three years of storage. Acorns with a moisture content of 46% after 6 months of storage at − 4 °C germinated at a frequency of 40% (that before storage was 90%) in the study of Schönborn (1964). Zajceva (1950) demonstrated a lack of resistance of pedunculate oak seeds to temperatures below − 5 °C, while 0 °C was suggested as the optimal temperature for acorn storage. Holmes and Buszewicz (1956) pointed out that at a temperature of − 12 °C, seeds lost their viability in a very short time, regardless of seed moisture content. Schönborn (1964) tested a lower temperature and found that seeds desiccated in acorns to a moisture content of 25% lost their germinability when frozen to − 14 °C. Guthke (1993) observed the frost resistance of acorns under natural conditions and found that seedlings emerged from 60% of the seeds after winter when the ground temperature decreased periodically to − 9 °C, with an air temperature of − 15 °C. These results support the hypothesis of a threshold temperature for frozen acorns of approximately − 10 °C, which is also the temperature we observed in our experiment. Effective under mild freezing conditions (to a minimum of − 10 °C), a high acorn moisture content seems to prevent seeds from dying.

Similar thresholds can be found in other Quercus species. Boese et al. (1985) determined a threshold for Q. velutina Lam. of approximately − 7 °C, and other authors measuring the exotherm profiles for Q. robur L. seeds found a critical freezing temperature of approximately − 9 °C for acorns at a moisture content of approximately 42% (Chmielarz 1997). For other oak species, a threshold temperature for ice crystallization in seeds was found for Q. pyrenaica Willd. at − 4.8 °C, for Q. faginea Lam. at − 7.5 °C, for Q. ilex L. at − 9.5 °C and finally for Q. coccifera L. at − 14 °C. For these species, the results were consistent with those of polyelectrolyte leakage analysis, where Lt50 values arranged Quercus species in a very similar way (Esteso-Martínez and Gil-Pelegrín 2004).

4.2 Desiccation sensitivity

During desiccation of acorns, which contain recalcitrant seeds sensitive to desiccation, toxic oxidation products and free radicals are formed in the cotyledons earlier than in the embryonic axes (Finch-Savage et al. 1992). This feature has also been used during cryopreservation of the pedunculate oak axis apical meristem (plumule), where all other parts of the seed were cut off before plumule drying (Chmielarz et al. 2011). Suszka (2002) found that drying whole acorns to a moisture content below 38% decreased seedling emergence, and seeds of acorns dried to a moisture content below 22% did not germinate (Suszka, 2002). We observed that the drying of acorns decreased the 3-month-old seedling mass, regardless of the temperature applied. However, temperatures lower than − 10 °C could increase this effect. Lower seedling mass has further consequences because it is known (Grossnickle 2012) that smaller seedlings have a lower survival rate. It was also noticed that in our drying experiment, at the final stage of acorn drying to a moisture content of approximately 30%, greater dispersion of extreme individual moisture content values occurred for the whole dried sample compared with the differences observed at the beginning of drying. Finch-Savage (1992) suggested that in this critically low moisture content of acorns, all ‘free’ (unbound) cellular water is lost. Furthermore, he suggested that viability is primarily related to the moisture content of the cotyledons, which subsequently causes a decline in viability in the embryonic axis. As the acorn drying time increases, the average time required for seed germination and seedling emergence also increases (Chmielarz 1997). In our current experiments, seeds significantly started to lose their germinability and seedling emergence when desiccated to a moisture content of 31.0%. For the same seed lot, a moisture content of 32.9% was still safe. However, for the second seed lot studied here, characterized by a higher moisture content after harvesting, the critical moisture content was 35.7%, while a moisture content of 38.2% was still safe. This indicates that the critical moisture content at which oak seeds begin to lose their ability to germinate and produce seedlings can depend largely on their moisture content when they fall from the tree. Furthermore, the range of critical threshold moisture contents when seeds start to lose their germinability is very narrow. In both experiments, germination and seedling emergence laboratory tests showed some differences, especially in some critical variants, which reassured us that the seedling emergence test could be applied to assess the true viability of seeds in order to produce properly growing plants. However, it is important to note that in many experiments, seedling emergence assays are not performed.

4.3 To what extent can desiccation increase the frost resistance of recalcitrant seeds?

Research carried out on orthodox seeds (Chmielarz 2009, 2010b) or plumules of embryonic axes (Chmielarz et al. 2011) of recalcitrant seeds shows that the freezing sensitivity of seeds or their parts increases with an increased moisture content (Chandel et al. 1995; Tompsett and Pritchard 1998; Daws and Pritchard 2008). We found in our experiment that for whole pedunculate oak seeds, which belong to the recalcitrant category, a reduction in the moisture content does not always lead to higher freezing tolerance. At temperatures of − 3 °C and − 5 °C, drying to a moisture content below 40% (the optimum level for long-term storage) did not significantly improve seed survival. Our results strongly connect the loss of recalcitrant seed viability during cooling with a decrease in moisture content. However, slight drying of acorns to a moisture content below 40% improves seed germinability at temperatures of − 7 °C and − 9 °C. The positive effect of further acorn drying (down to a moisture content of 30% compared with a moisture content of 40%) was observed in our experiments at − 11 °C and − 13 °C. However, here, the loss of seed viability, because of freezing damage, exceeded the benefits of acorn drying.

Thus, whole recalcitrant seeds from pedunculate oaks respond to freezing temperatures in a desiccated state in a different way than seeds from the orthodox category. Similar observations were published by Xin et al. (2010) on the dehydration of intact Q. acutissima seeds. The authors showed that freezing sensitivity increased when seeds were dried compared with that of seeds with a higher moisture content. This drying behaviour is the opposite of that for orthodox seeds. We demonstrated in our earlier epigenetic research (Michalak et al. 2015) performed on acorns stored with an optimal moisture content at − 3 °C for up to 18 months that global demethylation is significantly related to seed ageing, and Finch-Savage claimed (1992) that rapid ageing in recalcitrant seeds is similar to desiccation damage, causing a loss of viability in oak seeds. Connor and Sowa (2002) reported that seeds of Q. pagoda and Q. nigra, which were dried prior to storage, had lower germination than those stored fully hydrated. Pammenter et al. (1994) concluded that the inability to dry recalcitrant seeds might be due to a breakdown in the coordination of metabolism, leading to uncontrolled free-radical-mediated oxidative damage. Other authors claimed that acorns of pedunculate oak adjusted to a moisture content of 46% and stored for 6 months had the highest germination at 76%, while the controls and those adjusted to a moisture content of 37% had the lowest value of 17% (Özbingöl and O’reilly 2005). We can confirm the difficulties of drying pedunculate oak seeds, especially in the context of increasing their freezing tolerance.

5 Conclusions

Our study focused on determining the frost resistance of partially desiccated pedunculate oak seeds frozen at different temperatures down to − 18 °C for 2 weeks. The results demonstrated that there are many factors determining the survival of pedunculate oak seeds at temperatures below 0 °C. These factors include the weather conditions during seed maturation on a tree and, as a consequence, the moisture content of the acorns when falling from the tree, as well as the acorn moisture content during freezing and, finally, the freezing temperature. The survival benefit of seed drying during cooling below 0 °C is very limited. This indicates that for pedunculate oak seeds, the rule that is obvious for orthodox seeds—the lower the moisture content is, the higher the frost resistance—does not apply. Our research showed that only a slight reduction in acorn moisture content below 40% increases seed survival at − 7 °C or − 9 °C. We also identified the limiting temperature (− 10 °C) below which pedunculate oak seeds rapidly lose their ability to germinate and emerge. It was also observed that the severe drying and freezing of acorns, interacting together or separately, can lead to abnormal seedlings, characterized by a significantly lower dry mass.