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Article

Seed Preparation Methods for Increasing the Germination of Sour Cherry (Prunus cerasus L.)

by
Marek Szymajda
1 and
Robert Maciorowski
2,*
1
Department of Horticultural Crop Breeding, The National Institute of Horticulture Research, 96-100 Skierniewice, Poland
2
The National Institute of Horticulture Research, 96-100 Skierniewice, Poland
*
Author to whom correspondence should be addressed.
Forests 2025, 16(3), 516; https://doi.org/10.3390/f16030516
Submission received: 23 January 2025 / Revised: 12 March 2025 / Accepted: 13 March 2025 / Published: 14 March 2025
(This article belongs to the Special Issue Topicalities in Forest Ecology of Seeds, 2nd Edition)
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Abstract

:
Poor seed germination obtained in sour cherry breeding programs results in a limited number of seedlings. This makes breeding inefficient: the resulting hybridization is low in relation to human labor input. That is why a study was conducted to investigate the influence of different methods of treating seeds of three sour cherry cultivars—‘Wanda’, ‘Wroble’, and ‘Lutowka’—on their germination and the growth of the obtained seedlings under greenhouse conditions. The tested methods of seed treatment included different durations of the stratification period at 5 °C, and several variants of removing the sources of germination inhibitors present in the stones (endocarps), seed coats, and endosperm, and in the cotyledons of embryos. The highest number of germinated seeds/embryos was obtained by removing the seed coat attached to the endosperm and subjecting the exposed embryos to a temperature of 20 °C after stratifying them for a period of 90 days. The percentage of germinated seeds/embryos obtained by this method was as high as 80%–90%, and their germination occurred within 10–15 days, whereas with the traditional stratification of seeds in endocarps at 5 °C, a large number of seeds did not germinate, even after 150 days of stratification. This method produced 20–25 cm tall seedlings within five months. By contrast, the final germination percentage of the seeds in the Control Treatment was from 16.4% to 54.4%, and a large proportion of seeds had still not germinated after five months. Seedlings obtained from seeds stratified for 90 days grew better than those obtained from seeds stratified for a shorter time. The developed method makes it possible to obtain a larger number of sour cherry seedlings, thus increasing the efficiency of creative breeding. Moreover, obtaining a higher germination percentage over a shorter period shortens the breeding cycle, which contributes to reducing the costs of sour cherry breeding.

1. Introduction

The deep physiological dormancy of sour cherry (Prunus cerasus L.) seeds, as well as other species of the genus Prunus, is caused by the presence of germination inhibitors, mainly abscisic acid (ABA), found in varying concentrations within the endocarp and various parts of the seed: the seed coat and endosperm, and in the embryo itself [1,2,3,4] Therefore, when extracted from the fruit, the seeds are not able to germinate, even if the environmental conditions, i.e., temperature, substrate humidity, and oxygen availability, are suitable [5,6,7].
The endocarp and the seed coat, not only protect the embryo contained in them, but also constitute a mechanical barrier during the growth of the radicle. The endocarp also hinders the access of water to the seed inside [8] and the leaching of germination inhibitors from the seed coat and embryo [2]. Consequently, removing the endocarp reduces the time required for stratification and increases the number of germinated seeds of many species of woody plants, including sweet cherry, mahaleb cherry, peach, and small-fruited cherry [9,10,11,12].
Research has shown that the growth of the radicle in the seeds of species of the genera Prunus and Malus is prevented by inhibitors contained in the seed coat and endosperm. By removing the seed coat, together with the endosperm, it has been possible to obtain, within a dozen or so days, germinated embryos of sweet cherry [9,13], peach [14,15], and sour cherry [2], without subjecting them to stratification. However, seedlings obtained from embryos not exposed to low temperatures produce severely shortened shoot internodes and are characterized by very stunted growth, although their root system develops normally [16,17]. Studies conducted on peach and sour cherry seeds have shown that the inhibition of the growth of the apical part of the embryonic axis of seedlings obtained from unstratified embryos is caused by the presence of growth inhibitors contained in the cotyledons [2,18].
It is well known that the dormancy of seeds of species of the genus Prunus is effectively broken during the stratification period [19,20,21], during which chemical compounds called germination inhibitors undergo decomposition. The ABA content of seeds subjected to stratification is reduced, whereas the concentration of GA4 in the embryos of non-dormant seeds is higher than in those of dormant seeds [1]. During stratification, seeds should be subjected to a temperature suitable for the species, and sometimes even for the genotype, and have access to water and oxygen [22,23].
Prolonged seed stratification extends the breeding cycle. However, even after several months of stratification, not all seeds germinate. This is a serious problem, especially in the creative breeding of sour cherries, because their fruit has only one stone, which contains only one, not always fully developed, seed. Therefore, crossbreeding programs generally produce very few seeds relative to the number of pollinated flowers and, consequently, a small number of hybrid seedlings. This makes the creative breeding of sour cherries inefficient: the resulting hybridization is low in relation to human labor input. For this reason, research is still being undertaken to optimize germination uniformity and shorten the stratification time of seeds of various fruit tree species [24,25].
The aim of this study was to assess the influence of combining different stratification durations with the removal of the endocarp, the seed coat and endosperm, and parts of the embryo cotyledon on seed/embryo germination and the initial growth of the obtained sour cherry seedlings.

2. Material and Methods

2.1. Research Material

The objects studied were seeds of three sour cherry cultivars—‘Wanda’, ‘Wroble’, and ‘Lutowka’ (Polish names: ‘Wanda’, ‘Wróble’, ‘Łutówka’)—with different fruit-ripening times. In central Poland, the fruit of the cultivar ‘Wanda’ ripen most often in the first 10 days of July, those of the cultivar ‘Wroble’ around the middle of July, and those of ‘Lutowka’ around the 20th of July. Three cycles of research were conducted at the end/beginning of 2013/2014, 2014/2015, and 2015/2016. The trees from which the seeds were obtained grew in breeding plots in the Experimental Orchard in Dąbrowice near Skierniewice (central Poland—altitude 126 m, latitude 51°57′ N, longitude 20°09′ E), managed by the National Institute of Horticultural Research. The seeds for testing were obtained successively as the fruit ripened, at the stage of their harvest maturity. The fruit of each cultivar from which the stones were extracted were collected randomly from three trees, 350–450 from each tree, from different sides of the crown, and from a height of 1.0–1.8 m. The extracted stones were cleaned of pulp remnants, dried in ambient conditions at a temperature of about 20 °C, and then stored in paper bags under the same conditions for a period of 3–6 months until the testing began. The tests were conducted at the Horticultural Plant Breeding Department of the National Institute of Horticultural Research in Skierniewice.

2.2. Experimental Treatments

Within each genotype, 10 experimental treatments were applied (Figure 1). The Control Treatment consisted of stratified stones (seeds with endocarps) that had been disinfected by soaking (24 h) in a 0.1% solution of Captan 50 WP suspension fungicide (50% captan, Arysta Life Science North America Co., San Francisco, CA, USA) before stratification. Stones floating on the surface of the solution after 24 h of disinfection were not considered as containing fully developed seeds and were thus eliminated from the study. Before stratification (Treatments 2–10), the endocarp was removed from the dried stones and the seeds were extracted. Poorly developed seeds were eliminated from the tests. The obtained seeds were disinfected by soaking for 24 h in a 0.1% solution of Captan 50 WP. Then, the seeds were mixed with sterile and moist perlite and packed into plastic bags and stratified at 5 °C in an ‘MIR-554’ refrigerated incubator (‘SANYO’, Moriguchi, Japan). Each experimental treatment was performed in three replications, and for each replication the germination capacity of 20 stones, seeds, or embryos was assessed. Inspections to check substrate moisture and seed ventilation were conducted after 30, 60, and 90 days of stratification.
In each year, after 30 days (Treatments 5 and 6), 60 days (Treatments 7 and 8), or 90 days (Treatments 9 and 10) of stratification, the seed coat and endosperm were removed from the seeds (Figure 2a), and in Treatments 6, 8, and 10, the embryos additionally had their cotyledons shortened by 2/3 of their length (Figure 2b). In Treatment 4, after 24 h of disinfection in a 0.1% solution of Captan 50 WP suspension fungicide and the swelling of unstratified seeds, as in Treatments 6, 8, and 10, the cotyledons of the embryos were shortened by 2/3 of their length. The prepared embryos in Treatments 4–10 were again disinfected for 30 min in a 0.1% solution of Captan 50 WP, then mixed with moist sterile perlite and packed into plastic bags. A 0.1% solution of Captan 50 WP was also used to moisten the substrate. The bags with embryos were placed in ambient conditions at 19–21 °C and protected from light. In the seeds/embryos in Treatments 1–3, cold stratification was continued at 5 °C for 150, 120, and 90 days, respectively.
Starting from 15 January (2014–2016), inspections were carried out during which germinating seeds/embryos were selected and counted. The first 7 inspections were performed every 5 days, and the subsequent 3 every 10 days. In each treatment, seed germination was carried out for 60 days. Seeds/embryos with a 5–15 mm long radicle were considered as having germinated. In the Control Treatment, after 150 days of stratification, the endocarp was removed from ungerminated seeds (stones) and the number of developed seeds was counted. The percentage of germinated seeds in that experimental treatment was calculated based on the number of fully developed (viable) seeds.

2.3. Seedling Growth Assessment

Germinating seeds/embryos were planted out into plastic pots with a capacity of approx. 350 cm3, filled with a sterile substrate consisting of a mixture of peat substrate and washed sand at a volume ratio of 3:1 (Figure 3a,b).
To be able to compare the growth of the seedlings in all of the experimental treatments, the germinated seeds and embryos obtained during the first four seed inspections were kept in plastic bags filled with moist perlite in a refrigerator at 3 °C until February 4. Then, these seeds/embryos, along with those that germinated between 30 January and 4 February (Inspection 5), were planted out into pots. In some treatments, due to the insufficient number of obtained plants, seeds/embryos that had germinated by 14 February (Inspections 6 and 7) were also used for further study. In this way, all the seeds/embryos were planted out at a similar time in each year of the study, i.e., 4–14 February. Seedlings obtained from seeds/embryos that germinated after February 14 were not included in the measurements.
The pots with the seeds/embryos were placed in a heated greenhouse (20°/18 °C day/night, 16/8 h day/night), where the seedlings were cultivated for three months until the beginning of May. After planting out into pots, the seeds/embryos were watered twice with a 0.1% solution of fungicide Aliette 80 WG (80% aluminum fosetyl, Bayer CropScience AG, Monheim, Germany). They were first watered immediately after planting and again 5 days later.
Each experimental treatment included 21 seedlings (three replicates of 7 seedlings each), except for the treatments where too few seeds had germinated. These were seedlings of the cultivars ‘Wanda’—Treatments 1 and 4 (9–16 or 18–21 seedlings, respectively, depending on the year); ‘Wroble’—Treatments 3 and 4 (18–21 seedlings in each treatment); and ‘Lutowka’—Treatments 1, 3 and 4 (18–21 seedlings in each treatment). The experiment was arranged in a completely randomized design. For each treatment, the height of the seedlings was measured after 30, 60, and 90 days of their growth in the greenhouse.

2.4. Statistical Analysis

The detailed assumptions of the statistical methods used to analyze seed/embryo germination under the influence of the examined treatments were presented in an earlier paper [26]. The semi-parametric Cox proportional hazard model was applied, with year (Y), genotype (G), seed/embryo treatment, and their interactions as covariates.
The statistical significance of the final model forms and their components was tested using the Wald test. The germination functions for each genotype and treatment method were constructed using nonparametric Kaplan–Meier estimates. According to Onofri et al. [27], the term germination probability replaces survival probability, which is normally used in medical and epidemiological research. Germination probabilities measure the proportion of seeds that are still ungerminated (and thus at risk of germination) at each time step of seed inspection. The comparisons of germination functions were performed by means of a Peto–Peto test at p = 0.05 with Holm–Bonferroni adjustment for multiple comparisons.
Seedling growth data were analyzed using a classical linear model with year, genotype, treatment, and their interactions as the descriptors and the time of evaluation as a repeated-measurement factor. Prior to the analysis, the normality of the data and homogeneity of variance were verified by means of the Lilliefors and Levene tests, respectively. The sphericity assumption was evaluated with Mauchly’s test. If sphericity was violated, adjustments were performed with the Greenhouse–Geisser correction. Post hoc comparisons were made with Tukey’s HSD test at p = 0.05.
All calculations were performed using STATISTICA v. 13 package Dell Inc. [28].

3. Results

3.1. Seed Germination

The effects of individual factors on the number of germinated sour cherry seeds and the course of their germination described by the Cox proportional hazard model were found to be significant, except for the highest-order interactions (Table 1). The method of treating seeds/embryos was clearly the most important, i.e., both the presence and absence of the sources of germination inhibitors found in the endocarp, seed coat and endosperm, and the cotyledons of embryos, as well as the duration of the stratification period had an effect. The different responses of the tested genotypes to the methods of treating seeds/embryos were also clearly evident. Statistically significant differences, although with similar trends, were found in relation to the interaction of the year of study with the experimental factors: seed/embryo treatment method and genotype.
The course of the seed germination of the cherry cultivars tested is shown in Figure 4 in the form of germination percentage, while the estimated germination functions, which display the probability of seeds not germinating in time, are shown in Figure S1 (Supplementary Materials). The final germination percentage (60th day of germination assessment) of the ‘Wanda’, ‘Wroble’, and ‘Lutowka’ sour cherry seeds/embryos in the Control Treatment, despite stratification for as long as 150 days, was 16.4%, 54.4%, and 33.0%, respectively (Figure 4). Although the seeds in Treatment 2 (continuous stratification of seeds without endocarps for 60 days) were stratified for a period 30 days shorter than those in the Control Treatment (continuous stratification of seeds in endocarps for 120 days), the removal of the endocarp had a positive effect on their germination capacity, although in the case of ‘Lutowka’, the difference was not confirmed statistically. For this treatment, the obtained percentage of germinated seeds was from 42.4% for ‘Lutowka’ to 61.7% for ‘Wroble’.
The highest number of germinated seeds/embryos was obtained by stratifying the seeds for 90 days and then removing their seed coats and exposing the embryos to a temperature of 20 °C (Treatments 9 and 10). The percentage of germinated seeds/embryos obtained in these treatments was over 65.0% for ‘Wanda’, approx. 85.0% for ‘Wroble’, and approx. 90.0% for ‘Lutowka’. With the exception of ‘Wanda’, where the method of Treatment 9 did not differ considerably from that of Treatment 8 (seeds stratified for 60 days), these differences in relation to the remaining treatments and cultivars were significant.
The first germinated seeds of the cultivar ‘Wroble’ in the Control Treatment were obtained after 95 days of stratification (5th day of assessment), and not earlier than 120 and 130 days of stratification (30th and 40th day of assessment) for the seeds of ‘Lutowka’ and ‘Wanda’, respectively (Figure 4; Figure S1—Supplementary Materials). Seeds without endocarps of the cultivar ‘Wroble’ (Treatment 2) germinated after 85 days (25th day of assessment), while embryos (Treatment 3) germinated after 55 days (25th day of assessment). Seeds of the cultivars ‘Wanda’ and ‘Lutowka’ germinated after 90 and 100 days (Treatment 2), and after 60 and 70 days (Treatment 3), respectively. Thus, removing the endocarps shortened the stratification time required for the first seeds to germinate by 10–40 days, depending on the cultivar, compared with the Control Treatment, while removing the endocarps together with the seed coats shortened this time by as many as 40–70 days.
As the results of the study show, unstratified embryos, when placed at a temperature of 20 °C, germinated at a lower percentage than embryos stratified for 30, 60, or 90 days (Treatments 5–10). However, the number of germinated embryos increased with the duration of stratification of the seeds from which the embryos had been isolated. Seeds chilled for 90 days, except for seeds of the cultivar ‘Wanda’ (Treatments 8 and 9), germinated at a significantly higher percentage than seeds chilled for 60 days. Likewise, seeds of the tested sour cherry cultivars chilled for 60 days germinated at a significantly higher percentage than seeds stratified for 30 days.

3.2. Seedling Growth

In the linear ANOVA model, the influence of all the tested experimental factors and their interactions on the growth of the obtained sour cherry seedlings proved to be significant (Table 1). As expected, the strongest effect was found for the method of treating seeds/embryos, with a weaker effect for the influence of genotype and year of study. It is worth noting that the interactions of the investigated experimental factors with the year of study, although significant, had little impact on the growth and development of the seedlings. The tall seedlings of each cultivar were obtained from seeds stratified for at least 60 days (Figure 5). On each measurement date, these seedlings were significantly taller than those obtained from seeds stratified for 30 days. However, after 90 days of growth, the tallest seedlings were those obtained from embryos stratified for 90 days. With the exception of the cultivar ‘Wanda’ (Treatments 10 and 7), these differences were significant in relation to the seedlings obtained from embryos stratified for 60 days. Tall seedlings were also obtained from seeds of the cultivar ‘Wroble’ in the Control Treatment. They were slightly shorter than those in Treatments 9 and 10, but significantly taller than the seedlings obtained from seeds stratified for 60 days (Treatments 7 and 8).
The results of the study show that seedlings obtained from embryos without shortened cotyledons (Treatments 5, 7, and 9) grew markedly taller than seedlings obtained from embryos with shortened cotyledons (Treatments 6, 8, and 10). This correlation was most evident in seedlings obtained from embryos stratified for 60 days (Treatments 7 and 8) or 90 days (Treatments 9 and 10). The differences, after 90 days of growth, for seedlings of the cultivars ‘Lutowka’ and ‘Wanda’ (Treatments 9 and 10) and ‘Wroble’ (Treatments 7 and 8) were statistically significant. A similar tendency was maintained in all the tested cultivars for seedlings obtained from seeds stratified for 30 days, but the differences were not confirmed statistically.
As was to be expected, seedlings obtained from unstratified seeds showed the weakest growth, regardless of the cultivar. These seedlings had severely shortened internodes and were characterized by stunted growth. This stunted growth of seedlings became less pronounced if the seeds from which they were obtained had been refrigerated for at least 30 days. However, the seedlings obtained in this way were significantly shorter than those obtained from seeds stratified for a period of 60 or 90 days.

4. Discussion

4.1. Seed Germination

The obtained results showed a positive effect of removing the endocarp and the seed coat with the endosperm on seed germination. This effect has previously been observed in peach by du Toit et al. [8] and Martinez-Gomez and Dicenta [16], in sour cherry by Jensen and Kristiansen [2], and in the Yoshino cherry (Prunus × yedoensis) by Kim [29]. These treatments not only increased the number of germinated seeds, but also shortened the time before they germinated. A positive effect of endocarp removal on the germination of small-fruited cherry seeds was obtained by Rostamikia et al. [12]. The inhibitory effect of the endocarp and seed coat on the germination of sour cherry seeds results from the fact that they constitute a mechanical barrier for the radicle during seed germination and limit the access of water to the embryo inside. The presence of the endocarp and seed coat makes it difficult for the germination inhibitors (including ABA) to leach out from the embryo [8], and, moreover, they themselves are also a source of germination inhibitors [1,29]. It is possible that in the Control Treatment (seeds in endocarps), in Treatment 2 (seeds without endocarps), and also in Treatment 3 (embryos), more seeds/embryos would have germinated if the stratification had lasted for a longer period of time and the period of chilling had thus been extended, under the influence of which germination inhibitors decompose, but this would have significantly extended the breeding cycle.
After placing embryos at 20 °C, the unstratified embryos germinated at the lowest percentage. The number of germinated embryos increased with the length of the stratification period of the seeds from which the embryos had been isolated. This was particularly evident in Treatments 5–10. With the exception of seeds of the cultivar ‘Wanda’ (Treatments 8 and 9), embryos chilled for 90 days germinated at a significantly higher percentage than embryos chilled for 60 days. Likewise, seeds chilled for 60 days germinated at a significantly higher percentage than those chilled for 30 days. A similar correlation was obtained by Stein et al. [30] in a study conducted on sweet cherry seeds. These authors’ findings and those obtained in this study indirectly confirm that during stratification there is a decrease in the concentration of germination inhibitors present in the embryo. Earlier research by Chen et al. [1] on Taiwan cherry (Prunus campanulata) seeds showed that during stratification, under the influence of cold, the concentration of abscisic acid in the embryos decreased. Therefore, embryos that were stratified for longer at 5 °C started the germination process faster after being transferred to a higher temperature. Also, in the case of Treatments 1–3, the number of germinated seeds and embryos increased with the increased number of stratification days, so the reduction in the concentration of inhibitors probably also occurred in the endocarps, seed coats, and endosperm. The decreasing abscisic acid content in the endocarps and seed coats of sour cherry seeds under the influence of cold was demonstrated in the study by Chen et al. [1].
An important factor stimulating the germination of embryos was the removal of 2/3 of their cotyledons (Figure 2). The effect was most pronounced in seeds stratified for either 30 or 60 days, although the differences were not always statistically significant. The results indicate that after 30 days of stratification, the embryos had not fully broken their dormancy, and the inhibitors contained in them had not yet completely decomposed under the influence of the cold. Also, in the study by Jensen and Kristiansen [2], dormant sour cherry embryos with part of their cotyledons cut off germinated at a higher percentage than embryos with their cotyledons left intact. The already-mentioned research by Chen et al. [1] showed that during stratification, the abscisic acid content in Taiwan cherry embryos decreased. In our study, cutting off part of the cotyledons removed the inhibitors contained in them. Therefore, embryos with shortened cotyledons germinated at a higher percentage compared with those whose cotyledons remained intact.
Seeds without their seedcoat and endosperm (embryos) were unable to germinate when kept at 5 °C. To break dormancy and acquire the ability to germinate, they had to undergo stratification for a period of at least 55 days for the cultivar ‘Wroble’, and even as long as 70 days for the cultivar ‘Lutowka’. In their study, Mehanna and Martin [14] proved that the lack of germination ability of dormant peach seeds was caused by the mechanical obstacle presented to the growing radicle by the seed coat. However, in the present study, the removal of endocarps and also seed coats and endosperm from sour cherry seeds did not help embryos to germinate at 5 °C. This was probably caused by the presence of inhibitors inside them.
The factor that prevented the germination of embryos was undoubtedly too low a temperature to which the embryos had been exposed. In the study by Jensen and Kristiansen [2], sour cherry embryos not treated with cold were able to germinate at a temperature of 20 °C, while in the study by Martinez-Gomez and Dicenta [15], peach embryos germinated within one week at a temperature of 25–30 °C. Also, in the present study, embryos of each cultivar, both unchilled and those stratified for 30 days, germinated only after having been subjected to a temperature of 20 °C. However, the germination of embryos subjected to low-temperature treatment always occurred at a higher percentage and in a shorter time compared with the germination of unchilled embryos. The same correlation was obtained in research conducted on apricot and peach embryos [26].
The present study also showed a significant impact of genotype on seed germination capacity. Seeds of the cultivars ‘Lutowka’ and ‘Wroble’ germinated at a significantly higher percentage than seeds of the cultivar ‘Wanda’. Among the cultivars analyzed in this study, ‘Wanda’ is the cultivar with the earliest fruit ripening time [31]. It is therefore not surprising that the seeds of this cultivar have a lower germination capacity than the seeds of the cultivars ‘Wroble’ and ‘Lutowka’. It is known from the literature that trees of many Prunus species with early fruit ripening time produce seeds that are not fully ripe and not able to germinate under traditional stratification conditions, or their ability to germinate is significantly reduced [32,33,34].

4.2. Seedling Growth

The growth of sour cherry seedlings was stronger the longer the seeds from which the seedlings had been obtained were subjected to a low-temperature treatment. The best growing seedlings were those obtained from seeds stratified for 90 days (Figure 2). The growth of seedlings obtained from seeds stratified for 60 days was considerably poorer, but it was significantly better than that of the seedlings obtained from seeds stratified for 30 days. These results indicate that in the seeds/embryos stratified for 30 or 60 days, the inhibitors inhibiting seedling growth had not completely decomposed under the influence of the cold. The research by Chen et al. [1] on Taiwan cherry seeds also showed that during stratification, under the influence of cold, the abscisic acid content in the embryos decreased.
After 90 days of growth, seedlings obtained from embryos without shortened cotyledons were much taller than seedlings obtained from embryos with shortened cotyledons. The weaker growth of seedlings obtained from embryos with shortened cotyledons was probably caused by the fact that, together with the removed part of the cotyledons, some of the nutrients used by young developing plants before they become fully self-feeding had also been removed.
The shortest seedlings of each cultivar were obtained from unstratified embryos. These seedlings had severely shortened internodes and were characterized by very stunted growth. Studies conducted on peach showed that developmental disorders in seedlings obtained from unstratified seeds decreased with the extension of the duration of the cold treatment of the seeds [15]. Stunted growth of the seedlings was no longer evident if the seeds had been stratified for at least four weeks at 7 °C. However, these seedlings were significantly shorter than those obtained from seeds stratified for a period of 5–14 weeks [15]. Likewise, Stein et al. [30] obtained dwarf seedlings from unstratified sweet cherry seeds, but the stunted growth did not occur if the seeds had been stratified for four weeks. The presented results also showed that sour cherry seedlings obtained from seeds stratified for 30 days did not exhibit stunted growth, but they were significantly shorter than the seedlings obtained from seeds stratified for 60 or 90 days.
The stunted growth of the seedlings produced in our study from unstratified embryos is not consistent with the results of the research by Flemion [18] on peach embryos and seedlings, nor with those by the Danish researchers Jensen and Kristiansen [2] for sour cherry. After shortening the cotyledons of embryos, these authors obtained normally growing seedlings within a few weeks of sowing the seeds. It is possible that if a larger portion of the cotyledons had been removed in the present study, and thus a larger proportion of the growth inhibitors contained in them had been removed, normal growth of the seedlings would also have been achieved. The inhibitory effect of compounds found in cotyledons has been demonstrated by experiments performed on peach embryos [35], in which the addition of an extract from dormant seeds (i.e., mainly from cotyledons) to the nutrient medium caused the induction of seedling dwarfism. The research by Tang et al. [36] showed that the concentration of inhibitors in the outer part of the cotyledons of sour cherry embryos is higher than in the part close to the embryonic axis. Perhaps soaking embryos in water for more than 30 min, combined with gentle shaking to wash out the growth inhibitors, would also be helpful. In the present study, after removing the seed coat and endosperm from the seeds and shortening the cotyledons, the embryos were disinfected in a solution of Captan suspension fungicide for only 30 min.

5. Conclusions

The present research showed that the best way to obtain a high percentage of germinated seeds/embryos for each of the three sour cherry cultivars tested was by the removal of the seed coat and endosperm, and exposure of the embryos to a temperature of 20 °C after stratification for a period of 90 days at 5 °C (Treatment 9). The percentage of germinated seeds/embryos of each sour cherry cultivar obtained by this method was 80%–90%, and their germination occurred within a few or a dozen or so days after placing them at room temperature (19–21 °C), whereas under continuous cold stratification, the obtained percentage of germinated seeds ranged from only 16.4% in cv. ‘Wanda’ to 54.4% in cv. ‘Wroble’. Within five months, this method produced 20–25 cm tall seedlings, whereas with the traditional stratification of seeds in endocarps, a large proportion of them had still not germinated after five months. Seedlings obtained from seeds chilled for 90 days grew much better than seedlings obtained from seeds chilled for a shorter time. The developed method makes it possible to obtain a larger number of sour cherry seedlings, thus increasing the efficiency of breeding this species. It is also possible to obtain a higher percentage of germinated seeds within a shorter period, which shortens the breeding cycle and reduces breeding costs.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16030516/s1.

Author Contributions

M.S.: Conceptualization, methodology, investigation, writing—original draft, funding acquisition, project administration, formal analysis. R.M.: statistical analysis, review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed by the Polish Ministry of Education and Science [Project No. 1.1.1 “Effect of various methods of post-harvest treatment of seeds of selected Prunus species on their germination and growth of obtained seedlings”].

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials. Further inquiries can be directed to the corresponding author (R.M.) at robert.maciorowski@inhort.pl.

Acknowledgments

We thank Mieczysław Paszt for making valuable suggestions for improving the manuscript.

Conflicts of Interest

The authors declare conflicts of interest.

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Figure 1. Experimental treatments shown on a time scale.
Figure 1. Experimental treatments shown on a time scale.
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Figure 2. Sour cherry embryos: (a) whole embryo; (b) embryo with shortened cotyledons.
Figure 2. Sour cherry embryos: (a) whole embryo; (b) embryo with shortened cotyledons.
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Figure 3. Young sour cherry seedlings of cv. ‘Lutowka’ growing from embryos with whole cotyledons (a) and from embryos with shortened cotyledons (b) (planted out into pots in a greenhouse).
Figure 3. Young sour cherry seedlings of cv. ‘Lutowka’ growing from embryos with whole cotyledons (a) and from embryos with shortened cotyledons (b) (planted out into pots in a greenhouse).
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Figure 4. Treatment-dependent cherry seed germination percentages. The table inside the figure shows the results of comparisons of germination functions obtained using Kaplan–Meier estimators for each seed treatment. Comparisons were made with the Peto–Peto test at p = 0.05, adjusted for multiple comparisons by means of the Holm–Bonferroni method.
Figure 4. Treatment-dependent cherry seed germination percentages. The table inside the figure shows the results of comparisons of germination functions obtained using Kaplan–Meier estimators for each seed treatment. Comparisons were made with the Peto–Peto test at p = 0.05, adjusted for multiple comparisons by means of the Holm–Bonferroni method.
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Figure 5. Seedling growth of three sour cherry genotypes depending on treatment. Means averaged over the three years of the study. HSD0.05—honestly significant difference calculated according to the Tukey procedure at p = 0.05 for comparison of the treatments on each day of measurement.
Figure 5. Seedling growth of three sour cherry genotypes depending on treatment. Means averaged over the three years of the study. HSD0.05—honestly significant difference calculated according to the Tukey procedure at p = 0.05 for comparison of the treatments on each day of measurement.
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Table 1. Results of statistical verification of components of the linear models used for data analysis.
Table 1. Results of statistical verification of components of the linear models used for data analysis.
EffectGermination DynamicSeedling Height
dfWald apdfF bp
Year (Y)243.9<0.001217.6<0.001
Genotype (G)236.8<0.001283.9<0.001
Seed Treatment (T)91105.2<0.0019233.4<0.001
Y × G417.6<0.00143.60.007
G × T18138.7<0.0011811.0<0.001
Y × T1856.5<0.001188.5<0.001
Y × G × T3641.10.255364.0<0.001
Measuring Time (M)---1.5 c7041.5<0.001
M × Y---3.1 c14.7<0.001
M × G---3.1 c33.8<0.001
M × T---13.8 c105.8<0.001
M × Y × G---6.1 c5.8<0.001
M × G × T---27.5 c3.4<0.001
M × Y × T---27.5 c5.5<0.001
M × Y × G × T---55.0 c1.90.001
a Wald test statistic, b Fisher test statistic, c degree of freedom adjusted with the Greenhouse–Geisser correction.
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Szymajda, M.; Maciorowski, R. Seed Preparation Methods for Increasing the Germination of Sour Cherry (Prunus cerasus L.). Forests 2025, 16, 516. https://doi.org/10.3390/f16030516

AMA Style

Szymajda M, Maciorowski R. Seed Preparation Methods for Increasing the Germination of Sour Cherry (Prunus cerasus L.). Forests. 2025; 16(3):516. https://doi.org/10.3390/f16030516

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Szymajda, Marek, and Robert Maciorowski. 2025. "Seed Preparation Methods for Increasing the Germination of Sour Cherry (Prunus cerasus L.)" Forests 16, no. 3: 516. https://doi.org/10.3390/f16030516

APA Style

Szymajda, M., & Maciorowski, R. (2025). Seed Preparation Methods for Increasing the Germination of Sour Cherry (Prunus cerasus L.). Forests, 16(3), 516. https://doi.org/10.3390/f16030516

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