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Article

Exploring the Morpho-Physiological Dormancy and Germination Potential of Paeonia peregrina Mill. Seeds In Vitro

by
Virginia Sarropoulou
,
Eleni Maloupa
and
Katerina Grigoriadou
*
Hellenic Agricultural Organization (ELGO)—DIMITRA, Institute of Plant Breeding and Genetic Resources, Thermi, 57001 Thessaloniki, Greece
*
Author to whom correspondence should be addressed.
Seeds 2025, 4(1), 7; https://doi.org/10.3390/seeds4010007
Submission received: 4 November 2024 / Revised: 14 January 2025 / Accepted: 17 January 2025 / Published: 29 January 2025
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Versions Notes

Abstract

:
Herbaceous peonies, specifically the Balkan–Anatolian Paeonia peregrina Miller, are species with various uses such as ornamental and garden purposes, or they can be cut as flowers or potted, or they can be eaten or used for medicinal purposes due to the rich nutritional content of their seeds. However, conventional propagation methods, including rhizome division, grafting, and layering, are slow, while seed propagation is challenging due to double morpho-physiological dormancy. This study therefore evaluated the in vitro germination potential of P. peregrina seeds in darkness under different culture conditions, including different temperature regimes (constant at 15 °C or alternating from 22 °C to 15 °C); incubation periods [120 days: 22 °C (14 d) to 15 °C (0–105 d); 120 days: 22 °C (33 d) to 15 °C (0–87 d); 90 or 140 days at 15 °C]; seed cold storage period (none; 30 days; 3 months; or 2, 5, and 8 years); and gibberellic acid (GA3) concentrations (0, 250, 500, 750, and 1000 mg L−1), as dormancy release methods. The results indicated that 60-day-stored seeds (30 d at 15 °C and 30 d cold-storaged at 4–5 °C) exhibited 100% germination within an 80-day culture under 250 mg L−1 GA3 at 15 °C. A lower and constant temperature of 15 °C, a shorter 30-day seed cold storage period, and the lowest GA3 concentration of 250 mg L−1 comprised the most effective combination treatment for dormancy release and germination acceleration. An understanding of the underlying mechanisms of seed dormancy removal is imperative for successful germination, growth rate and seedling establishment, shortened breeding cycles, and germplasm conservation, leading to the field cultivation and economic production of these peony plants.

1. Introduction

Herbaceous peony species have demonstrated high ornamental [1], edible [2], medicinal [3], economic, and ecological usefulness [4], as garden, cut flower, and potted plants [5], as they are long-lived and relatively disease- and pest-resistant [6]. The economic benefits of herbaceous peonies are increased [7] due to the high content of their seeds in unsaturated fatty acids (UFAs) (>90%), protein, and secondary metabolites (i.e., stilbenes—resveratrol derivatives) for the production of health products and medicines [8,9,10]. Approximately 30% of seed dry biomass corresponds to the seed testa, the primary co-product in peony seed oil manufacture [11,12,13]. Non-viable wild collecting pressures [14], the trade of wild herbaceous peonies, and the escalating demand for oil production from their seeds have attracted the attention of scientists and industries for further exploitation [15].
Seed germination, rhizome or crown division, the division of the shrub, layering, and grafting are reported as propagation methods for herbaceous peonies [16,17]; however, the rate of vegetative propagation is very slow [16]. Herbaceous peony plants can also be reproduced through cuttings consisting of underground rhizomes, nodal stems [18], and green shoots; however, the rooting percentage of green shoot cuttings is low (<20–30%) [17], and the time needed for new plants obtained through root cuttings to be replanted in a terminal field is 30 years [19]. The division of the shrub can be applied every 2–6 years, depending on species, cultivar, and environmental conditions [17]. Rhizome division, used for the preservation of covetable traits [20], needs two years in the field for the installation of a robust plant capable of further division [17] and three years to obtain double the number of stock plants [21], as the propagation coefficient is extremely low. Only 5–8 new plants can be acquired within 4–5 years after the division of a mother plant [20]. Even though the propagation of herbaceous peonies via layering is rare, the combined application of vertical layering with rooting chemicals such as Ukorzeniacz B2 (i.e., a blend of plant growth regulators) has been demonstrated to be highly effective [22], while stem layering can be repeated every 3–4 years [19].
The sexual propagation of peonies through seeds is a difficult and complex process due to low and spatially narrow or confined seed dispersal in nature [23]; the slow maturation, ripening, and dispersal of seeds in late summer and autumn [24]; and very low germination rates in diverse natural conditions extending up to 24 months [25], which makes the subsequent cultivation of herbaceous peonies even more difficult [6]. Peonies generally have a prolonged germination period; for instance, P. lactiflora seeds require 6–7 months under controlled conditions [26], 8–9 months in nature with low seedling survival [27], and 10–16 months for wild peonies in Russia, extending to 2 years in unfavorable conditions [4,28]. Germination refers to the development of an embryo from the seed by initiating a variety of synthesis and decomposition activities, such as respiration, protein synthesis, and the motility of food reserves and thus water uptake [29]. Seed dormancy could be due to seed coat thickness, the imbalance of seed hormone concentrations, and undeveloped embryos [30]. Abscisic acid (ABA) prompts seed maturation and enhances dormancy, whereas gibberellins compete with ABA and boost germination [31]. The ability of seeds to germinate and overcome dormancy is crucial for adaptation to challenging environmental conditions, contributing to the conservation of the plant gene pool on Earth [4].
Herbaceous peonies nourish and flourish in mild and chill environments [4] and produce seeds presenting combined deep, simple, and double morpho-physiological dormancy as a key safeguard conservation mechanism for withstanding the low temperatures of the winter after their dispersal from the mother plant [4,32]. Double seed dormancy can be eliminated by exposure to temperature fluctuations via the several stages of the germination cycle, mechanical, and/or chemical stratification to facilitate seed coat opening and embryo permeability to water and gases [25], the application of gibberellic acid (GA3) [33] or other hormones such as ethylene, dry storage (after ripening) [34], or warm and cold stratification [16,34,35]. Warm stratification is required for embryo growth prior to radicle protrusion followed by cold stratification for epicotyl growth, notwithstanding GA3 treatment reinforcing embryo growth and the subsequent germination rates of peony seeds [23,28].
Germination ecology patterns and dormancy release techniques are highly dependent and varied in plant species of the same family, species of the same genus, and different ecotypes of a species [36], locality (altitude, shading, etc.), plant position at the locality, year [37], physical and morphological properties of the seed (number, size, shape, and color), seed collection period ranging from July to the end of October [24,25], temperature, light (quality, intensity, and photoperiod), seed endogenous factors (i.e., plant hormones, expression of genes and enzymes), the developmental status and the water absorption capacity (moisture content) of the seed embryo, the interdependence levels of plant growth regulators (GA3 and ABA) inside the seed, seed dormancy level (intermediate or non-deep for herbaceous peonies) [6], and seed dormancy category (exogenous physical and endogenous morpho-physiological in herbaceous peonies) [6,34,37]. A decrease in germination has been associated with infertility and not enough mature peony seeds when collected earlier than the optimum (i.e., initiation of follicle opening and seed testa darkening) or a higher thickness of the seed coat when seeds are collected later than the optimum, though this is species-dependent, for instance, P. tenuifolia, P. cambessdesii, and P. veitchii mature earlier, while P. peregrina, P. banatica, P. mascula, P. officinalis, P. sinjiangensis, P. anomala, P. emodi, P. obovata, and P. sterniana mature later [24,25]. Larger, physically dormant seeds become water permeable earlier (i.e., have a higher water content) than smaller ones, which explains why they show faster dormancy release and germinate faster [38]. The storage method of seeds after their collection is of vital importance for their viability, seeing that increasing seed age can diminish germination due to deterioration of the seed metabolic system, ending up with sluggish, very poor germination, weak seedling development, and lower establishment for old seeds; in other words, seeds age and lose their vitality after a period of storage [39]. Fresh peony seeds are introduced for top-notch germination results because of expeditious germination without any pre-soaking treatment before planting [40]. Moisture content (up to 10%) is an essential factor affecting seed viability, especially after long-term conservation in the refrigerator at 4–6 °C [25,41].
Despite the drawbacks of the propagation methods for herbaceous peonies, the increase in temperature during the last 25 years, especially during the winter months, has led to changes in synchronization and successfulness of germination [42] and to the appearance of an increased number of irregular seedlings [34]. Some plants’ insufficient ability to adapt to temperature shifts and translocate to northern regions and/or higher altitudes has resulted in their disappearance from natural populations, rendering the species rare or endangered [15,42]. Among the herbaceous peonies, Paeonia peregrina Miller (Paeoniaceae) is a native/non-range-restricted Balkan–Anatolian geophyte that thrives in woodlands and scrub habitats, characterized as a least concern species according to the IUCN category and until now not under protection by the Greek Presidential Decree 67/1981 [43]. Therefore, in vitro culture is considered the ideal propagation method due to the wide range of benefits it offers, including the mass, facile, and speedy production of new plants under controlled environmental conditions, germplasm conservation, shortened reproductive cycle, high propagation rates, speeding up of breeding evolution, and the prompt launching of new varieties, unraveling the difficulties related to conventional breeding methods for the manufacturing and trade of herbaceous peonies [35,44].
This study aimed to evaluate the dormancy release and in vitro germination potential of Paeonia peregrina seeds under the influence of different factors, including temperature, incubation period, seed dry-storage period in a dehumidifying chamber, seed cold-storage period in the refrigerator, and GA3 concentrations, assessing the evolution course of germination at subsequent time intervals throughout the whole culture period.

2. Materials and Methods

2.1. Plant Material and Culture Conditions

The plant material used was seeds collected from the field from mother plants in mid–late July, maintained at the collection of the Balkan Botanic Garden of Kroussia (BBGK), Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization—DIMITRA (ELGO–DIMITRA) (Figure 1a–f). Upon taxonomic identification, the seeds received the unique International Plant Exchange Network (IPEN) accession number of 98,281. Afterwards, they were placed in a drying–dehumidification chamber at a temperature of 15 °C and relative humidity (RH) of 15% for dry storage and short-term conservation [45,46,47]. Numerous treatments have been proposed to minimize infection from seeds during disinfection before the germination test, including a few fungicides [48], ethanol [49], and NaOCl (dilute household bleach) [50] without a significant adverse effect on seed germination. After preliminary testing of different disinfection methods [51], the following method was used: Before in vitro establishment, the seeds were hydrated for 24 h with distilled water at room temperature in the dark and disinfected with immersion in a fungicide solution for 1 h (0.1 g Signum/100 mL ddH2O) [commercial formulation Signum 26.7/6.7 WG consisted of 26.7% w/w boscalid, 6.7% w/w pyraclostrobin and 65.3% w/w suitable excipients in the form of suspendable granules, BASF HELLAS SA, VIPETH Sindos, Thessaloniki, Greece], then in 70% ethanol (5 min) and 5% sodium hypochlorite (NaOCl) solution (30 min).
As peony seeds are considered relatively large, their germination process relies less on light [52], and in this study, they were left to germinate in darkness. Four germination assays were carried out under controlled in vitro laboratory conditions. In the first, second, and third germination assays, the nutrient culture medium used was MS [53] supplemented with 250 mg L−1 GA3 and 20 g L−1 sucrose, and solidified using 6 g L−1 Plant Agar as a coagulant agent (pH 5.8), while in the fourth assay the composition of the medium was the same but comprised different GA3 concentrations (0, 250, 500, 750, and 1000 mg L−1). The seeds were placed in glass borosilicate tubes (25 mm width × 100 mm height). The culture media were sterilized in an autoclave at 121 °C for 20 min.
The first germination assay included 10 seeds as replicates, and the second assay included 35 seeds. In the third assay, 30 seeds of an 8-year cold-storage period, 24 seeds of a 5-year cold-storage period, 30 seeds of a 2-year cold-storage period, and 27 seeds of a 3-month cold-storage period were used. The fourth germination assay included five GA3 concentration treatments with 10 seeds (i.e., replicates) per treatment. The variation in the number of replicates (i.e., seeds) used for each treatment is due to infection losses by fungi- and/or bacteria, resource limitations dependent on the time of period each germination assay took place, and/or availability of seeds in the seed gene bank of BBGK.
All chemicals used, such as Murashige and Skoog (MS) medium basal salt mixture including vitamins, sucrose, GA3, and Plant Agar, were purchased from Duchefa Biochemie B.V., Haarlem, The Netherlands.

2.2. In Vitro Seed Germination

2.2.1. In Vitro Germination of 20-Day-Stored Seeds at 15 °C and 15% RH Within 120 Days of Total Culture Under Transition Temperature (22 °C for 14 Days and 15 °C for 106 Days)

In the initial—first assay, 10 seeds, aged 20 days, were cultured at 22 °C in continuous darkness. After 14 days of no germination, they were moved to 15 °C in darkness. Germination started after 34 days at a lower temperature. The culture period was 120 days, with the first 14 days at 22 °C and the subsequent 106 days at 15 °C.

2.2.2. In Vitro Germination of 30-Day-Stored Seeds at 15 °C and 15% RH Within 120 Days of Total Culture Under Transition Temperature (22 °C for 33 days and 15 °C for 87 Days)

In the second assay, 35 seeds, aged 30 days, were cultured in glass borosilicate tubes at 22 °C in darkness. Since no germination was observed after 33 days of culture, the seeds were moved to 15 °C in darkness. Germination began after 23 days at a lower temperature. The culture period was 120 days, with the first 33 days at 22 °C and the subsequent 87 days at 15 °C. The evolution process of seed germination was evaluated within a total culture period of 120 days divided into the first 33 days at 22 °C and the other 0–87 days at 15 °C.

2.2.3. In Vitro Germination Within 140 Days of Total Culture (Dark; 15 °C) Using Seeds of Different Cold-Storage Periods (8 Years, 5 Years, 2 Years, and 3 Months)

The third assay explored the effect of different cold-storage periods (8 years, 5 years, 2 years, and 3 months). Seeds collected from mother plants were initially stored in a drying chamber at 15 °C and 15% RH for 30 days (short-term), followed by long-term conservation in a refrigerator at 4–5 °C and RH < 5% and their germination was compared with eight-year-old, five-year-old, two-year-old, and three-month-old seeds maintained at the gene bank of BBGK (4–5 °C; RH < 5%). After 140 days of in vitro culture (15 °C; dark), the percentages of germination and infection (fungi and bacteria) were recorded.

2.2.4. In Vitro Germination of 60-Day Storage Seeds (15 °C, 15% RH, 0–30 Day and 4–5 °C, RH < 5%, 31–60 Day) Within 90 Days of Culture (Dark; 15 °C) Under Different GA3 Concentrations

In the fourth assay, the effect of gibberellic acid (GA3) concentrations (0, 250, 500, 750, and 1000 mg L−1) added into the basal culture medium and combined with 30-day cold storage (4–5 °C, RH < 5%) of the seeds as pretreatment was studied. Fifty (50) seeds, aged 60 days, underwent storage in a drying chamber at 15 °C and 15% RH for the initial 30 days, followed by 30 days in a refrigerator at 4–5 °C. The experiment comprised five treatments with 10 seeds each, and the germination process was assessed at intervals over a 90-day culture period.

2.3. In Vitro Seed Germination Metrics

The visible breaking of the top of the hard outer seed coat, the protrusion—mild expansion of the radicle (hypocotyl) (>2 mm) from it, and/or the sprout (epicotyl) emergence (>2 mm) were considered as germination criteria. At successive time intervals, the following three germination parameters were evaluated including total germination percentage (%) [=number of germinated seeds (only radicle protrusion without sprout and both radicle and sprout emergence)/total number of seeds × 100], germination percentage (%) with only radicle protrusion [=number of germinated seeds with only radicle protrusion without sprout emergence/total number of seeds × 100], and germination percentage (%) with both radicle and sprout emergence [=number of germinated seeds with simultaneous radicle and sprout emergence/total number of seeds × 100]. In addition to germination percentages, other basic parameters evaluated were the maximum total germination percentage (%), day of maximum total germination percentage, germination onset day, germination rate (GR) = n1 + n2 + … + ni/total number of seeds × 100%, where ni is number of seeds germinate on i-day [54], mean germination time (MGT) (day) = ∑(n × d)/N, where n = number of seeds germinated on each day, d = number of days from the beginning of the test, and N = total number of seeds germinated at the termination of the experiment [55], germination speed index (GSI) = number of germinated seeds with both radicle and sprout emergence on 1st count/(day of the 1st count) + … + number of germinated seeds on final count/day of the final count [55], germination energy (GE) percentage (%) = number of germinated seeds with both radicle and sprout emergence at the time of the first day of counting/total number of germinated seeds at the end of the test × 100 [56], germination energy period (in days), and t50 as the days needed to reach 50% of the final germination percentage [57].

2.4. Statistical Analysis

The experimental layout was completely randomized. The means were subjected to analysis of variance (ANOVA) and compared using the Duncan multiple-range test (p < 0.05) using the statistical program SPSS 17.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was applied for means comparison related to germination percentages among the different culture period treatments in all four germination assays and the different GA3 concentrations per culture period in the 4th assay. A two-way ANOVA was performed to test the effect of the different combinations [culture periods x seed cold-storage periods (3rd assay), culture periods × GA3 concentrations (4th assay)] on germination percentages. In the 3rd assay (i.e., seed cold-storage period, culture period in days) and 4th assay (i.e., culture period in days, GA3 concentration), the main effect of factors and their interactions were determined by the General Linear Model.

3. Results

3.1. In Vitro Germination of 20-Day-Stored Seeds at 15 °C and 15% RH Within 120 Days of Total Culture Under Transition Temperature (22 °C for 14 Days and 15 °C for 106 Days) Conditions

In the first assay, germination onset with only radicle protrusion (Figure A1a) was defined on the 48th day of culture (14 days at 22 °C and 34 days at 15 °C). After a total culture period of 120 days (14 days at 22 °C and 106 days at 15 °C), there was 100% disinfection success and a 60% total germination percentage. On the 102nd day (14 days at 22 °C and 88 days at 15 °C), the total germination percentage peaked at 60%, with 50% exhibiting both radicle and sprout development (Figure A1b). By the 120th day, the total germination percentage remained at 60%, with a preference for seeds showing both radicle and sprout (50%) over those with only radicle protrusion (10%) (Figure A1c). Radicle protrusion began on the 48th day, and simultaneous radicle and sprout emergence occurred at 20% on the 89th day (14 days at 22 °C and 75 days at 15 °C). Considering higher total and combined radicle and sprout germination percentages, lower radicle germination, and a shorter culture period, it seems that 102–120 days of total culture (14 days at 22 °C and 88–106 days at 15 °C) are optimal for the in vitro germination of 20-day-stored seeds. The impact of 15 °C on germination percentages was significant (p-value = 0.000 < 0.05) (Figure 2; Table S1).

3.2. In Vitro Germination of 30-Day-Stored Seeds at 15 °C and 15% RH Within 120 Days of Total Culture Under Transition Temperature (22 °C for 33 Days and 15 °C for 87 Days) Conditions

Since no germination was observed after 33 days of culture at 22 °C, test tubes were placed at a lower temperature (15 °C), and the onset of germination (Figure A2a) was determined on the 56th day after disinfection and initial in vitro seed establishment (33 days at 22 °C and 23 days at 15 °C). After 110 days of culture (33 days at 22 °C and 77 days at 15 °C), there was a 51.43% total germination percentage. Among the germinated seeds, 27.78% showed only radicle protrusion (Figure A2a), while 22.86% exhibited both radicle and sprout development (Figure A2b) as complete plants (Figure A2c). Radicle protrusion began on the 56th day, and simultaneous radicle and sprout emergence occurred at 22.86% on the 110th day of total culture (33 days at 22 °C and 77 days at 15 °C). Therefore, 110 days of total culture (33 days at 22 °C and 77 days at 15 °C) are necessary for the optimized in vitro germination of P. peregrina seeds. The impact of the culture period at 15 °C on germination percentages was found to be significant, as before (p = 0.000 < 0.05) (Figure 3; Table S2).

3.3. In Vitro Germination Within 140 Days of Total Culture in the Dark at 15 °C Using Seeds of Different Cold-Storage Periods (8 Years, 5 Years, 2 Years, and 3 Months)

In the third germination assay, after 140 days of in vitro culture, an average of 92.5% disinfection success and 7.5% infection percentage were recorded. The disinfection percentage ranged between 80% and 100% among the four seed cold-storage periods (Table S3).
The combined statistical analysis among the different seed cold-storage periods showed a maximum germination of 74.07% for 3-month cold-stored seeds after 87 days of culture with radicle and sprout development (Figure 4 and Figure A3a; Table S4). For 8-year-old seeds, germination onset was observed after 73 days with a 6.67% percentage, reaching 26.67% on the 140th day (Table S4). The germination percentage for 5-year-old seeds ranged from 4.17% to 16.67% between the 80th and 140th culture day (Table S4). Two-year-old seeds showed a 3.33% germination on the 30th day, peaking at 46.67% on the 140th day (Table S4). For 3-month-old seeds, germination onset occurred on the 30th day (14.81%), with the highest percentage of 74.07% on the 87th day (Table S4). A negative correlation was observed between seed age and germinability, with decreasing and delayed germination responses as follows: 3-month-old seeds > 2-year-old > 5-year-old > 8-year-old (Figure 4 and Figure A3b; Table S4).

3.4. In Vitro Germination of 60-Day Storage Seeds (15 °C, 15% RH, 0–30 Day and 4–5 °C, RH < 5%, 31–60 Day) Within 90 Days of Culture (15 °C; Dark) Under Different GA3 Concentrations

After 80 days of culture, the maximum seed germination percentage (100%) occurred with GA3 at 250 mg L−1, followed by 83.33% with 500 mg L−1 GA3, and 66.67% and 62.5% with 1000 mg L−1 and 750 mg L−1 GA3, respectively. Germination onset was on the 22nd day for 1000 mg L−1 GA3 and the 41st day for lower GA3 concentrations. In the GA3-free medium, the lowest germination percentage was 25%, even after 90 days (germination onset day). Continuous germination progression was noted for 250 mg L−1 GA3, especially from the 41st to the 80th day (42.86–50–75–100%). In other treatments, the maximum germination percentage was reached on day 62 for 750 mg L−1 GA3 (62.5%), and on day 66 for 500 mg L−1 (83.33%) and 1000 mg L−1 GA3 (66.67%) (Figure 5; Table S5). Breakage of the hard outer seed coat and radicle protrusion occurred in all GA3-supplemented media. However, complete plants with parallel radicle and sprout development were observed only under 1000 mg L−1 GA3 (Figure A4a–e). The best germination percentage (100%) of 60-day-stored seeds occurred after an 80-day culture (15 °C; dark) in medium with 250 mg L−1 GA3 (Figure 5; Table S5). The effect of the culture period, GA3 concentration, and their interaction on germination percentage was significant at a 0.1% level (p = 0.000 < 0.05) (two-way ANOVA/General Linear Model). The germination percentage was significantly affected by the main effect of the culture period for each GA3 concentration separately (p = 0.000 < 0.05) and by the main effect of GA3 concentration for almost every culture period (p = 0.000 < 0.05), except for the two shorter culture periods of 5 and 15 days (p = 1.000 > 0.05) (one-way ANOVA) (Table S5).

3.5. Optimum Outcome per Evaluated Parameter Irrespective Germination Assay

The highest total germination percentage (100%) and germination speed index (0.769) were found for 30-day cold-stored seeds cultured in 250 mg L−1 GA3-supplemented medium at 15 °C. The earliest 62nd culture day of maximum total germination percentage and the lowest t50 value (21 days) were observed for 30-day cold-stored seeds cultured in 750 mg L−1 GA3-supplemented medium at 15 °C. The earliest germination onset (22nd day) and the shortest germination energy period (24 days) were observed for 30-day cold-stored seeds cultured in 1000 mg L−1 GA3-supplemented medium at 15 °C. The highest germination rate (53.09%) was noticed in the case of 3-month cold-stored seeds cultured at 15 °C in 250 mg L−1 GA3-supplemented medium. The lowest mean germination time (48 days) was recorded by non-cold-stored seeds cultured at 22 °C for 14 days and subsequently at 15 °C for another 106 days in 250 mg L−1 GA3-supplemented medium. The highest germination energy percentage (100%) was evidenced by non-cold-stored seeds cultured at 22 °C for 33 days and subsequently at 15 °C for another 87 days in 250 mg L−1 GA3-supplemented medium (Table S6).

4. Discussion

In this study’s in vitro germination assays, P. peregrina seed cultures were incubated in the dark, aligning with the preference for dark conditions in peony seed germination [58]. Most herbaceous peony species undergo subterranean seed germination, resulting in underground cotyledons [19,23]. Before disinfection and in vitro establishment, P. peregrina seeds were soaked in distilled deionized water for 24 h at room temperature in the dark, as previous immersion stimulates the induction germination process, activating respiratory metabolism and transcriptional activities [41]. The advantages of seed soaking, including reduced time, accelerated germination, and improved uniformity, are particularly beneficial for coarse seed species like P. peregrina, aiding in repairing damage caused by the seed ripening stages [59,60,61]. Water-soluble inhibitors, such as ABA, are released from the shell or embryo during soaking, which is particularly effective when dealing with dormant seeds [62]. Hydropriming benefits germination by initiating biochemical changes such as hydrolysis, enzyme activation, DNA replication, and increased RNA and protein synthesis. This enhances embryo growth and reduces metabolite leakage by speeding up water absorption [63]. Recommendations for soaking herbaceous peony seeds vary; the European Peony Society [64] suggests 3–4 days at room temperature, while others propose up to 7 days [24], 1 day for P. tenuifolia [65], and 2 days for P. lactiflora [26] and P. emodi [40].
In this study, a 60% maximum total in vitro germination percentage (10% radicle only and 50% complete seedlings) was achieved on the 120th culture day in 250 mg L−1 GA3-supplemented medium, germination onset (only radicle protrusion) occurred on the 48th day (14 d—22 °C and 34 d—15 °C), and simultaneous radicle and sprout emergence onset occurred after 89 days (14 d—22 °C and 75 d—15 °C) (Figure 2; Table S1). These findings align with a previous study where GA3 supplementation led to increased embryo size, rapid hypocotyl dormancy release, and removing barriers associated with the surrounding endosperm [65]. Low temperatures induce ABA accumulation, initiating dormancy, while warm temperatures lead to ABA degradation and increased GA3, promoting dormancy release and germination [66].
The prolonged dormancy and slow in vitro germination of P. peregrina and other Paeonia species are attributed to several factors. These include increased ABA content hindering germination, underdeveloped embryos due to weak differentiation and low fermentation activity, high lignin content in the seed peel preventing water penetration, and low peroxidase enzyme activity [4,34,67]. In the presented germination assay, a 51.43% maximum total germination percentage (28.57% radicle only and 22.86% complete seedlings) was achieved after 110 days in 250 mg L−1 GA3-supplemented medium for 30-day-stored P. peregrina seeds, germination onset (only radicle protrusion) was on the 56th day, and simultaneous radicle and sprout emergence onset occurred on the 110th day (Figure 3; Table S2). Although similar germination percentages were observed on the 120th day for both 20- and 30-day-stored seeds under the same conditions, the percentage of fully developed seedlings (50%) was nearly double in seeds exposed to a shorter period at a higher temperature (22 °C for 14 days) followed by a much more extended period at a lower temperature (15 °C for 106 days) compared to the other alternating temperature regime (Figure 2 and Figure 3; Tables S1 and S2). These differences suggest that the temperature range and exposure duration influence germination rates in P. peregrina, as reported in previous studies [52]. In various Paeonia species, early exposure to warm stratification (15–25 °C) followed by cold stratification (0–10 °C) promotes dormancy breaking and germination [23,52,68]. The same principle applies to P. peregrina, explaining the higher total germination percentage (60%; 102nd day), earlier onset (48th day), and a more significant percentage of fully developed seedlings (50%) under the transition temperature regime with a shorter higher-temperature period followed by a more prolonged lower-temperature period (Figure 2; Table S1). The germinability of peony species depends on multiple factors, including temperature, seed age, genotype, and dormancy-breaking treatments. For instance, fresh, perennial P. corsica seeds exhibit less than a 10% final germination rate even after one week, emphasizing the complex interplay of factors influencing germination [52].
Alternating temperatures or the transition of seeds from a lower to higher constant temperature or vice versa during the whole germination evolutionary culture period have been proposed to be more favorable for germination than only constant temperatures because seeds are subject to frequent variations under natural conditions [34]. The transition of peony seeds herein during their culture from a shorter incubation period (14 days) at a higher temperature (22 °C) to a more extended incubation period (106 days) at lower temperature (15 °C) versus 33 days at 22 °C and 87 days at 15 °C resulted in a higher total germination percentage and germination rate, earlier germination onset day, t50, and day of maximum germination, and a lower mean germination time (MGT), but a lower germination speed index (GSI) and germination energy percentage, and more extended or delayed energy period (Table S6). Temperature, in addition to its impact on germination percentage and germination energy metrics, exerts a substantial role in germination onset day, mean germination speed, and germination vigor [69] as an adaptation mechanism to align with germination time and growth and following the development of the seedlings under auspicious junctures [70]. Seeds stored at higher temperatures have higher metabolic activity, resulting in a lower germination ability, while seeds stored at lower temperatures maintain a relatively higher germination ability due to lowered metabolic activity [71]. Similarly, the storage period is the main factor affecting seed germination [72]. Storage at room temperature often leads to low seed germination, seed deterioration, and loss of viability, which are natural phenomena during storage [73]. The moisture content of seeds is an essential factor influencing their germination percentage during storage, i.e., the moisture content and germination percentage of stored seeds gradually decrease with an increase in storage period [74].
The natural lifespan of peony seeds is around 3 years, but storage conditions significantly affect germination rates [75]. Prolonged germinability is observed with dry storage in a refrigerator or soaking in water, especially in wet refrigeration conditions [76]. This study compared different cold-storage periods as treatments for P. peregrina seeds and showed that 3-month-old seeds exhibited the highest germination percentage of 74.07% after 87 days in 250 mg L−1 GA3-supplemented medium with both radicle and sprout development. There was a negative correlation between seed age and germinability, with 3-month-old seeds showing the most effective germination response (Figure 4; Table S4). Factors like soaking in cold water, gibberellin treatment, and controlled storage conditions are crucial in breaking seed dormancy and enhancing germination rates, especially after long-term conservation [76]. Advancing the cold-storage period of P. peregrina seeds herein led to increases in the mean germination time (MGT) and t50, delays in the germination onset day, and reductions in germination rates (Table S6); therefore, seed viability loss could be ascribed to seed coat features, increased production of carbon dioxide in the glass sealed container due to the trivial exchange of gases and moisture with the external environment, physicochemical seed deterioration due to senescence, lipid peroxidation, the generation of free radicals and reactive oxygen species as toxic byproducts, denaturation of proteins and enzymes, suffocating conditions due to lack of oxygen, and the inability to activate “enzymatic and non-enzymatic” antioxidant defense mechanisms to amend the induced damage [77,78]. Generally, increasing seed age based on the storage period is accompanied by a lower germination energy and germination percentage due to temperature and atmospheric relative humidity shifts occurring during seed storage in containers (i.e., low temperature maintains seed moisture content, with the latter factor controlling all cellular activities); nevertheless, the lower the temperature and/or humidity, the more extended storage is possible [79,80,81,82]. In this study, despite achieving 100% germination for 30-day cold-stratified seeds on the 80th day, 3-month-old seeds under the same conditions demonstrated 74.07% total germination on the 97th day (Figure 4; Table S4), emphasizing that a shorter cold-storage period results in higher and faster germination but delays the onset and appearance of seedlings with both radicle and sprout emergence compared to a more extended storage period. The study highlights the intricate interplay of storage temperature, duration, and seed vigor in cultivated peony species, as evidenced by significant variations in germination rates under different storage conditions as reported in previous studies [83]. Seed storage behavior is a means of determining whether the seeds of a species successfully retain their viability after a long-, medium-, or short-term period [84] to be able to germinate, a reference clue vital for the development of suitable ex situ conservation strategies [85].
Under a constant 15 °C, 100% in vitro germination with radicle protrusion was observed for 30-day cold-stored P. peregrina seeds after 80 days in the medium with the lowest GA3 concentration (Figure 5; Table S5). The positive influence of GA3 on dormancy inhibition and germination acceleration is attributed to factors like cold stratification, increased gibberellin biosynthesis gene activity, replacement of low temperature by GA3, stimulation of seed storage sources, water uptake, altered hormone balance, increased RNA polymerase activity, and enhanced synthesis of hydrolyzing enzymes and proteins [61,63]. Optimal concentrations for radicle emergence in herbaceous peonies were 250–300 mg L−1 GA3 [26,52]. Complete seedling development occurred herein only under 1000 mg L−1 GA3 (Table S5, Figure 4Ae), consistent with previous findings. Exogenous GA3 positively influenced epicotyl germination in herbaceous peonies [86]. For P. peregrina, the most favorable germination percentage outcome (100%) was achieved with 30 days of cold storage followed by 250 mg L−1 GA3 treatment for 80 days at a constant 15 °C (Table S5), surpassing the 51.43–60% germination of the non-cold-storage seeds under the same GA3 concentration treatment but with temperature transition (Tables S1 and S2); thus, combining cold storage with GA3 treatment proved more effective for dormancy release and higher seed germinability than individual treatments. GA3 is reported to be a more potent dormancy-breaking treatment than low-temperature or cold storage [87], as GAs can substitute seed requirements for light, temperature, or cold for germination [88]. Cold storage near 4 °C is considered the most effective for the germination of peony species [89], with wild tree peonies favoring temperatures between 10 and 15 °C [83]. In this study, possible explanations for the better germination metrics of peony seeds cultured in the GA3-supplemented media (15 °C; dark), including an earlier germination onset day and t50, lower mean germination time (MGT), shorter germination energy period, higher germination speed index (GSI), germination rate (GR), and germination energy (GE) percentage, as compared to seeds cultured in GA3-free medium, are the mobilization of storage reserves through enzyme activation caused by GA3 [78], as well as the increase in both proteins and sugars needed for embryo growth, the decrease in seed oil content, and the increase in moisture absorption induced by seed storage [90], in the case of the 3-month cold-stored peony seeds herein prior to their cultivation in GA3 supplemented media. The simultaneous occurrence of higher germination speed index (GSI, as the best indicator of the dormancy depth), higher germination rate (GR), and germination energy (GE) values accompanied by a lower t50 and mean germination time (MGT) values has been positively correlated with better seed germination performance, efficient and uniform germination, and lower viability loss [91,92,93]. Germination percentage (%) is the most critical parameter in determining the suitability of a seed lot for commercial use, but germination rate influences the uniformity of emergence in nurseries [94]. Generally, seeds with higher germination energy percentages and minimum germination energy periods are ideal for producing uniform and high-quality seedlings [95]. In line with our findings, a previous study conducted on P. peregrina, as herein, showed that the immersion of seeds for 2 days in GA3 solution (150–350 mg L−1) accelerated dormancy release and augmented the seed germination rate, being optimized under 200 mg L−1 GA3 treatment [96].
Previous studies have demonstrated the beneficial outcome of various seed dormancy release and germination promotion treatments under in vitro conditions, including warm stratification followed by cold stratification in P. corsica [52] and P. lactiflora [68]; low temperature in P. ostii [10], P. caucasica [66], and P. rockii [87]; GA3 in tree peony species [76], herbaceous peonies [86], P. ostii [65], P. rockii [87], and P. peregrina [96]; soaking in cold water and controlled storage conditions in tree peony species [76]; complete darkness compared to a light/dark photoperiod regime in P. suffruticosa [58]; and temperature and duration of storage in P. rockii and P. szechuanica seeds [83]; as well as hydropriming treatments for dormancy removal and enhancement of in vivo germination for P. veitchii [24], P. lactiflora [25], and P. tenuifolia [65] seeds based on the European Peony Society [64]. Only one study has been carried out in P. peregrina attempting to define the effect of seed immersion in different GA3 concentrations (up to 2 days before culture) on in vitro germination performance [96]; however, in our study on the same species, GA3 did not apply as a short-term presoaking treatment but was exogenously incorporated into the culture medium and maintained through the whole culture period extending from 90 to 140 days. Therefore, the novelty of our findings regarding previous studies on seed germination under similar conditions is the evaluation of different factors either individually or in combination (i.e., constant temperature, transitioning from higher to lower temperature, seed age based on their dry-storage period prior to culturing, seed cold-storage period, a variety of incubation culture periods, GA3 concentrations, and different application methods of GA3) for the first time in this particular herbaceous peony (P. peregrina) that could add depth and relevance to its germination preferences and requirements, which depend on the interplay among multivariate coefficients and constantly change due to alterations occurring in the natural environment prior their collection from mother plants.

5. Conclusions

This study highlights the crucial factors influencing the in vitro germination of Paeonia peregrina seeds, including temperature, incubation period, cold storage duration, and GA3 concentration in a dependent and combinational way. The 60-day storage seeds (30 d at 15 °C, RH 15% and 30 d at 4–5 °C, RH < 5%) showed optimal results with a 100% germination percentage on the 80th day of culture at 15 °C in the dark, under the influence of 250 mg L−1 GA3. The lower and constant temperature of 15 °C was more effective for dormancy release and germination acceleration than the transition from 22 °C to 15 °C. A reverse relationship was observed between germination success and the seed cold-storage period. Additionally, the lowest GA3 concentration of 250 mg L−1 proved the most beneficial.
From the results of this study, it is recommended to use either a short period (from 30 days up to 3 months) of cold storage at 4–5 °C and RH < 5% of P. peregrina seeds before culturing at a constant lower temperature (15 °C) in the case of an available seed gene bank for short-term ex situ conservation or a 20- to 30-day shorter period of seed dry storage at 15 °C and RH 15% in a dehumidifying chamber when no seed gene bank is available and then culture under a transitioning temperature regime simulating the spring or autumn seasonal environmental conditions in nature; nevertheless, the GA3 treatment in both cases is imperative.
Understanding the underlying mechanisms of the seed storage period, temperature, moisture, light, hormones, and various pretreatments for seed dormancy removal and germination enhancement is crucial for the effective propagation, breeding, and cultivation of diverse herbaceous peony species. The upcoming view extending the depth of this study will be the dormancy release and germination enhancement of P. peregrina seeds sown in a peat–vermiculite substrate under in vivo greenhouse conditions, applying the in vitro experimental germination assays described in this research. Another goal is the establishment and development of a complete micropropagation protocol from seed disinfection to ex vitro acclimatization of in vitro plantlets using different plant growth regulators (cytokinins, auxins, and GA3) in a stage-dependent manner. Future perspectives concern the in vitro culture of adventitious roots or hairy roots transformed after Agrobacterium rhizogenes inoculation in bioreactors within liquid nutrient media under the influence of various biotic and abiotic elicitors for the production of secondary metabolites.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/seeds4010007/s1, Table S1: In vitro germination percentage (%) of 20-day dry storage Paeonia peregrina seeds (sealed glass containers; 15 °C; 15% RH) as evolved within a 120-day total incubation period in the dark while changing the temperature from 22 °C in the first 14 days to 15 °C for the following 106 days. Table S2: In vitro germination percentage (%) of 30-day dry storage Paeonia peregrina seeds (sealed glass containers; 15 °C; 15% RH) as evolved within a 120-day total incubation period in the dark while changing the temperature from 22 °C in the first 33 days to 15 °C for the following 87 days. Table S3: Effect of seed cold-storage period at 4–5 °C and RH < 5% (8 years, 5 years, 2 years, and 3 months) on the number and percentage of seeds with infections (fungi, bacteria, and fungi plus bacteria) and without infections, after 140 days of in vitro culture in the dark at 15 °C on MS medium enriched with 20 g L−1 sucrose, 250 mg L−1 GA3 (pH 5.8), and 6 g L−1 Plant Agar in Paeonia peregrina. Table S4: Development course of in vitro germination of Paeonia peregrina within 140 days culture (15 °C; dark) in MS medium with 20 g L−1 sucrose, 250 mg L−1 GA3 (pH 5.8), and 6 g L−1 Plant Agar using seeds of different cold (4–5 °C; RH < 5%) storage periods (8 years, 5 years, 2 years, and 3 months). Table S5: In vitro germination percentage (%) of 60-day storage Paeonia peregrina seeds, the first 30 days in a dehumidification chamber (15 °C; 15% RH) and the next 30 days in a refrigerator (4–5 °C; RH < 5%; cold storage), after 90 days of culture in MS medium with 20 g L−1 sucrose, 6 g L−1 Plant Agar, pH 5.8, and different GA3 concentrations (0, 250, 500, 750, and 1000 mg L−1) (15 °C; dark). Table S6: In vitro maximum total germination (only radicle and both radicle plus sprout) percentage (%), day of maximum germination percentage, germination rate (GR) (%), germination onset day, mean germination time (MGT) (in days), germination speed index (GSI), germination energy percentage (GE %), germination energy period (in days), and t50 value (i.e., the time needed to achieve the 50% of total germination percentage) of Paeonia peregrina seeds under the different experimental conditions (1st, 2nd, 3rd, and 4th assays) described in this study.

Author Contributions

Conceptualization, V.S. and K.G.; methodology, V.S. and K.G.; software, V.S.; validation, V.S. and K.G.; formal analysis, V.S.; investigation, V.S.; resources, E.M. and K.G.; data curation, V.S.; writing—original draft preparation, V.S.; writing—review and editing, V.S. and K.G.; visualization, V.S.; supervision, K.G.; project administration, K.G.; funding acquisition, K.G. and E.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the European Regional Development Fund of the European Union and Greek National Funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH—CREATE—INNOVATE (project code: Τ2EΔΚ-02927, MIS 5069915) entitled “Development and optimization of in vitro culture methods in bioreactors to produce repeatable and excellent quality plant material for extracts used in food supplements and cosmetics” (Acronym: BIOREACT).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article or Supplementary Materials. The original contributions presented in the study are included in the article/Supplementary Materials, and further inquiries can be directed to the corresponding author.

Acknowledgments

The authors acknowledge the permanent staff of the Institute of Plant Breeding and Genetic Resources of ELGO-DIMITRA for providing administrative and technical support during the experiments’ implementation.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Appendix A

Seeds 04 00007 g0a1
Figure A1. Evolution course and progression stages of in vitro germination [120 days: 22 °C (14 days) and 15 °C (0–106 days)] of Paeonia peregrina 20-day-stored seeds (15 °C; 15% RH) (1st assay): (a) breaking of the top of the hard outer seed coat and radicle protrusion; (b) radicle expansion and sprout emergence; (c) establishment of whole plants with simultaneous radicle and sprout development.
Figure A1. Evolution course and progression stages of in vitro germination [120 days: 22 °C (14 days) and 15 °C (0–106 days)] of Paeonia peregrina 20-day-stored seeds (15 °C; 15% RH) (1st assay): (a) breaking of the top of the hard outer seed coat and radicle protrusion; (b) radicle expansion and sprout emergence; (c) establishment of whole plants with simultaneous radicle and sprout development.
Seeds 04 00007 g0a1
Seeds 04 00007 g0a2
Figure A2. Evolution course and progression stages of in vitro germination [120 days: 22 °C (33 days) and 15 °C (0–87 days)] of Paeonia peregrina 30-day-stored seeds (15 °C; 15% RH) (2nd assay): (a) breaking of the top of the hard outer seed coat and radicle protrusion; (b) radicle expansion and sprout emergence; (c) establishment of whole plants with simultaneous radicle and sprout development.
Figure A2. Evolution course and progression stages of in vitro germination [120 days: 22 °C (33 days) and 15 °C (0–87 days)] of Paeonia peregrina 30-day-stored seeds (15 °C; 15% RH) (2nd assay): (a) breaking of the top of the hard outer seed coat and radicle protrusion; (b) radicle expansion and sprout emergence; (c) establishment of whole plants with simultaneous radicle and sprout development.
Seeds 04 00007 g0a2
Seeds 04 00007 g0a3
Figure A3. In vitro germination of Paeonia peregrina (15 °C; dark) in MS medium with 20 g L−1 sucrose, 250 mg L−1 GA3 (pH 5.8), and 6 g L−1 Plant Agar using seeds of different cold (4–5 °C; RH < 5%) storage periods (8 years, 5 years, 2 years, and 3 months): (a) development of complete seedling from a 3-month-storage seed after 87 days of culture; (b) radicle protrusion and/or sprout emergence using 8-, 5-, and 2-year, and 3-month cold-stored seeds, after 140 days of culture.
Figure A3. In vitro germination of Paeonia peregrina (15 °C; dark) in MS medium with 20 g L−1 sucrose, 250 mg L−1 GA3 (pH 5.8), and 6 g L−1 Plant Agar using seeds of different cold (4–5 °C; RH < 5%) storage periods (8 years, 5 years, 2 years, and 3 months): (a) development of complete seedling from a 3-month-storage seed after 87 days of culture; (b) radicle protrusion and/or sprout emergence using 8-, 5-, and 2-year, and 3-month cold-stored seeds, after 140 days of culture.
Seeds 04 00007 g0a3
Seeds 04 00007 g0a4
Figure A4. In vitro germination of 60-day-stored seeds (30 d, 15 °C, 15% RH and 30 d, 4–5 °C, RH < 5%) of Paeonia peregrina after 80 days of culture (15 °C; dark) in MS medium with 20 g L−1 sucrose and 6 g L−1 Plant Agar, pH 5.8, under different GA3 concentrations: (a) 0 mg L−1; (b) 250 mg L−1; (c) 500 mg L−1; (d) 750 mg L−1; and (e) 1000 mg L−1.
Figure A4. In vitro germination of 60-day-stored seeds (30 d, 15 °C, 15% RH and 30 d, 4–5 °C, RH < 5%) of Paeonia peregrina after 80 days of culture (15 °C; dark) in MS medium with 20 g L−1 sucrose and 6 g L−1 Plant Agar, pH 5.8, under different GA3 concentrations: (a) 0 mg L−1; (b) 250 mg L−1; (c) 500 mg L−1; (d) 750 mg L−1; and (e) 1000 mg L−1.
Seeds 04 00007 g0a4

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Figure 1. (a) Full red-colored blossom mother plants of Paeonia peregrina in the soil during summer; (b) Ripen pericarps in the mother plant; (c) Desiccation of mature pericarps and breaking the capsules for seed release; (d) Collected seeds of different maturity level based on color; (e) Mature black-colored seeds for experimentation; (f) Seed size (0.6–0.9 cm length × 0.5 cm width × 0.3 cm thickness).
Figure 1. (a) Full red-colored blossom mother plants of Paeonia peregrina in the soil during summer; (b) Ripen pericarps in the mother plant; (c) Desiccation of mature pericarps and breaking the capsules for seed release; (d) Collected seeds of different maturity level based on color; (e) Mature black-colored seeds for experimentation; (f) Seed size (0.6–0.9 cm length × 0.5 cm width × 0.3 cm thickness).
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Figure 2. In vitro germination percentage (%) of Paeonia peregrina seeds after 20 days of storage (15 °C; 15% RH) within a total culture period of 120 days in the dark and transition temperature from 22 °C (the first 14 days) to 15 °C (for another 0, 34, 39, 43, 50, 57, 67, 75, 81, 88, 92, and 106 days) corresponding to 14, 48, 53, 57, 64, 71, 81, 89, 95, 102, 106, and 120 days of total culture, respectively. Different lowercase letters of the same color per germination percentage (total, only radicle, and radicle and sprout) denote significant differences (one-way ANOVA, Duncan’s test, p ≤ 0.05).
Figure 2. In vitro germination percentage (%) of Paeonia peregrina seeds after 20 days of storage (15 °C; 15% RH) within a total culture period of 120 days in the dark and transition temperature from 22 °C (the first 14 days) to 15 °C (for another 0, 34, 39, 43, 50, 57, 67, 75, 81, 88, 92, and 106 days) corresponding to 14, 48, 53, 57, 64, 71, 81, 89, 95, 102, 106, and 120 days of total culture, respectively. Different lowercase letters of the same color per germination percentage (total, only radicle, and radicle and sprout) denote significant differences (one-way ANOVA, Duncan’s test, p ≤ 0.05).
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Figure 3. In vitro germination percentage (%) of Paeonia peregrina seeds after 30 days of storage in a drying chamber (15 °C; 15% RH) with 24 h-ddH2O hydration pretreatment, within a 120-day total culture period in the dark and transition temperature from 22 °C (the first 33 days) to 15 °C (for another 0, 23, 30, 40, 48, 54, 61, 65, 77, and 87 days) corresponding to 33, 56, 63, 73, 81, 87, 94, 98, 110, and 120 days of total culture, respectively. Different lowercase letters of the same color per germination percentage (total or only radicle or radicle and sprout) denote significant differences (one-way ANOVA, Duncan’s test, p ≤ 0.05).
Figure 3. In vitro germination percentage (%) of Paeonia peregrina seeds after 30 days of storage in a drying chamber (15 °C; 15% RH) with 24 h-ddH2O hydration pretreatment, within a 120-day total culture period in the dark and transition temperature from 22 °C (the first 33 days) to 15 °C (for another 0, 23, 30, 40, 48, 54, 61, 65, 77, and 87 days) corresponding to 33, 56, 63, 73, 81, 87, 94, 98, 110, and 120 days of total culture, respectively. Different lowercase letters of the same color per germination percentage (total or only radicle or radicle and sprout) denote significant differences (one-way ANOVA, Duncan’s test, p ≤ 0.05).
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Figure 4. Evolution course (23–140 days) of in vitro germination percentages (total, only radicle, and radicle and sprout) (%) of Paeonia peregrina (15 °C; dark) seeds in MS medium with 20 g L−1 sucrose, 250 mg L−1 GA3 (pH 5.8), and 6 g L−1 Plant Agar, originated from different cold-storage periods (8 years, 5 years, 2 years, and 3 months). Different blue-colored lowercase letters per the total germination percentage (%); different red-colored lowercase per the germination percentage (%)—only radicle; and different green-colored lowercase letters per germination percentage (%)—radicle and sprout denote significant differences among all 60 combinations: 15 subsequent culture period intervals (23, 30, 35, 43, 48, 55, 62, 73, 80, 87, 97, 104, 115, 122, and 140 days) x 4 seed cold-storage periods (two-way ANOVA, Duncan’s test, p ≤ 0.05).
Figure 4. Evolution course (23–140 days) of in vitro germination percentages (total, only radicle, and radicle and sprout) (%) of Paeonia peregrina (15 °C; dark) seeds in MS medium with 20 g L−1 sucrose, 250 mg L−1 GA3 (pH 5.8), and 6 g L−1 Plant Agar, originated from different cold-storage periods (8 years, 5 years, 2 years, and 3 months). Different blue-colored lowercase letters per the total germination percentage (%); different red-colored lowercase per the germination percentage (%)—only radicle; and different green-colored lowercase letters per germination percentage (%)—radicle and sprout denote significant differences among all 60 combinations: 15 subsequent culture period intervals (23, 30, 35, 43, 48, 55, 62, 73, 80, 87, 97, 104, 115, 122, and 140 days) x 4 seed cold-storage periods (two-way ANOVA, Duncan’s test, p ≤ 0.05).
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Figure 5. Evolution course of in vitro germination percentage (%) of 60-day storage Paeonia peregrina seeds, the first 30 days in a dehumidification chamber (15 °C; 15% RH) and the next 30 days in a refrigerator (4–5 °C; RH < 5%; cold dry storage) within a 90-day total culture period (5, 15, 22, 29, 41, 49, 55, 62, 66, 80, and 90 days) in MS medium with 20 g L−1 sucrose, 6 g L−1 Plant Agar, pH 5.8, supplemented with different GA3 concentrations (0, 250, 500, 750, and 1000 mg L−1) (15 °C; dark). Different black-colored uppercase letters denote significant differences among all 55 combinations (11 culture periods x 5 GA3 concentrations) (two-way ANOVA, Duncan’s test, p ≤ 0.05). Different lowercase letters of the same color denote significant differences among the 11 subsequent culture period intervals for each GA3 concentration separately (one-way ANOVA, Duncan’s test, p ≤ 0.05).
Figure 5. Evolution course of in vitro germination percentage (%) of 60-day storage Paeonia peregrina seeds, the first 30 days in a dehumidification chamber (15 °C; 15% RH) and the next 30 days in a refrigerator (4–5 °C; RH < 5%; cold dry storage) within a 90-day total culture period (5, 15, 22, 29, 41, 49, 55, 62, 66, 80, and 90 days) in MS medium with 20 g L−1 sucrose, 6 g L−1 Plant Agar, pH 5.8, supplemented with different GA3 concentrations (0, 250, 500, 750, and 1000 mg L−1) (15 °C; dark). Different black-colored uppercase letters denote significant differences among all 55 combinations (11 culture periods x 5 GA3 concentrations) (two-way ANOVA, Duncan’s test, p ≤ 0.05). Different lowercase letters of the same color denote significant differences among the 11 subsequent culture period intervals for each GA3 concentration separately (one-way ANOVA, Duncan’s test, p ≤ 0.05).
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Sarropoulou, V.; Maloupa, E.; Grigoriadou, K. Exploring the Morpho-Physiological Dormancy and Germination Potential of Paeonia peregrina Mill. Seeds In Vitro. Seeds 2025, 4, 7. https://doi.org/10.3390/seeds4010007

AMA Style

Sarropoulou V, Maloupa E, Grigoriadou K. Exploring the Morpho-Physiological Dormancy and Germination Potential of Paeonia peregrina Mill. Seeds In Vitro. Seeds. 2025; 4(1):7. https://doi.org/10.3390/seeds4010007

Chicago/Turabian Style

Sarropoulou, Virginia, Eleni Maloupa, and Katerina Grigoriadou. 2025. "Exploring the Morpho-Physiological Dormancy and Germination Potential of Paeonia peregrina Mill. Seeds In Vitro" Seeds 4, no. 1: 7. https://doi.org/10.3390/seeds4010007

APA Style

Sarropoulou, V., Maloupa, E., & Grigoriadou, K. (2025). Exploring the Morpho-Physiological Dormancy and Germination Potential of Paeonia peregrina Mill. Seeds In Vitro. Seeds, 4(1), 7. https://doi.org/10.3390/seeds4010007

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