Effect of cold stratification on the temperature range for germination of Pinus koraiensis

Germination at low spring temperatures may offer a competitive advantage for the growth and survival of plant species inhabiting temperate forest ecosystems. Pinus koraiensis is a dominant species in temperate forests of northeastern China. Its seeds exhibit primary morphophysiological dormancy following dispersal in autumn, limiting natural or artificial regeneration: direct seeding and planting seedlings in spring. The aim of this study was to determine the optimum cold stratification temperature that induces germination to increase towards lower temperatures. Seeds from two populations (Changbaishan and Liangshui) were cold stratified at 0, 5 and 10 °C. Germination to incubation temperatures (10/5, 20/10, 25/15 and 30/20 °C; 14/10 h day/night) were determined after 2 and 4 weeks, and 5.5 and 6.5 months of cold stratification. After 5.5 months, approximately 68–91% of seeds from both populations germinated at incubation temperatures of 25/15 °C and 30/20 °C, regardless of cold stratification temperatures. When the cold stratification temperature was reduced to 0 °C and the period increased to 6.5 months, germination at 10/5 °C significantly improved, reaching 37% and 64% for the Changbaishan and Liangshui populations, respectively. After 6.5 months of cold stratification, there was a significant linear regression between cold stratification temperatures and germination at 10/5 °C. The range in temperatures allowing for germination gradually expanded to include lower temperatures with decreasing cold stratification temperatures from 10 to 5 °C and further to 0 °C.


Introduction
Seed germination is a critical process throughout a plant's life cycle, playing a critical role in the establishment of populations (Donohue et al. 2010). Germination during spring or early summer in temperate regions allows seedlings to reach maturity, thus avoiding frost damage during winter (Vandelook et al. 2009). The ability to germinate at low temperatures is essential for efficient and successful regeneration of temperate forest species during spring (Schütz 1997).
Following seed dispersal, treatments that simulate natural overwintering conditions can release the seeds from dormancy (Chien et al. 2011;Fernández-Pascual et al. 2015. The seeds of tree species and spring-germinating Abstract Germination at low spring temperatures may offer a competitive advantage for the growth and survival of plant species inhabiting temperate forest ecosystems. Pinus koraiensis is a dominant species in temperate forests of northeastern China. Its seeds exhibit primary morphophysiological dormancy following dispersal in autumn, limiting natural or artificial regeneration: direct seeding and planting seedlings in spring. The aim of this study was to determine the optimum cold stratification temperature that induces germination to increase towards lower temperatures. Seeds from two populations (Changbaishan and Liangshui) were cold stratified at 0, 5 and 10 °C. Germination to incubation temperatures (10/5, 20/10, 25/15 and 30/20 °C; 14/10 h day/ summer annuals and perennials in temperate regions require stratification at cold temperatures (1-10 °C) to break dormancy (Deng et al. 2016;Finch et al. 2019;Chen et al. 2020). The range of temperatures usually used for stratification of seeds of some pine species is between 2 and 5 °C Bower et al. 2011;Saeed et al. 2016;Houšková et al. 2021), thus allowing germination to occur at temperatures above 15 °C. However, whether these cold stratified pine seeds can germinate at temperatures below these ranges, thereby increasing the range of germination temperatures, is generally unknown. An increase in the length of the cold stratification period was found to improve seed germination in several woody plant species (Tang and Tian 2010;Farhadi et al. 2013;Porceddu et al. 2013;Al-Hawija et al. 2014) and in herbs (Carta et al. 2016;Bandara et al. 2019;Soltani et al. 2019) under low temperature conditions. In addition, a reduction in cold stratification temperature may also lower the base temperature for germination, as reported for Aesculus hippocastanum L. seeds by Steadman and Pritchard (2004). However, few studies have considered the effects of different cold stratification temperatures on the range of germination temperatures of pine species. If a cold stratification temperature of 2-5 °C is insufficient, a lower temperature may be able to expand the range in germination temperatures of pine seeds to include lower temperatures. However, this has not been previously investigated.
The ceiling temperature for successful cold stratification is 10 °C but is rarely used compared to 5 °C. Some studies suggest that the effects of higher cold stratification temperatures (up to 10 °C) on breaking seed dormancy differ depending on the species. For example, cold stratification at 9 °C was more effective than 4 °C in breaking seed dormancy in Abies nordmanniana (Stevan) Spach (Kirdar and Ertekin 2008). However, for several herbs, 5 °C was more effective than 10 or 11 °C (Brändel 2005;Chen et al. 2020). Thus, considering the interspecific differences in cold stratification temperature requirements, determining the specific effects of increasing cold stratification temperatures on the range in germination temperatures of pine seeds in temperate regions is needed. In addition, cold-adapted species with limited dispersal ability and obligate seeding (Bell 2001;Bond and Midgley 2001) are more likely to be threatened by climate change (Vaughn et al. 2022). The current scenario of global change may affect plant distribution and survival through early life stage processes (Walck et al. 2011;Ferreira et al. 2022). Since germination is the first step in producing plants (Kaye et al. 2018), seedlings are extremely vulnerable to climate change (Cochrane et al. 2011). For spring-germinating summer annuals and perennials of temperate ecosystems, germination and subsequent seedling growth are largely determined by low temperatures following seed dispersal under natural conditions. Increasing warmer winter temperatures under climate warming will likely have a strong impact on the breaking of seed dormancy. Data on the effects of increasing cold stratification temperatures (e.g., to 10 °C) on the range in germination temperatures would contribute to predicting the potential responses of populations to future global climate change.
Pinus koraiensis Sieb. et Zucc., commonly known as Korean pine, is an evergreen species of the Pinaceae family (Editorial Committee of Flora of China, Chinese Academy of Sciences 1978). Korean pines are slow growing and produce seeds at the age of 15 years (Han et al. 2008;Ning et al. 2022). The seeds ripen in late September and early October and are approximately 1.6 cm long × 1.0 cm wide. Mixed-broadleaved Korean pine forests cover a large area between northeastern China and the Russian Far East, and Korean pine is a dominant species of these forests, playing a key role in maintaining ecosystem functions (Wu et al. 2004). Large-scale industrial logging and disturbance by other anthropogenic activities have resulted in smaller, fragmented areas of these (Tian et al. 2009). More than 60% of the original mature mixed-broadleaved Korean pine forests have been changed to secondary forests and other land-use types (Li et al. 1997). Therefore, measures to restore this forest ecosystem are necessary. Determining the key limiting factors during germination and regeneration of Korean pine is crucial for improving the future viability of these forests, especially under the current scenarios of global change. Upon dispersal in autumn, fresh Korean pine seeds exhibit an embryo-based primary morphophysiological dormancy (MPD), which impedes subsequent germination in early spring (Song et al. 2018(Song et al. , 2022. Although the results of a previous study on Korean pine seeds suggest that cold stratification at both 1 °C and 5 °C could improve germination at 25/16 °C (Song et al. 2020), the germination capacity of cold stratified seeds at low temperatures remains unclear. The present study investigates the effects of three cold stratification temperatures on the range in temperatures for germination of Korean pine seeds, including 0 °C (lower cold stratification temperature), 5 °C (generally used cold stratification temperature) and 10 °C (relatively higher cold stratification temperature). The hypothesis was tested that the range in germination temperature gradually expands to include colder temperatures as cold stratification temperatures are decreased to 0 °C. In addition, whether the germination performance would be affected by an increased cold stratification temperature of 10 °C was determined.

Seed collection
A pool of freshly fallen seeds from at 50 plants was randomly selected from two populations of Korean pine from Changbaishan (CBS) and Liangshui (LS) in late September 2019. The natural environment characteristics of the sampling sites are shown in Fig. 1. After collection, the seeds were stored at − 20 °C to maintain dormancy and then used for subsequent cold stratification and initial germination tests. Before storage at − 20 °C, the viability of the seeds from the two populations was determined using a tetrazolium test (Moore 1966). Seed viability tests were performed on three replicates of approximately 100 seeds each. Seeds without seedcoats were stained with 1% 2,3,5 tetrazolium chloride (TTC) for 24 h at 30 °C in the dark. Embryos stained red by TTC were considered viable.

Cold stratification treatment
On November 5, 2019, seeds were removed from the − 20 °C storage and kept at room temperature (approximately 15 °C) for several hours. Afterwards, the seeds were soaked in water for 7 days at room temperature, and the water changed every day. On November 12, 2019, the imbibed seeds were cold stratified at 0, 5, and 10 °C. Four sampling times, 2 weeks, 4 weeks, 5.5 months, and 6.5 months, were set for each cold stratification temperature. For each combination treatment of cold stratification temperature and sampling time, there were two replicates of 150 seeds each. Each 150 seed batch was mixed with moist sand, and each mixture of seeds and sand placed in a bag of parafilm. Each seed bag corresponded to one replicate, for a total of 24 seed bags and placed in three refrigerators. The temperature of each refrigerator was set at 0, 5, and 10 °C for the three cold stratification treatments. For the germination tests, two bags of seeds were removed from each refrigerator after 2 weeks, 4 weeks, 5.5 months, and 6.5 months.

Germination tests
Laboratory germination tests were conducted in a growth chamber (MGC-350BP-2; Bluepard, Shanghai, China). The temperature regimes included four alternating daily treatments: 10/5 °C (representing mid-spring/late-autumn), 20/10 °C (late-spring/early-autumn), 25/15 °C (early-summer) and 30/20 °C (mid-summer). Each alternating regime had a high and low temperature interval coupled to a 14-h light and 10-h dark period, respectively. For each test temperature, three Petri dishes were prepared in triplicates. Twenty seeds were placed in each 10-cm-diameter Petri dish on filter paper moistened with 12 mL of deionized water. The Petri dish was sealed with breathable parafilm to prevent desiccation and incubated at each germination test temperature. During incubation, the number of germinated seeds with radicle protrusion was counted every two days, and removed from the Petri dish. At the end of the experiment, the viability of non-germinated seeds was determined using a tetrazolium test. Viable seeds were used to calculate seed ge r m i n a t i o n p e r c e n t a ge . Germination percentage = Total number of germinated seeds Total number of germinated seeds and viable seeds × 100% . The mean germination time was calculated according to Ellis and Roberts (1981). Before storage at − 20 °C, the germination percentages of fresh seeds at the four germination test temperatures were determined.

Statistical analysis
The interactive effects of cold stratification temperatures and periods and incubation temperatures on germination percentage and mean germination time were tested using a general linear model of analysis of variance. The germination percentage data was square root transformed before statistical analysis to ensure homogeneity of variance. If there was a significant interaction, an independent-samples T test was used to test the difference in germination percentage and mean germination time between different cold stratification temperatures, cold stratification periods, and incubation temperatures. The linear regression relationship between cold stratification temperatures and germination percentage at each incubation temperature was analyzed for each seed population. Data analyses were performed using IBM SPSS Statistics 19.0 (Chicago, IL, USA).

Germination percentage of cold stratified seeds
Cold stratification temperatures and periods, and incubation temperatures had significant interactive effects on the germination percentage and mean germination time of CBS populations seeds and the germination percentage of the LS population seeds (Table 1).
Stratification at 10 °C for two weeks significantly reduced the germination percentage of CBS population seeds at 25/15 °C compared to stratification at 0 °C (Fig. 2a, c). Otherwise, cold stratification temperatures did not significantly affect subsequent seed germination from the CBS populations at 25/15 °C and 30/20 °C. When stratified seeds from the CBS population were incubated at 10/5 °C and 20/10 °C, stratification at 0 °C was comparable to 5 °C or 10 °C and lasting for 6.5 months and significantly improved germination percentage to 37% and 63%, respectively ( Fig. 2a, b, c). Seeds from the LS population stratified at 10 °C for 6.5 months showed a significantly lower germination at 25/15 °C compared with seeds that underwent the same period of stratification at 0 °C (Fig. 2d, f). However, germination of seeds from the LS population at 20/10 °C and 30/20 °C was not significantly changed by variations in cold stratification temperatures (Fig. 2d, f). Germination of seeds from the LS populations at 10/5 °C significantly increased to approximately 64% after a treatment of 6.5 months of cold stratification at 0 °C compared to 5 °C or 10 °C (Fig. 2d,e,f).
As the stratification period at each cold temperature was extended to 5.5 months, germination increased at the four germination test temperatures (Fig. 2). After 6.5 months, cold stratification did not significantly (P > 0.05) improve germination at 20/10, 25/15 and 30/20 °C.
After the seeds were cold stratified for different periods at three different cold temperatures, they germinated at a significantly lower percentage at 10/5 °C than at 25/15 °C and 30/20 °C (Fig. 2). The exceptions are seeds from the CBS and LS populations which were cold stratified at 0 °C for 6.5 months, for which there was no significant difference in germination between 10/5 °C and 30/20 °C (Fig. 2a, d).

Mean germination time
Following transfer to 10/5, 25/15 and 30/20 °C incubation conditions, mean germination times of seeds from both populations were not significantly changed by cold stratification over 6.5 months at 10 °C compared to 0 °C (Fig. 3). There was a clear decline in mean germination times of seeds from both populations at the four germination temperatures after 5.5 months of cold stratification at all three cold stratification temperatures. When seeds of CBS or LS populations exposed to 0 °C, 5 °C or 10 °C of cold stratification were incubated at 25/15 °C and 30/20 °C, their mean germination times were significantly reduced to 16-19 days and 18-24 days, respectively (Fig. 3).
For seeds from both populations, higher mean germination times were obtained for germination temperature of 10/5 °C (Fig. 3).

Relationship between cold stratification temperature and germination
After 5.5 and 6.5 months of cold stratification, there was a significant linear relationship between cold stratification temperature and germination for seeds from the CBS population incubated at 10/5 °C (Fig. 4c, d). Cold stratification temperature significantly affected germination at 20/10 °C of CBS population seed only after 6.5 months Capital letters indicate differences in significance (P < 0.05) among incubation temperatures under the same cold stratification temperature and period. Lowercase letters indicate differences in significance (P < 0.05) among cold stratification temperatures under the same incubation temperature and cold stratification period. Error bars represent one standard error of cold stratification (Fig. 4h). For the LS population, a linear relationship between cold stratification temperature and germination at 10/5 °C was also found for seeds kept for 6.5 months of cold stratification (Fig. 5d). At relatively higher incubation temperatures (25/15 °C and 30/20 °C), seeds from the CBS and LS populations did not show significant relationships between cold stratification temperatures and germination irrespective of length of stratification (Figs. 4, 5), except for seeds from the CBS population that were cold stratified for two weeks and germinated at 25/15 °C (Fig. 4i), seeds from the LS population that were cold stratified for 6.5 months and germinated at 25/15 °C (Fig. 5l), and seeds from the LS population that were cold stratified for four weeks and germinated at 30/20 °C (Fig. 5n).

Discussion
Korean pine seeds do not germinate immediately after dispersal from the parent tree. They require a period of cold stratification to acquire germination capacity. For temperate species, cold stratification is a standard procedure for breaking dormancy (Yao and Shen 2018;Tang et al. 2021;Chen et al. 2022). Based on the laboratory data, germination of Korean pine seeds at the four germination temperatures gradually increased over the extended cold stratification period, regardless of cold stratification temperatures. In fact, studies have shown, for example, that cold stratification had a beneficial effect on subsequent germination in A. hippocastanum L. seeds, P. brutia Te. seeds, Pistacia vera L. seeds and Juniperus polycarpos (K. Koch) seeds (Skordilis and Thanos 1995;Pritchard et al. 1999;Daneshvar et al. 2016;Einali and Valizadeh 2017). The longer the treatment, the greater the effect. The results of this investigation show that the range in germination temperatures of Korean pine seeds gradually increased towards 10/5 °C with an increasing 0 °C cold stratification period. Therefore, it is suggested that 6.5 months of stratification at 0 °C could improve germination at a wider range of temperatures than stratification at 5 °C and 10 °C. Based on the laboratory germination results of Korean pine seeds at low temperatures (10/5 °C and 20/10 °C), it may be concluded that the dormancy of Korean pine seeds was successfully broken by cold stratification at 0 °C, whereas Relationship between cold stratification temperature and germination percentage of Korean pine seeds from the Changbaishan population at each germination temperature and cold stratification period 5 °C was the least effective, and 10 °C the least successful. The broadening of the range of germination temperatures induced by longer periods of cold stratification and lower temperatures is consistent with results obtained for recalcitrant seeds of A. hippocastanum (Steadman and Pritchard 2004). The excellent low temperature germination (10/5 °C) of cold stratified seeds from the LS population indicates that Korean pine seeds from higher latitudes may be more adaptable to cold stratification. Similarly, for recalcitrant seeds of Acer saccharum Marsh. (sugar maple) those originating from the northern range, compared to seeds from the southern range, had higher germination at lower temperatures after two months of stratification at 3 °C (Solarik et al. 2016). The seeds of Betula pendula Roth (silver birch) from higher latitudes also germinated well at lower temperatures following a 21-day cold stratification at 4 °C (Midmore et al. 2015). Similar results have not yet been reported for pine species in China. The germination requirement of Korean pine seeds at low temperatures was satisfied by long periods of cold stratification at low temperatures. This response favors seed germination in spring, reflecting a climate-adapted strategy. Moreover, in temperate climates, seeds require low temperatures to break dormancy, a physiological mechanism that has been observed in summer annuals and most temperate perennials (Finch-Savage and Footitt 2017;Blandino et al. 2022). In natural environments, such a cold stratification requirement can prevent germination during autumn conditions that are Relationship between cold stratification temperature and germination percentage of Korean pine seeds from the Liangshui population at each germination temperature and cold stratification period only temporarily suitable for seedling establishment, thus coordinating seedling emergence with favorable seasons and preventing frost damage to seedlings (Wang et al. 2017). Since seedling emergence and establishment are the most vulnerable life phases for plants, breaking dormancy following winter ensures species survival as a seed.
Korean pine seeds have MPD upon dispersal (Song et al. 2022). Common forest nursery practice is to place fresh Korean pine seeds at low temperatures for approximately six months to break the physiological dormancy part of MPD (Sun et al. 1994;Kong et al. 2002;Zhang 2011;Song and Zhu 2016). These moist seeds, which are already nonphysiological dormant, are exposed to higher temperature conditions or in the sun (warm stratification) to release the morphological dormancy part of MPD (Sun et al. 1994;An et al. 2003;Sun 2012). There are two shortcomings in the method of MPD release in Korean pine seeds. First, if the temperature increases because of poor air movement under cold stratification, the seeds are likely to decay. Second, if the moisture content of the seed is not well regulated during warm stratification, the time of morphological dormancy release would be asynchronous, such as some seed coats may crack prematurely, whereas others remain intact. Hence, finding better methods is important, not only to prevent seed decay during cold stratification and pre-germination during warm stratification, but also to ensure seed germination at low temperatures. The results of the present study show that all three cold stratification temperatures broke physiological dormancy and improved germination at high temperatures. However, the germination performance at 10/5 °C was only improved by cold stratification at 0 °C for 6.5 months. Thus, approximately 6.5 months of stratification of moist Korean pine seeds at 0 °C is recommended to break physiological dormancy in the nursery. In addition, a lower cold stratification temperature of 0 °C did not result in Korean pine seed decay. After Korean pine seeds are released from physiological dormancy at 0 °C, they can be directly placed in low temperature conditions to initiate germination. Exposure to relatively higher temperature conditions (e.g., sun) is thus avoided to prevent pre-germination during this period. Seed germination of Korean pine at low temperatures in early spring could result in sufficient seedling growth to obtain a competitive advantage for survival and establishment. Together, cold stratification at 0 °C for 6.5 months could be considered as an alternative method for raising Korean pine seedlings in nurseries.
Under the scenario of global change, a rise in temperature may affect dormancy dynamics in seeds of temperate species (Hoyle et al. 2013;Vaughn et al. 2022). The impact of climate warming on seed dormancy is especially important for seeds that require cold stratification to break dormancy for germination. According to meteorological data over the past 50 years , the rate of temperature increase has been fastest in northeastern China (Ju et al. 2007). Under projected climate scenarios, there may be a 1.2-2.7 °C increase in minimum winter temperatures from 2021 to 2030 across northeastern China (Jiang et al. 2004). Moreover, this region has a greater rise in temperature during winter than summer. Thus, Korean pine seed dormancy release, subsequent seed germination and seedling establishment could be strongly affected by increasing winter temperatures. The main finding of the present study was that cold stratification at 0 °C facilitated Korean pine seed germination at low temperatures, germination capacity gradually decreased with increasing cold stratification temperatures from 0 to 5 °C, and further to 10 °C. Thus, the loss of dormancy of Korean pine seeds in natural environments through overwintering may be less effective if winter temperatures increase. However, this speculation was based only on two seed populations. Moreover, the cold stratification experiment was carried out with seeds collected only in one year. Further studies on germination performance in relation to cold stratification temperatures are necessary to reasonably predict population dynamics in a changing climate.

Conclusions
Germination of Korean pine seeds at low temperatures gradually increased with decreasing cold stratification temperatures from 10 to 5 °C and further to 0 °C. The temperature range gradually expanded to include lower temperatures with decreasing cold stratification temperatures. A temperature of 0 °C for 6.5 months could be considered as an alternative method for culturing Korean pine seedlings in nurseries. The loss of dormancy of Korean pine seeds in natural environments through cold overwintering may be less effective in the context of increasing winter temperatures in northeastern China.