Non-deep physiological dormancy and germination characteristics of Primula florindae (Primulaceae), a rare alpine plant in the Hengduan Mountains of southwest China

Introduction

The timing of germination is a crucial event in the life cycle of a plant, and it significantly influences the growth and survival of seedlings (Donohue et al., 2010). This phenomenon is especially true in alpine regions, where seedlings must attain a critical size by the end of the short growing period to be able to survive the long, harsh winter (Wang et al., 2017). Thus, alpine plants have evolved a series of mechanisms to prevent the germination of freshly shed seeds in autumn and to allow seeds to germinate at the first opportunity in spring, so that seedlings grow under optimal environmental conditions and maximize the length of the growing season (Jaganathan, Dalrymple & Liu, 2015). Several mechanisms control the timing of germination, including seed internal (e.g., dormancy) and external (e.g., temperature, moisture, and light) factors, with seed dormancy being a major mechanism (Finch-Savage & Leubner-Metzger, 2006).

Seed dormancy prevents germination in a specified period, under any combination of environmental factors that otherwise favor germination for non-dormant seeds (Baskin & Baskin, 2004). Seed dormancy is a very common phenomenon in arctic-alpine plant species, and more than 70% of them produce primary dormant seeds, exhibiting mainly physiological dormancy (PD) (Baskin & Baskin, 2014; Schwienbacher et al., 2011). Its production can prevent seed germination during cold autumn/winter seasons and also be seen as an environment that is not long enough to support seedling growth (Ndihokubwayo, Thang & Cheng, 2016). Seeds with PD have fully developed embryos that have a physiological inhibiting mechanism (high abscisic acid (ABA)/gibberellic acid (GA) ratio) in the freshly shed seeds (Baskin & Baskin, 2014), which prevents germination at any temperature or allows it only at high temperatures (conditional dormancy). ABA induces dormancy during ripening, and GA plays an important role in the liberation of dormancy and the promotion of germination (Kucera, Cohn & Leubner-Metzger, 2005), so it demonstrated that artificial addition of gibberellic acid-3 (GA₃) could break PD in many plants (Baskin & Baskin, 2014). Further, PD is classified into two subclasses and seven levels, non-deep PD (Level 1) is by far the most common, which also includes two sublevels and six types (Baskin & Baskin, 2021). Non-deep PD seeds can be released dormant when they are dry-stored at room temperature, a phenomenon researchers called after-ripening (DAR) (Baskin & Baskin, 2020). Cold-wet stratification (CS) is also regarded to be an important way to release non-deep PD, especially in seeds of most alpine plants (Fernandez-Pascual, Jimenez-Alfaro & Bueno, 2017; Peng et al., 2021; Shimono & Kudo, 2003; Yang et al., 2020), because this is similar to winter cryogenic stimulation seeds that can induce dormant broken seeds to germinate in spring (Margreiter et al., 2020). To some extent, as a mediate, CS can promote GA biosynthesis via enhanced expression of some specific transcription factors (Penfield et al., 2005). As dormancy-break progresses during cold stratification, the ABA/GA ratio decreases (Finch-Savage & Leubner-Metzger, 2006), the range of temperatures at which seeds will germinate, as well as germination rate and percentages, increase (Baskin & Baskin, 2004; Porceddu et al., 2013).

In addition to being dormant and having a temperature requirement, the responses of seeds to light can also control the timing of germination in alpine regions (Zhang et al., 2014). Moreover, the interplay between light requirement and non-deep PD also plays a critical role in regulating the timing of germination (Nur et al., 2014; Jaganathan, Dalrymple & Liu, 2015; Peng et al., 2021). Light requirement prevents germination in deep snow cover or soil and forms a soil seed bank (Milberg, Andersson & Thompson, 2007; Wang et al., 2017), whereas, the light requirement would be substantially reduced during the low winter temperature in the field or simulating the overwintering environment (CS) in the laboratory, accompanied by dormancy-release (Peng et al., 2021; Wang et al., 2017). Non-dormant seeds without a light requirement would germinate simultaneously soon after snow melting in spring. Therefore, the timing of germination is regulated through dormancy mechanisms and a light environment, thereby protecting seedling survival and growth.

Primula spp., a famous alpine flower belonging to the Primulaceae family, consists of approximately 430 species in the world (Richards, 1993). China is the main origin center of Primula with approximately 309 species; most are found in high-elevation regions in the Hengduan Mountains (including eastern Tibet, north-western Yunnan, and south-western Sichuan) (Yang, 2005). Germination of Primula does not generally involve complex dormancy (Baskin & Baskin, 2004). Freshly shed seeds of many alpine Primula species possess non-deep PD and require special temperature and light combination for germination; these dormancy and germination characteristics delay germination until the following spring and represent an advantageous ecological adaptation toward the unpredictable alpine environments (Hitchmough, Innes & Mitschunas, 2011; Peng et al., 2019a; Yang et al., 2020). Primula florindae is an early-flowering perennial forb in subalpine and alpine regions endemic to eastern Tibet, SW China, growing at 2,600 to 4,200 m a.s.l. on streamside, bog margins, wet areas in glades of Picea forests or Rhododendron thickets. It is a wild ornamental plant, having large, yellow umbels. The high development value of P. florindae highlights the increasing need for an investigation of seed germination. Previous studies have focused on reproductive strategies from the flowering to the fruiting stage (Zhang et al., 2017; Zhang et al., 2023; Zhang, 2017); however, few studies have investigated the dormancy and germination of this species.

In this study, we aimed to investigate seed dormancy and germination characteristics of P. florindae. Based on the timing of seed germination of Primula species in the alpine regions (Peng et al., 2019a; Wang et al., 2017; Yang et al., 2020), we hypothesized that the dormancy mechanism and/or environmental factors (e.g., low temperature and dark) prevent freshly shed seeds of P. florindae from germinating after dispersal in autumn, and allow seeds germinate only at the beginning of the growing season in spring. We predicted that winter low temperatures might break dormancy and reduce the high temperature/light requirement for germination, resulting in seed germinating earlier in spring. To test these hypotheses, we asked the following questions: (1) Are freshly shed seeds dormant, and if so, how do dormancy break treatments (GA₃, DAR, and CS) release dormancy? (2) How do temperature and light affect germination in different states of dormancy? Based on the seed dormancy and germination responses to GA₃, temperature, and light measured in the laboratory, we would provide some valuable data for the cultivation of P. florindae.

Materials & Methods

Results

Effect of GA₃ and light condition on germination of fresh seeds

For two populations, germination of fresh seeds in water in light was significantly higher than it was in dark at 15/5 and 25/15 °C (Fig. 1). GA₃ concentration of 20 mg L⁻¹ significantly (p < 0.01) improved germination, except the SJL in light and the LL in dark at 15/5 °C. GA₃ concentration of 200 mg L⁻¹ had the highest germination (>80%) at both temperatures, regardless of light condition; moreover, there was no difference between germination in light and dark (Fig. 1). GLMs showed that germination of fresh seeds was significantly stimulated by GA₃ concentration (p < 0.001), light (p < 0.001) and the interaction between light and GA₃ concentration (p < 0.001), except light × GA₃ concentration at 15/5 °C for LL (Table 2).

Table 2:

GLM analysis of light condition and GA₃ concentrations and their interactions on final germination percentages of Primula florindae (LL and SJL) at two alternating temperatures.

Population code	LL				SJL
Treatments	15/5 °C		25/15 °C		15/5 °C		25/15 °C
	df	p	df	p	df	p	df	p
Light condition (L)	1	<0.000	1	<0.000	1	<0.000	1	<0.000
GA3 concentration (G)	2	<0.000	2	<0.000	2	<0.000	2	<0.000
L×G	2	0.426	2	<0.000	2	<0.000	2	<0.000

DOI: 10.7717/peerj.15234/table-2

Notes:

Extremely significant difference: P < 0.001; No significant difference: P > 0.05.

Effects of temperatures and light condition on germination of fresh seeds

Fresh seeds from the two P. florindae populations germinated well (LL: >60%; SJL: >70%) at the warmer temperatures (20, 25, and 25/15 °C) in light, but only about 40% at 10 and 15/5 °C or even to 0% germination below 10 °C (Fig. 2). Fresh seeds incubated in dark did not germinate as effectively as in light, and germination was lower than 30% or even 0% at the temperature at which the test was performed (1, 5, 15, 25, 5/1, 15/5 and 25/15 °C; Figs. 3 and 4).

Discussion

P. florindae is a perennial herb endemic to the alpine habitat in eastern Tibet, but information on seed dormancy and germination of this species is scarce. Hitchmough, Innes & Mitschunas (2011) reported that surface sowing (a thin layer of soil covering) was sufficient to stimulate germination of P. florindae in dry-stored seed, but they did not provide any information about seed dormancy and germination. In this study, we determined how GA₃, light, temperature, CS, and DAR treatments affect seed germination by conducting a series of laboratory experiments. We found that the autumn-dispersal seeds did not germinate or germinate rarely at low temperatures, especially in dark condition (Figs. 2–4). After overwintering, dormancy release and non-dormant seeds germinate at a wide range of temperatures, regardless of light condition (Fig. 4). Our results showed that freshly-matured seeds with PD and light requirements for germination, synchronized the timing of germination of P. florindae in the alpine habitats to early spring. This finding confirms that P. florindae seeds have PD and supports earlier observations of other alpine species (Peng et al., 2017; Peng et al., 2018; Peng et al., 2019a; Peng et al., 2019b).

In regular PD, there are three levels (non-deep, intermediate, and deep), and there are six types of non-deep PD (Baskin & Baskin, 2021). The dormancy type of P. florindae has been demonstrated in three steps. First, freshly-matured seeds had fully developed embryos, and germination was significantly improved by GA₃ at 15/5 and 25/15 °C (Fig. 1). These results suggested that shed seeds may exhibit PD and supported earlier observations of other Primula species (Baskin & Baskin, 2014; Hitchmough, Innes & Mitschunas, 2011). Second, a period of DAR and CS treatments could break dormancy (Figs. 3 and 4). Thus, these results indicated that seeds appeared to have non-deep PD. Further, fresh seeds could germinate well at higher temperatures but not at lower temperatures. As dormancy break treatments (DAR and CS) in the seed populations continue, seeds gained the ability to germinate over a wide range of temperatures (Figs. 3 and 4), especially decreasing in the minimum temperatures in dark at which seeds could germinate (Figs. 3 and 4), indicating that freshly shed seeds of P. florindae have type 2 non-deep PD (Baskin & Baskin, 2014). Seeds with type 2 non-deep PD first enter conditional dormancy and germinate to high percentages only at high temperatures, and the lowest temperature at which they will germinate decreases as dormancy break progresses (Soltani, Baskin & Baskin, 2017).

Matured seeds of P. florindae with type 2 non-deep PD had high temperatures temperature requirements for germination (Figs. 3 and 4). So, freshly shed seeds likely failed to germinate immediately after dispersal in autumn due to low soil temperatures. However, the timing of germination in this species was regulated by seasonal temperature changes. Non-deep PD in fresh seeds was gradually broken in CS treatments (3 and 6 months) at winter temperatures under snow (1 °C, Fig. 4), resulting that the lowest temperature at which they will germinate decreases in the field during winter (Fig. 4). Therefore, dormancy break at low temperature is completed by late winter, and a high percentage of them germinate in a short period over the widest range of temperatures (Fig. 4) in spring. Non-dormant seeds can germinate soon after snowmelt at the beginning of the short growing season, which maximizes the length of the seedling’s growing season.

In addition to dormancy break at low temperatures, germination of P. florindae seeds also depends on the light condition (Figs. 3 and 4). Hitchmough, Innes & Mitschunas (2011) reported that dry-storage seeds of P. florindae could not be germinated well when sown in the soil at 10 mm depth, indicating that the light requirement prevents seed germination in deep soil. However, we do not know how light requirement change with dormancy broken in P. florindae. Our study showed that fresh seeds incubated in dark did not germinate as well as those in light (Figs. 3 and 4). Dry-storage (DAR) for 3 and 6 months broken seed dormancy and increased seed germination percentages in light, but DAR did not significantly improve germination in dark except at warmer temperatures (25 and 25/15 °C) for LL (Fig. 3 and Table 3). The light requirement for germination varies with the season (Pons, 2000) in many alpine plants with non-deep PD, and it can be reduced by CS treatments (Peng et al., 2021; Shimono & Kudo, 2005). By comparing germination between the daily photoperiod and continuous darkness, we found that the light requirement changed as seeds of P. florindae came out of dormancy. CS treatment for 3 and 6 months (Fig. 4), treatment with >20 mg L⁻¹ GA₃ (Fig. 1) changed the light requirement for germination of P. florindae seeds. Thus, the seeds can germinate in dark but only in spring during a short period of the year (Wang et al., 2017). Moreover, most of the non-dormant seeds (CS seeds) of both populations could germinate in dark, thus P. florindae should not be able to form a large persistent soil seed bank. The similar light sensitivity in the germination of seeds of the two populations may be a result of adaptive selection in species exposed to similar micro-habitat in the alpine regions.

Based on our results, we suggest the following two scenarios for the timing of P. florindae germination in nature. First, at the time of dispersal in autumn (late September-October), seeds of P. florindae can germinate well at relatively warmer (>20 °C) temperatures (Fig. 2). However, temperatures in its high elevation habitat are too low (see Figure 5a in Wang et al. (2017)) for germination. During winter, cold stratification reduces the temperature requirement for germination. Thus, the germination temperature of cold-stratified seeds widens from high to low temperature (Fig. 4). Such temperature requirements for germination would prevent P. florindae seeds germinating in autumn because the high temperature requirement for germination is not being met, and allows them rapidly germination soon after snowmelt in spring, which undoubtedly increases the chance for survival of seedlings during their first establishment year. Second, P. florindae have relatively small seeds (Table 1); there are approximately 1,500 seeds per gram. So, fresh seeds were easily covered by soil or a deep layer of litter and snow. Moreover, fresh seeds had strict light requirement, especially at lower (1−5 °C) temperatures; thus, any seeds in the absence of light could not germinate in autumn (Fig. 3). CS treatments at winter temperatures reduced the light requirement for germination (Fig. 4) and promoted germination of a high number of buried seeds in spring. The ability of P. florindae seeds to germinate over the wide range of temperatures without a strict light germination in early spring ensures a sufficient length of the growing season for seedling recruitment and the plants to grow to a large enough size to survive over winter.

Conclusions

Matured seeds of P. florindae have type 2 non-deep PD and light requirement for germination, which prevent seeds from germinating after dispersal in autumn. Seeds lost dormancy and light requirement for germination in winter temperatures and many of them germinated at low temperatures in spring when temperature, light, and moisture conditions are favorable for seedling establishment and growth, enabling plants to take full advantage of the short, unpredictable alpine growing season. Thus, our hypothesis that seed dormancy and special germination requirement restrict germination to the beginning of the growing season is supported. Moreover, from farm cultivation, it is suggested that seeds germinate following DAR and CS treatments at higher temperatures (20−25 °C) in light conditions.

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