Introduction

External factors (such as habitat destruction, exploitation, and pollution) and internal factors (such as ecophysiological adaption, reproduction, and growth rates) are associated with decreased biodiversity ( Kang et al ., 2010 ). According to International Union for Conservation of Nature (IUCN), 5,727 plant species and 3,325 plant species were listed as endangered and critically endangered, respectively, in 2020. In Korea, a total of 88 species were listed as endangered species in 2020, with 11 species listed as endangered I and 77 listed as II (National Institute of Biological Resources ( NIBR), 2021 ). There are four endangered Iris species native to Korea. Among these, Iris dichotoma Pall. is an endangered species distributed on the Daechung Island and islands off the west coast of Korea. Presently, as this species and its habitat are restricted, ex situ conservation is important to conserve I. dichotoma. Research studies on its propagation and habitat restoration are warranted due to insufficient previous studies.

Propagation using seed germination provides a large number of plant individuals at once. However, since each species has different germination characteristics, further research is needed. This is especially true for seeds that re- quire a specific environment during dormancy, e.g., cold temperature or hormone treatment. Such seeds will not germinate until particular conditions are met, even if they are in a favorable environment (Finch-savage & Leubner- Metzger, 2006). Seed dormancy types are classified into five main categories: 1) physical dormancy, 2) physiological dormancy, 3) morphological dormancy, 4) morphophysiological dormancy, and 5) combinational dormancy (physical dormancy + physiological dormancy). These five categories of seed dormancy are subdivided based on the depth and pattern of dormancy ( Baskin & Baskin, 1998; Finch-savage & Leubner-Metzger, 2006 ). Seed dormancy plays a decisive role in controlling germination timing and adaption of seeds to natural environments ( Geneve, 2003 ). However, seeds of species in the same genus can exhibit various types of dormancy and germination characteristics ( Hidayati et al ., 2000; Park et al ., 2019 ). In a research study conducted by ( Park et al . 2019 ), four Lonicera species native to Korea showed different germination characteristics and dormancy types under four temperature treatments, although they were the same species native to different regions. Seeds of eight Veronica species native to Korea also exhibited interspecific germination responses that varied under identical temperature treatments ( Song et al ., 2018 ). Thus, classification of seed dormancy and investigation of germination characteristics are needed to provide insight into how each species adjusts to different natural habitats ( Park et al ., 2019 ).

In previous studies, seeds of species in the Iris genus showed physiological dormancy. Such seed dormancy is a typical characteristic of plant species native to the Northern Hemisphere ( Baskin & Baskin, 1998 ). Seeds with physiological dormancy can germinate when gibberellic acid concentration in seeds increases due to particular conditions, such as a cold or a warm stratification treatment. Seed dormancy of I. suaveolens Boiss can be broken via stratification treatments, especially with 2 weeks of cold followed by 6 weeks of warmth ( Hajyzadeh et al ., 2019 ). NaOH scarification and stratification can effectively break seed dormancy and improve seed germination of I. lactea Pall ( Sun et al ., 2006 ). Although previous research has examined germination rates of I. dichotoma and I. setosa Pall according to seed storage method ( Lee et al ., 2015 ), studies about seed dormancy type of irises native to Korean seeds are insufficient.

Thus, the objective of the present study was to investigate germination characteristics under various temperatures (4/1°C, 15/6°C, 20/10°C, and 25/15°C) and cold stratification treatment (4/1°C at 4 weeks). Seed morphology was observed and seed dormancy was classified for I. dichotoma. Results of this study can improve our understanding of seed ecophysiological mechanisms in a natural habitat. They could be used for mass propagation of I. dichotoma.

Materials and Methods

Plant materials

study was carried out at the Endangered Resources Center in Yeong -yang, Korea. Fruits of I. dichotoma were collected in 2020 from plants growing at the center’s greenhouse. These seeds were dehisced, and dried at an ambient room temperature (~25°C) for 2 weeks, and then stored at 4°C until further analysis.

Temperature treatments

To investigate seed germination characteristic and type of dormancy of I. dichotoma, three replicates of 50 seeds were placed in 10-cm-diameter Petri dishes on top of two sheets of filter papers moistened with distilled water. All Petri dishes were sealed with Parafilm to prevent water loss during the experiment. Temperature- and light-controlled multiroom chambers were used in this study. These chambers were set at 4/1°C, 15/6°C, 20/10°C, and 25/15°C, respectively. Cool white florescent lamps were set to an alternating 12-h/12-h light/dark photoperiod.

Germination investigation was carried out once per week. Germinated seeds were removed from Petri dishes. Distilled water was frequently supplied to Petri dishes to prevent water loss. Rotten seeds were excluded from the calculation of germination rate. Measured traits included final germination rate (FGR) and mean germination time (MGT). They were calculated with the following equations:

FGR = (N/S) × 100 MGT = Σ(t i × n)/N

where N was the total number of germinated seed, S was the total number of seed sown, t i was the number of days from sowing, and n was the number of seeds newly germinated at time t.

Seed morphology

To investigate morphological characteristics, seed morphology characterization was carried out on June 8, 2021. Iris seeds were incubated at 25/15°C. These seeds were halved using a surgical blade (stainless blade; Feather Safety Razor Co., LTD). Lengths of the embryo of each seed at dispersal and just before germination were measured using a USB microscope (AD7013MZT Dino-Lite; AnMo Electronics Co., Taiwan). The embryo–seed ratio (E:S ratio) was then calculated and compared.

Cold stratification

Cold stratification treatment was conducted to investigate its effect on seed germination. Seeds were placed in 10-cm-diameter Petri dishes on two filter papers moistened with distilled water. These seeds were stored in a chamber set to 4/1°C with a 12-h alternating light and dark cycle for 4 weeks. They were moved to the chamber at 25/15°C after 4 weeks of cold stratification. Germinated seeds were counted every week and removed from Petri dishes. Distilled water was frequently supplied to Petri dishes to prevent water loss. Rotten seeds were excluded from the calculation of germination rate.

Statistical analysis

Statistical analysis software version 9.4 (SAS Institute Inc., Cary, NC, USA) was used for all statistical analyses of the data. Differences between mean FGRs of seeds under four temperature treatments were assessed using Tukey’s honestly significant difference tests. Differences between E:S ratios of seeds at dispersal and just before germination were assessed using paired t-tests. Results with p values < 0.05 were considered statistically significant.

Results

The seed morphology experiment showed that seeds of I. dichotoma had fully linearly developed embryos at dispersal (Fig. 1). This embryo type is well-presented in gymnosperms, monocots, and dicots ( Martin, 1946 ). E:S ratios of seeds were 0.70 ± 0.03 and 0.75 ± 0.02 at dispersal and just before germination, respectively (Fig. 2). There was no significant difference in the E:S ratio between dispersal and just before germination. In general, seeds with underdeveloped embryos have morphological dormancy or morphophysiological dormancy ( Baskin & Baskin, 1998; 2004 ). Seeds with morphological dormancy and morphophysiological dormancy will disperse with underdeveloped embryos. Their embryos then show additional growth until just before germination within 30 days. Seeds of I. dichotoma have no morphological dormancy. Thus, they will germinate without additional growth of the embryo within 30 days.

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Fig. 1.
Internal seed morphology of Iris dichotoma Pall. Fully developed embryos at seed dispersal (A) and at just before germination (B) are shown. pr, pericarp; sc, seed coat; en, endosperm; em, embryo. Scale bars are 0.5 mm.
PNIE2-4-2-f1.jpg
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Fig. 2.
Embryo:seed ratio (E:S ratio) in the seeds of Iris dichotoma Pall at seed dispersal and at just before germination. The seeds of I. dichotoma were incubated at 25/15°C. The vertical error bars represent SE (n = 10). Each E:S ratio at dispersal and just before germination was compared using paired t test.
PNIE2-4-2-f2.jpg
Temperature treatments

At 15/6°C, seeds of I. dichotoma started to germinate at 3 weeks after sowing. At 5 weeks after sowing, the FGR of seeds was 81.3%. At 20/10°C, freshly matured seeds started to germinate at one week after sowing. At 5 weeks after sowing, the FGR of seeds was 75.3%. At 25/15°C, seeds started to germinate at one week after sowing. At 2 weeks after sowing, the FGR was 80.0% (Fig. 3). Its seeds did not germinate when they were incubated at 4/1°C. Final germination rates showed no significant differences among all temperature treatments except for 4/1°C. Seeds germinated within 2 weeks. According to Baskin and Baskin (2004), seeds with physiological dormancy will not germinate within ~30 days even in an optimal environment. To break the physiological dormancy, seeds need particular conditions, such as a cold or a warm stratification, or treatment with germination-improving chemicals such as GA 3 , KNO 3 , and so on. Several previous studies have reported that seeds of the Iris genus exhibit a typical physiological dormancy. They germinate when germination inhibitors are removed. They also need optimal conditions of light, temperature, and so on ( Lashkarian et al ., 2012; Lee et al ., 2015 ). Seeds of I. lactea require NaOH scarification and cold stratification to break dormancy and experience greater rates of germination than the control ( Sun et al ., 2006 ). Combined alternate cold-warm stratification could effectively break the seed dormancy of I. suaveolens ( Hajyzadeh et al ., 2019 ). In this study, however, seeds of I. dichotoma germinated within 30 days after all temperature treatments except for the treatment at 4/1°C. These results indicate that the seeds of I. dichotoma have no physiological dormancy, unlike other species of the Iris genus.

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Fig. 3.
Cumulative germination rate in seeds of Iris dichotoma Pall at four various temperatures (4/1°C, 15/6°C, 20/10°C and 25/15°C). The vertical error bars represent SE (n = 3). The different letters represent statistically significant differences, as determined by Tukey’s HSD tests ( p < 0.05).
PNIE2-4-2-f3.jpg

It is theorized that in the evolution of seed dormancy, morphological dormancy and morphophysiological dormancy belong to the primitive dormancy class. Seeds with morphophysiological dormancy evolved as nondormant seeds with larger embryo size and lost physiological dormancy ( Baskin & Baskin, 1998; FinchSavage & Leubner- Metzger, 2006; Nikolaeva, 2004 ). Therefore, nondormant seeds with fully developed embryo were hypothesized to have more evolved seed physiological characteristics. Thus, in this study, interspecific differences in seed dormancy type might have resulted from the evolution of seed dormancy in the Iris genus.

Seeds of I. dichotoma showed different MGTs under four temperature treatments (Fig . 4). MGTs of the seeds at 15/6°C, 20/10°C, and 25/15°C were 3.2, 2.1, and 1.5 weeks, respectively. There were significant differences in MGTs of these seeds under the four temperature treatments. As the temperature increased, the MGT decreased significantly. The MGT at 25/15°C was twice as fast as that at 15/6°C. MGT is not the only the parameter to predict the rate of germination. Plant growth post germination (e.g., root length, stem length, etc.) is also predictive ( Demir et al ., 2008; Matthews & Hosseini, 2006 ). In particular, MGT is highly correlated with mean emergence time (MET) and higher temperature ( Demir et al ., 2008; Demirkaya et al ., 2020 ). In maize (Zea mays L.), there was a high correlation between MGT and MET. Root length was higher at lower MGT ( Matthews & Hosseini, 2006 ). In a study on seeds of eight Veronica species native to Korea, MGT was found to be decreased with a higher temperature ( Song et al ., 2018 ). Thus, METs of seeds of I. dichotoma in laboratory and natural habitat can be predicted depending on MGT and the temperature of the environment. In natural habitats with relatively higher temperatures, seeds of I. dichotoma are expected to germinate faster than in habitats with relatively moderate temperatures. These results are helpful for mass propagation of I. dichotoma for the purpose of restoration.

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Fig. 4.
Mean germination time of Iris dichotoma Pall under four temperature treatments. The vertical error bars represent SE (n = 3). The different letters represent statistically significant differences, as determined by Tukey’s HSD tests ( p < 0.05). An asterisk (*) indicates that no information was available for this field.
PNIE2-4-2-f4.jpg
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Fig. 5.
Germination response of Iris dichotoma Pall according to cold stratification treatment (4 weeks at 4/1°C). The vertical error bars represent SE (n = 3). The different letters represent statistically significant differences, as determined by Tukey’s HSD tests ( p < 0.05).
PNIE2-4-2-f5.jpg
Cold stratification

Cold-stratified seeds of I. dichotoma started to germinate at one week after the temperature was raised to 25/15°C. At 6 weeks after sowing, the FGR was 77.3%. There was no significant difference in the FGR be tween 0 week of cold stratification and 4 weeks of cold stratification (Fig . 5). Previous studies have shown that cold stratification can break physiological dormancy by removing germination inhibitors and improve germination improving hormone concentration (likely GA 3 ). However, in the present study, the temperature experiment showed that seeds of I. dichotoma had no physiological dormancy. Although cold stratification did not enhance the FGR, the highest FGR was maintained during cold stratification. The seed germination rate of I. dichotoma was the highest when seeds were stored at 2°C for 60 days among all treatments ( Lee et al ., 2015 ). Therefore, in natural habitats, seeds of I. dichotoma can disperse from October to November. These seeds can become cold stratified in the soil. As temperature increases in the following year, seeds can germinate without a decrease in germination rate.

Conclusion

Results of this study demonstrate that seeds of I. dichotoma have no seed dormancy, whereas seeds of other species in the Iris genus have physiological dormancy. This interspecific variation of seed dormancy in the same genus is considered to be due to the evolution of seed dormancy. I. dichotoma is considered to be a species that has differentiated or evolved more recently compared with other species of the Iris genus with seed dormancy. The FGR of I. dichotoma seeds was not affected by temperatures, although temperatures did affect MGT, which was the lowest at 25/15°C. In natural habitats, seeds of I. dichotoma can disperse with fully linearly developed embryos from October to November. These seeds then experience cold stratification in the soil. As temperature increases in the following year, seeds can germinate without a decrease in germination rate. Our results are helpful for understanding seed ecophysiological adaptions and mass propagation of I. dichotoma.

Conflict of Interest

The authors declare that they have no competing interests.

Acknowledgments

This research was supported by the National Institute of Ecology (NIE), NIE-C-2021-47.

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