Introduction
Hemerocallis was included in Asphodelaceae which is substituting for Xanthorrhoeaceae (APG IV 2016) and commonly called daylily (Zhao et al. 2018). Hemerocallis species are one of the most popular ornamental crops worldwide because of the large, conspicuous flowers and their adaptation to a wide range of soil types and climates (Li et al. 2020). Plants of Hemerocallis are distributed in East Asia (temperate and sub-tropical Asia), but the main diversity of the genus centered on Korea, China and Japan (Manole 2018;Rodriguez-Enriquez and Grant-Downton 2012). About 10 taxa are distributed in Korea, including H. hakuunensis and H. hongdoensis, which are endemic species (Lee 1980;Jang 2010). Most of them bloom from July to August (Hwang and Kim 2012). H. hakuunensis, H. hongdoensis, amd H. thunbergii are listed as LC (Least Concern species) in the national red list in Korea (KNA 2021). Therefore, for the ornamental use of these native Hemerocallis plants and the conservation of genetic resources, it is necessary to develop mass propagation technology using seeds.
Hemerocallis plants, commonly known as daylily, have been used for medicinal purposes in traditional medicine systems for centuries (Liu et al. 2017). The plants have been reported to possess several pharmacological properties such as antioxidant, anti-inflammatory, and antimicrobial activities (Li et al. 2022;Lim et al. 2012, 2013). Daylily has also been found to contain various phytochemicals, including phenolic compounds, flavonoids, and carotenoids, which contribute to their medicinal properties (Szewczyk et al. 2020). Therefore, Hemerocallis deserves more attention in the future due to its beneficial compounds on human health.
Seed dormancy restrains the germination of intact viable seeds during (temporary) favorable conditions in an unfavorable season (Bewley 1997). Seed dormancy depends on genetic factors with a substantial environmental influence and provides adaptation strategys to a diversity of habitats. Seed germination during a favorable season for plant growth increases the probability of seedling survival (Baskin and Baskin 1972, 1976, 2014). Therefore, seed dormancy is an important component of plant fitness (Donohue et al. 2005;Huang et al. 2010). Several seed dormancy types exist among plant species, which can be classified into physiological dormancy (PD), morphological dormancy (MD), morphophysiological dormancy (MPD), physical dormancy (PY) and combinational dormancy (PY + PD) (Baskin and Baskin 2004;Finch-Savage and Leubner-Metzger 2006).
The Hemerocallis spp. are known to have MPD (Baskin and Baskin 2014;Griesbach and Voth 1957). However, Kim et al. (2016) reported that seeds of H. thunbergii and H. hongdoensis showed the high germination rate without cold stratification, while germination in H. dumortieri was lower than 1% with 2 weeks of cold stratification. None of the above studies on Hemerocallis germination has examined internal embryo morphology and growth. These aspects are important to determine whether the seed has embryos that are fully developed or underdeveloped at maturity, and therefore, if the embryo must elongate inside the seed before germination. Such information is required to classify the class of seed dormancy found in the genus.
Responses to techniques for breaking seed dormancy depends on the species and dormancy type (Flores- Sánchez et al. 2022). The current study was conducted to infer the role of various seed dormancy-release treatments in improving seed germination of four Hemerocallis species native the Korean Peninsula. The focus of the research was to identify the type of four Hemerocallis seed dormancy, explore efficient methods to remove four Hemerocallis seed dormancy and promote as many healthy seeds as possible to germinate. We asked the following questions: (i) Does the embryo length:seed length (E:S) ratio increase prior to seed germination? (ii) What are the temperature and light/dark requirements for germination? (iii) What are the effects of GA3 and cold stratification on the emergence of the radicle?
Materials and Methods
Plant materials
Four species of native Hemerocallis in Korea were tested in this experiment. Seeds of H. dumortieri, H. hakuunensis, and H. hongdoensis were on September 1, 2020 at Korea National Arboretum, and H. thunbergii were harvested on October 3, 2020 at Hantaek Botanical Garden. The harvested seeds were dried in a laboratory at 20 - 23℃ for four weeks and seeds were stored at 0 ℃ in a refrigerator (DOI1815DOP, Winiamando, Gwangju, Korea) until use.
Analysis of internal and external seed morphology
To observe the internal and external morphology of seeds, the seeds were cut with a stainless-steel razor blade (Dorco, Korea), and the resultant cross sections were photographed at 50 - 100× magnification under a USB microscope (AM3111 Dino-Lite premier, AnMo Electronics Co., Taiwan). To determine whether the photographed seeds comprised immature embryos, embryos were investigated at the time of early detachment and just before germination, and the corresponding E:S ratio was calculated.
Water absorption
Water absorption was examined to identify the presence or absence of physical dormancy in seeds of the four Hemerocallis species. Twenty seeds of each specieswere placed in Petri dishes (90 × 15 mm; diameter × height) lined with two sheets of filter paper (ADVANTEC No. 1, Toyo Roshi Kaisha, Ltd., Tokyo, Japan), added with distilled water (10 mL), and then cultured at 20 - 23℃. The distilled water was added as needed to keep the seeds moist. The initial weight of the seeds before water absorption and final weights after 2, 4, 8, 12, 24, and 48 h of water treatment were measured. The water absorption rate was calculated as follows:
Where Ws is a relative weight ratio of seeds increased through water absorption, Wh is weight per culture time, and Wi is weight of seeds before water absorption.
Effect of temperature on germination
The seeds were sterilized in 1000 mg·L-1 diluted solution of benomyl (a fungicide) for 24 h. Petri dishes (90 × 15 mm) were lined with two sheets of filter paper (ADVANTEC No. 1, Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and 20 seeds were placed in each dish; three replicates were prepared per treatment. Distilled water (10 mL) was added to avoid drying out of seeds. The seeds were cultured in a multi-room incubator (HB-101-4, HANBAEK-Scientific, Bucheon, Korea) at 15/6, 20/10 and 25/15℃ for 12/12 h of day/night, and the photosynthetic photon flux density (PPFD) of 69.96 ± 8.62 μmol·m-2·s-1. Culturing at 4℃ was conducted in a growth chamber (HB-603CM; HANBAEK-Scientific, Bucheon, Korea) under slight light with the average photosynthetic photon flux density (PPFD) of 9.33 ± 0.53 μmol·m-2·s-1. The seeds were examined every week, and they were considered as germinated if the emerged radicle reached about 2 mm in length six weeks after. Subsequently, the germinated seeds were counted and removed from the Petri dish.
Effect of light on germination
The seeds were sterilized in 1000 mg·L-1 diluted solution of benomyl (a fungicide) for 4 h. Petri dishes (90 × 15 mm) were lined with two sheets of filter paper (ADVANTEC No. 1, Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and 20 seeds of each species were placed in a Petri dish. Distilled water (10 mL) was added to keep seeds moisture. At 25/15℃, seeds were cultured in a multi-room incubator (HB-101-4, HANBAEK-Scientific, Bucheon, Korea) for 12/12 h of day/night and the average PPFD of 63.46 μmol·m-2·s-1. For dark treatment, Petri dishes were wrapped in double aluminum foil to avoid exposure of seeds to light. The seeds were examined as described above.
Effect of cold stratification
The effects of cold stratification were determined to understand whether the dormancy could be broken by cold stratification. Twenty sterilized seeds per replicate, three replicates per treatment, were placed in Petri dishes (90 × 15 mm) lined with two sheets of filter paper (ADVANTEC No. 1) and transferred to a growth chamber at 4℃. Cold stratification at 4℃ was conducted for 0, 2, 4, 8, and 12 weeks in a growth chamber (HB-603CM, HANBAEK-Scientific, Bucheon, Korea) under PPFD of 0.93 ± 0.53 μmol·m-2·s-1. The cold-stratified seeds were incubated at 15/6 or 25/1 5℃ under light conditions.
Effect of GA3 treatment
To analyze the effects of GA3 on dormancy , seeds were soaked in 0, 10, 100, or 1000 mg·L-1 GA3 solution for 24 h. Then, seeds were rinsed with distilled water for 3 min and disinfected with benomyl wettable powder (1000 mg·L-1) for 8 h. The seeds were sown in Petri dishes as described above and incubated at 15/6 or 25/15℃.
Statistical analysis
Analysis of variance was performed on the collected data using SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Significant differences in embryo to seed length ratio (E:S ratio) at initial stage and seed coat split stage were determined by paired t-test at p < 0.05. To determine the statistical significance of the percentage germination between different treatments, Duncan's multiple range tests at p < 0.05 was used. The graphs were prepared using Sigma Plot 10.0 (Systat Software Inc., San Jose, CA, USA).
Results
Internal and external seed morphology
Four Hemerocallis seeds were black and wide oval in shape. The length of the seeds of the H. dumorieri, H. thunbergii, and H. hakuunensis were 4.9 - 5.4 mm and the width were 3.5 – 3.8 mm, but the seeds of the H. hongdoensis were 7.1 mm and 4.9 mm in length and width, respectively (Table 1). Hemerocallis species initial and prior to germination E:S ratios were 0.83 - 0.91 and 0.89 - 0.95, respectively, with no significant difference between two time points (Fig. 1).
Water absorption rate
Seeds of all tested species increased their weight after imbibed in water for 48 h (Fig. 2). However, the degree of mass increase was species-specific. The species that most increased their mass were H. dumorieri (100.7%) and H. hongdoensis (91.1%), while H. hakuunensis and H. thunbergii only increased their mass in 59.5% and 52.5%, respectively.
Effect of temperature on germination
The germination of all tested species was significantly affected by the temperature conditions (Fig. 3). All species was recorded the highest germination rate of more than 80% at 25/15℃. Seeds of H. hongdoensis germinated to more than 80% at 20/10℃ under light conditions after 6 weeks of incubation, but those of H. dumorieri, H. thunbergiiH. hakuunensis showed much lower germination rates at 20℃. All species showed a decrease in germination rate as the temperature decreased from 25/15 to 4℃.
Effect of light on germination
Seeds of H. dumorieri, H. thunbergii, and H. hakuunensis had high germination rates regardless of light and dark conditions (Fig. 4). However, germination rates of H. hongdoensis showed significant differences between light and dark conditions.
Effect of cold stratification
At 25/15℃, all tested species exhibited more than 80% germination rate regardless of the cold stratification period (Fig. 5). At 15/6 ℃, H. thunbergii without cold stratification did not germinate, and the germination rates of H. dumortieri and H. hakuunensis were less than 20%. However, in the case of cold stratification treatment, all species showed more than 80% germination rate at 15/6℃ regardless of the cold stratification period.
Effect of GA3 on germination
At a high temperature (25/15℃), in which the germination percentage of untreated seeds was high (> 80%), GA3 did not significantly increase germination percentage (Fig. 6). As majority of the species germinated well with distilled water. At a relatively low temperature (15/6℃), in which the germination percentage of seeds was low, GA treatment increased the germination rate or germination speed. However, this treatment did not improve germination rate as much as 25/15℃.
Discussion
Since mass of the four Hemerocallis seeds increased by approximately 30 % or more within 24 h compared with the initial mass after water absorption (Fig. 2), the seed coat was permeable to water (Baskin and Baskin 2003) and the seeds do not have PY.
When seeds have an immature embryo that grows to the critical length within 30 days under favorable germination conditions, they are considered to show MD (Baskin and Baskin 2014;Lee et al. 2018). As the differences in the E:S ratios between the initial point of seed separation and immediately before germination were not significantly different (Fig. 1), the embryo was considered to be in a mature state during separation. Therefore, seeds of four Hemerocallis do not have MD.
About 70% of the seed plants produce dormant seeds, and PD is the most common class of dormancy on earth (Soltani et al. 2017). Three levels of PD have been distinguished: nondeep, intermediate, and deep (Baskin and Baskin 2004, 2014; Nikolaeva 1977). Nondeep PD can be broken by relatively short periods (a few days to a few weeks) of warm or cold stratification (Baskin and Baskin 2014). The germination speed and rate of the four Hemerocallis seeds were increased through low-temperature stratification treatment (≥ 2 weeks), and the range of germination temperature was widened (Fig. 5). At a relatively low temperature of 15/6℃, the germination rate was as high as at 25℃ following cold stratification. The response of four Hemerocallis seeds to cold stratification suggests that they are under the state of conditional dormancy and thus have nondeep PD (Baskin and Baskin 2004;Soltani 2017). Conditional dormancy occurs in seeds with nondeep PD, and it represents an intermediate state between dormancy and nondormancy (Baskin and Baskin 1985;Baskin and Baskin 2014).
GA3 treatment is commonly used in various plant species to break physiological dormancy (Yao et al. 2015). In this study, although GA treatment did not dramatically improve the germination rate of the seeds at 15/6℃, it accelerated the germination speed or slightly increase the germination rate (Fig. 6). However, the GA3 treatment was found to be less effective as compared to cold stratification. It has been reported that cold stratification and exogenous GA treatment affect seed dormancy and germination by controlling the balance and sensitivity of GA/ABA (Finch-Savage and Leubner-Metzger 2006).
Conclusions
The seeds of the four Hemerocallis species had fully developed embryos upon dispersal from their parent plants. The germination of the four Hemerocallis seeds was promoted by warm temperature (25/15℃) and inhibited by relatively low temperature (20 - 4℃). Cold stratification extended the temperature ranges for germination in which ≥ 80% seeds germinated at 15/6℃. Therefore, it is determined that the four Hemerocallis species have nondeep PD.