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Article

Deep Simple Epicotyl Morphophysiological Dormancy in Seeds of Endemic Chinese Helleborus thibetanus

by
Xueyan Zhao
,
Fangyuan Wang
,
Li Wang
,
Qing Wang
,
Ancheng Liu
and
Yan Li
*
Shaanxi Engineering Research Centre for Conservation and Utilization of Botanical Resources, Xi’an Botanical Garden of Shaanxi Province (Institute of Botany of Shaanxi Province), Xi’an 710061, China
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(7), 1041; https://doi.org/10.3390/agriculture14071041
Submission received: 13 May 2024 / Revised: 11 June 2024 / Accepted: 27 June 2024 / Published: 29 June 2024
(This article belongs to the Section Seed Science and Technology)
Browse Figures
Versions Notes

Abstract

:
Helleborus thibetanus is an endemic species in China with important ornamental and medicinal value. However, the seeds have dormancy, and their germination percentage is low under natural conditions. This research was carried out to determine the seed germination requirements of H. thibetanus and to characterize the type of seed dormancy. The morphological post-ripening process of the seed embryo was studied according to the morphological anatomy, and the effects of temperature and gibberellic acid (GA3) on seed germination were investigated in H. thibetanus. The H. thibetanus seeds had a heart-shaped embryo at maturity. The embryo fully grew within the seed through warm stratification, and the embryo/seed ratio increased from 8.58% to 42.6%. The shortest time for a radicle to emerge (58.33 d) and the highest radicle emergence percentage (84.44%) were obtained at a temperature of 15 °C. The results showed that the H. thibetanus seeds had a morphophysiological dormancy. In addition, 300 mg/L GA3 treatments shortened the time of warm stratification and increased the radicle emergence percentage. Seeds with emerged radicles could not emerge epicotyl–plumule without cold stratification, which showed that the H. thibetanus seeds had epicotyl physiological dormancy. The length of the roots, cold stratification time, and GA3 markedly affected the release of the epicotyl physiological dormancy in H. thibetanus. The seeds with 2.5 cm roots required the shortest time to break their dormancy (50 d), and the epicotyl–plumule emergence percentage was the highest. Additionally, GA3 treatment also shortened the incubation time in cold stratification (5 °C) and successfully broke the epicotyl physiological dormancy. Our study showed that H. thibetanus seeds exhibited deep simple epicotyl morphophysiological dormancy. Temperature, GA3, and duration of stratification played vital roles in the seed germination of H. thibetanus. This research will provide valuable data for seed germination and practical dormancy-breaking techniques and will promote the cultivation and conventional crossbreeding of H. thibetanus.

1. Introduction

Helleborus thibetanus Franch is a perennial plant of Helleborus belonging to the family Ranunculaceae. H. thibetanus is an endemic species in China, and it is mainly distributed in the Shaanxi, Sichuan, and Gansu provinces [1]. H. thibetanus is a folk herb in China, and the roots and rhizomes are used to treat cystitis, urethritis, sores, and traumatic injury [2]. The genus Helleborus comprises 22 species [3]. Helleborus plants have become more popular due to their ornamental value and winter or early spring flowering [4]. H. thibetanus is an acaulescent plant, and its basal leaves are early deciduous, glabrous, and pedate with three primary segments. The outer two segments are each divided into three to four additional segments for a total of seven to ten. The segments are narrowly elliptical. The leaf margins are coarsely serrated to dentate. The flower comprises five petal-like sepals surrounding a ring of petals that are reduced to nectaries, a boss of stamens, and a central cluster of pistils. The flowers, which bloom in February and last until June, are pale pink with deep pink veins when they first bloom and then turn green. H. thibetanus has two—or, rarely, three—follicles, and the seeds are black [1]. In recent years, studies of this species mainly focused on its chemical constituents and pharmacology [5,6,7,8,9,10], its organ evolution and morphological anatomy [11,12,13], and the transcriptome of the leaf [14].
Seed dormancy is a biological adaptation of environmental conditions and seasonal changes obtained in the long-term evolution of plants [15]. Baskin and Baskin (2008) divided seed dormancy into physical dormancy, physiological dormancy, morphological dormancy, combinational dormancy, and morphophysiological dormancy (MPD) [16]. MPD is the most complex of the five types of plant seed dormancy; it includes morphological dormancy and physiological dormancy, that is, undeveloped embryos have physiological dormancy [17]. Our previous study showed that H. thibetanus seed displayed a serious morphological post-ripening phenomenon, and the embryo was in the heart-shaped embryo stage at the harvest period [18]. Similarly, the ripe seed of H. niger had a heart-shaped embryo [19]. The seeds of six Clematis species (C. apiifolia, C. heracleifolia var. urticifolia, C. heracleifolia, C. serratifolia, C. terniflora, and C. trichotoma) undergo morphological or morphophysiological dormancy. Warm temperatures were found to be beneficial to their embryo development. In addition, gibberellin was found to promote seed germination [20].
Plant hormones can regulate seed dormancy and germination, and gibberellin is considered to promote seed germination hormones [21]. GA3 increased the germination percentage and germination rate of Primula beesiana [22], and it regulated rice embryo growth and seed germination by regulating glutelin mobilization [23]. In addition, gibberellin also broke the physiological epicotyl dormancy of Paeonia ostii and Paeonia emodi seeds [24,25]. GA3 was required for seeds to overcome mechanical obstacles or other inhibitory effects caused by surrounding structures [26].
The undeveloped seed embryo of H. thibetanus is an important factor in seed germination difficulty [18]. As an important medicinal and ornamental plant, the low germination percentage and long germination period are the main reasons for the impedance of the further widespread cultivation of H. thibetanus. Nonetheless, dormancy breaking and germination of H. thibetanus seeds has not been studied. Therefore, the objectives of the present study were to determine the class and level of seed dormancy of H. thibetanus and the germination requirement of this species. These results will offer a reference for its large-scale cultivation, horticultural application, and crossbreeding.

2. Materials and Methods

2.1. Seed Materials

The freshly matured seeds of H. thibetanus were collected from several hundred plants growing in natural populations in the Qingling Mountains (33°48′ N; 108°32′ E, Xi’an, Shaanxi, China) during late May 2021. The seeds were not stored and were used directly for experiments. H. thibetanus was identified by Prof. Ming Yue, and a voucher specimen (ZY190528) was deposited in the Xi’an Botanical Garden Herbarium. The seeds were disinfected with 70% alcohol for 30 s. After rinsing with sterile water, the H. thibetanus seeds were further disinfected with 1% sodium hypochlorite for 10 min and washed with sterile water several times.

2.2. Seed Properties

The width, length, and thickness of H. thibetanus seeds were measured with a Vernier caliper. The weights of fresh seeds (10 replicates, 10 seeds/replicate) were measured. Subsequently, the weights of the seeds which dried in an oven for 48 h at 75 °C to a constant weight were measured. The moisture content of the fresh seeds was measured according to the following formula: [(fresh weight − dry weight)/fresh weight] × 100% [27]. Thereafter, 1000 freshly matured seeds were divided into 10 groups and selected randomly. The average seed mass was measured with an electronic balance. The average seed mass was used to calculate the thousand-seed weight [27]. Ten replicates were performed in this section.

2.3. Embryo Morphology Observations and Index Determination

First, 10 fresh seeds and 10 seeds that were about to sprout radicles were randomly selected, dissected, and photographed under a Leica EZ4W microscope (Wetzlar, Germany). The lengths of the embryos and seeds were measured with a Leica DM3000 microscope (Wetzlar, Germany), and the ratio of embryo to seed length was analyzed (n = 10). After the radicle emergence experiment, the embryo development stage of seeds that were incubated at 25 and 30 °C were observed under a Leica EZ4W microscope (Wetzlar, Germany).

2.4. Effects of Temperature and GA3 on Radicle Emergence

A factorial experiment in a completely randomized design was used in the radicle emergence experiment. Firstly, the seeds were tested at five constant temperature regimes (10, 15, 20, 25, and 30 °C) and one alternating temperature regime (15/20 °C, 12 h at 15 °C /12 h at 20 °C). The other sterilized seeds were soaked in 100 mg/L, 300 mg/L, and 500 mg/L GA3 (Sigma-Aldrich, St. Louis, MO, USA) solution for 24 h. After GA3 treatments, the seeds were incubated at 10, 15, 20, 25, 30 and 15/20 °C until the end of the experiment. In each temperature treatment, the seeds were incubated in continuous darkness. Each treatment contained 3 replicates of 30 seeds placed on Petri dishes with two moistened pieces of filter paper. In the present study, a seed was deemed to have an emerged radicle when at least 2 mm of the radicle was visible [28,29]. The duration of the seed radicle emergence experiment was 180 days. At the end of the radicle emergence period, the radicle emergence percentage was calculated according to the following formula: radicle emergence percentage (%) = (number of seeds with an emerged radicle ÷ number of seeds per sample) × 100. The radicle emergence percentage of each treatment was calculated by the average of the percentages of the three replications. The number of days for the first emerged seed radicle was determined as the radicle emergence starting time for each incubation temperature. The radicle emergence duration (d) = the number of days for the seed radicle emergence end − the number of days for the first seed emerged radicle. Observations were halted when no further seeds with an emerged radicle appeared for one month. After the experiment, the seeds which were incubated at 10 °C, 25 °C, and 30 °C were transferred to 15 °C, and whether the seeds could emerge radicle was observed.

2.5. Epicotyl Dormancy Release Test

Sand was sterilized at 121 °C for 1 h and then cooled to room temperature. The sterilized seeds were mixed with wet sand (water content: 40 ± 5%) and then were incubated in an artificial climate chamber at a constant temperature of 15 °C. The sand was kept moist throughout the experiment. The seeds from which a radicle had emerged were selected for the epicotyl dormancy release experiment. The duration of the epicotyl–plumule emergence experiment was 160 days. The epicotyl–plumule emergence percentage was calculated according to the following formulas: epicotyl–plumule emergence percentage (%) = (number of epicotyl–plumule-emerged seeds ÷ number of seeds from which a radicle emerged per sample) × 100. We used a factorial experiment in a completely randomized design in the epicotyl dormancy release experiment. The following treatments were set up.
Effect of root length on epicotyl dormancy: Seeds with root lengths of 0.5 cm, 1.5 cm, and 2.5 cm were selected, transferred to plastic boxes that contained disinfected sand, and then kept at 5 °C. There were 3 replicates per treatment with 30 seeds with a radicle that had emerged. During stratification, the seeds with an epicotyl–plumule that had emerged were taken out, and their epicotyl–plumule emergence was observed every 10 days. The epicotyl–plumule emergence initiation time was counted for each treatment, and the epicotyl–plumule emergence percentage was calculated for each treatment at 10-day intervals. Epicotyl–plumules that had grown to ≥5 mm were considered indicative of seed germination [28,30]. The epicotyl–plumule-emerged seeds were selected and transplanted under light conditions at 20–25 °C.
Effect of GA3 treatment on epicotyl dormancy: Seeds with different root lengths (0.5 cm, 1.5 cm, and 2.5 cm) were soaked in 400 mg/L GA3 for 2 h at room temperature. The subsequent experimental procedures were the same as those described for the experiment on the effect of root length on epicotyl dormancy. Each treatment contained 3 replicates of 30 seeds with a radicle that had emerged. Then, the epicotyl–plumule emergence percentage was calculated.

2.6. Data Analysis

In the seed radicle emergence experiment, one-way analysis of variance (ANOVA) was performed using the Tukey test to analyze the difference in the radicle emergence starting time, radicle emergence duration, radicle emergence percentage at 15 °C, 20 °C, and 15/20 °C, radicle emergence percentage among different GA3 concentrations (control, 100, 300, 500 mg/L) and epicotyl–plumule emergence percentage between control and GA3 treatment at the same root length (0.5, 1.5, 2.5 cm). Two-way ANOVA was used to evaluate the effects of incubation temperature (four levels; 10, 15, 15/20, 20 °C) and GA3 concentration (four levels; control, 100, 300, 500 mg/L) on seed radicle emergence. In the epicotyl dormancy release experiment, two-way ANOVA was used to evaluate the effects of GA3 (control, 400 mg/L) and cold stratification time (30, 60, 90, 120, 140 d) on epicotyl–plumule emergence percentage of seeds with root lengths of 0.5, 1.5, and 2.5 cm. Correlation analysis between root length and epicotyl–plumule emergence percentage was performed in the control and GA3 treatments. All statistical tests were performed with the IBM SPSS Statistics 19.0 software.

3. Results

3.1. Characteristics of Helleborus thibetanus Seeds

The mature seeds of H. thibetanus were black, elliptic, or oblate in shape with one longitudinal rib. The average length, width, and thickness of the seeds were 4.41 ± 0.21 mm, 3.02 ± 0.13 mm, and 3.02 ± 0.20 mm, respectively. The thousand-seed weight was 26.22 ± 0.45 g. The moisture content of the H. thibetanus seeds was 57.40 ± 0.56%.

3.2. Seed Embryo Growth during the Morphological Post-Ripening Process

The embryos of the freshly matured seeds were heart-shaped and embedded in a rich endosperm at the hilum position (Figure 1A,C). The length of the heart-shaped embryos was 0.39 ± 0.04 mm, and the average embryo/seed (E/S) ratio was 8.58 ± 0.85% (Figure 1A,E). Under warm stratification conditions (15 °C), the heart-shaped embryos gradually grew and developed into fully mature cotyledon embryos within the seeds (Figure 1C,D). The length of the cotyledon embryos was 1.92 ± 0.09 mm at the radicle protrusion stage, and the average E/S ratio increased to 42.6 ± 2.05% (Figure 1B,E). It took about 53 days for the heart-shaped embryo to fully develop at 15 °C. The seed embryos of H. thibetanus completed the process of morphological post-ripening, and the radicle was about to emerge at this stage (Figure 1B).

3.3. Effect of the Incubation Temperature on Radicle Emergence

The H. thibetanus seeds were tested at different stratification temperatures of 10, 15, 20, 25, 30, and 15/20 °C. Radicles emerged from the seeds after the heart-shaped embryos grew into mature cotyledon embryos. The seeds were considered to have a radicle that had emerged when at least 2 mm of the radicle was visible (Figure 2A). The seeds that were incubated at 15 °C, 20 °C, and 15/20 °C grew radicles, while none of the seeds had grown radicles at 10 °C, 25 °C, and 30 °C before the end of the experiment (180 d). Higher percentages (84.44% and 81.11%) were obtained at 15 and 15/20 °C, and the radicle emergence was completely inhibited at the higher (25 °C and 30 °C) and lower (10 °C) temperatures (Table 1). The embryos grew to the torpedo-shaped stage and ceased to develop at 25 °C and 30 °C. In addition, the inhibition of radicle emergence at 10 °C, 25 °C, and 30 °C was reversible, and seed radicles emerged when the seeds were transferred to the appropriate temperature for some time.
Similarly to the radicle emergence percentage, the temperature affected the initiation time and speed of radicle emergence. The shortest time for a radicle to emerge (58.33 d) was achieved at 15 °C, and the radicle emergence percentage increased to 84.44% after 135 d. Nevertheless, the longest mean radicle emergence time (79.33 days) was obtained in the seeds that were incubated at 20 °C. After 150 d of incubation, the seed radicle emergence percentage was 51.11% (Table 1, Figure 3). GA3, temperature (warm stratification), and their interaction had a noticeable effect on the radicle emergence percentage (Table 2). With the increase in the length of the incubation time, the radicle emergence percentage gradually increased at 15 °C, 20 °C, and 15/20 °C (Figure 3).

3.4. Effect of GA3 on Radicle Emergence

Compared with those of the corresponding control groups, the radicle emergence initiation times were earlier, and the radicle emergence rates increased after GA3 treatments at 10, 15, 15/20, and 20 °C. GA3 significantly affected the radicle emergence percentage at 10, 15, 15/20, and 20 °C (p < 0.01). However, a radicle did not emerge at 25 °C with or without the GA3 treatment. Of the different GA3 concentrations, 300 mg/L GA3 had the most significant effect on seed radicle emergence. After 60 days, the seed radicle emergence percentages reached 80.00% and 72.22% at 15 °C and 15/20 °C, respectively. In the corresponding groups at 15 °C and 15/20 °C, the radicle emergence percentages were 10.00% and 7.88%, respectively, after 60 days (Figure 3B,C). Interestingly, a radicle emergence percentage of 82.22% was obtained from the seeds treated with 300 mg/L GA3 at 10 °C after 135 days, but no radicles emerged in the control group (Figure 3A). In addition, the effect of GA3 on the seed radicle emergence rate and stratification time at 20 °C was not as significant as that under 10 °C, 15 °C, and 15/20 °C (Figure 3D).

3.5. Effects of Root Length and GA3 on the Epicotyl Dormancy of Seeds with Emerged Radicles

In the present study, a seed was only considered germinated when the epicotyl–plumule had grown to ≥5 mm (Figure 2B). In the control groups, the epicotyl–plumule emergence percentages of the seeds with root lengths of 2.5 cm and 1.5 cm were 92.22% and 82.22%, respectively, after cold stratification at 5 °C for 100 d, while the seeds with a root length of 0.5 cm roots did not have an emerged epicotyl–plumule (Figure 4). The results of the correlation analysis showed that there was a high correlation (0.860, p < 0.01) between root length and the final epicotyl–plumule emergence percentage in the control groups. GA3, cold stratification time and their interactions had apparent effects on the epicotyl–plumule emergence percentage (Table 3). Firstly, the influences of different root lengths on epicotyl–plumule germination were analyzed. The results (Figure 4) showed that the root length influenced the initiation time of epicotyl–plumule emergence and the emergence speed in H. thibetanus. The epicotyl–plumule emergence initiation time of a seed with a root length of 2.5 cm was 50 days, and the epicotyl–plumule emergence percentage was about 92.22% after incubation at 5 °C for 70 d. While the epicotyl–plumule emergence initiation time of seeds with root lengths of 1.5 cm and 0.5 cm were 60 and 110 days, the epicotyl–plumule emergence percentages were 91.11% and 52.22% after incubation for 110 d and 140 d. In addition, the effect of the number of days of cold stratification on epicotyl dormancy was analyzed. The epicotyl–plumule emergence percentage of H. thibetanus seeds with root lengths of 0.5 cm, 1.5 cm, and 2.5 cm gradually increased with the length of cold stratification (Figure 4). The epicotyl–plumule-emerged seeds were transferred to room temperature for growth. The seedlings lacked visible cotyledons, which stayed inside the seeds, and a three-lobed true leaf was expanded for photosynthesis (Figure 2C).
Then, the effect of GA3 on the epicotyl dormancy of H. thibetanus was analyzed. The results showed that GA3 shortened the epicotyl–plumule emergence initiation time and the emergence end time and increased the epicotyl–plumule emergence rate in H. thibetanus (Figure 4). In particular, the epicotyl–plumule emergence percentage of seeds with a root length of 0.5 cm was significantly enhanced, and it increased by 38.89%. The epicotyl–plumule emergence percentage was 52.22% in the control group, while it was 91.11% with the GA3 treatment. In addition, the epicotyl–plumule emergence initiation time was 50 d with the GA3 treatment, while it was 110 d in the control group. The epicotyl–plumule emergence initiation time was shortened by 60 d, and the cold stratification end time for epicotyl–plumule emergence was shortened by 70 d with the GA3 treatment. For seeds with root lengths of 1.5 cm and 2.5 cm, the epicotyl–plumule emergence rate increased slightly in the GA3 treatment, while the cold stratification time was considerably shortened compared to that in the control group. The cold stratification end time of epicotyl–plumule emergence was 60 d. All results indicated that GA3 affected the epicotyl dormancy release in H. thibetanus seeds. In GA3 treatments, the epicotyl–plumule emergence percentage of the seeds with a root length of 2.5 cm was 14.44% after cold stratification at 5 °C for 40 d, while the seeds with root lengths of 1.5 cm and 0.5 cm roots did not have an emerged epicotyl–plumule. After cold stratification at 5 °C for 50 d, the epicotyl–plumule emergence percentages of the seeds with a root length of 0.5, 1.5, and 2.5 cm were 11.11%, 71.11%, and 86.66%, respectively. The final epicotyl–plumule emergence percentages the seeds with a root length of 2.5, 1.5, and 0.5 cm were 98.86%, 93.33%, and 91.11%, respectively (Figure 4). The correlation coefficient between root length and the final epicotyl–plumule emergence percentage was 0.639, and there was no significant difference between them in the GA3 groups (p = 0.064).

4. Discussion

H. thibetanus is a shade-tolerant perennial with flowers blooming in winter or early spring, making it a valuable ornamental plant. Its low germination percentage and long germination time under natural conditions limit its large-scale artificial cultivation and application. Therefore, in the present study, the seed dormancy type and the requirements for seed germination of H. thibetanus were evaluated. The freshly matured seeds of H. thibetanus had an underdeveloped (heart-shaped) embryo. The E/S ratio was 8.58% (Figure 1). The radicles emerged from seeds only when differentiated embryos were fully developed and reached a certain E/S ratio or length [17]. In this work, the radicles emerged from H. thibetanus seeds after the embryos were fully grown, and the cotyledon embryo length was 1.92 mm. The E/S ratio increased from 8.58% (heart-shaped embryo) to 42.6% (cotyledon embryo) (Figure 1) before the radicle emerged. Similarly, the freshly matured seeds of other Ranunculaceae species, such as H. niger [19], Delphinium tricorne [31], Hepatica nobilis [32], Aconitum lycoctonum [33], Thalictrum rochebrunianum [34], Clematis hexapetala [27], Thalictrum squarrosum [35], and Thalictrum uchiyamae [36], also had small and underdeveloped embryos, and the embryos must reach a critical length within the seed before the radicle can emerge.
Both morphological dormancy and morphophysiological dormancy have incompletely developed embryos. A seed has morphological dormancy if the seed embryo begins to grow immediately at the appropriate temperature and the radicle emerges within 30 days. Morphophysiological dormancy in seeds involves both morphological dormancy and physiological dormancy. In addition, the seed radicle emerges after more than 30 days [17]. The results showed that the seeds of H. thibetanus had an underdeveloped embryo and grew radicles after about 58.33 days at 15 °C. Thus, the seeds of H. thibetanus exhibited morphophysiological dormancy.
Temperature is one of the main factors affecting the length of the seed dormancy period and germination [37,38]. The embryos of Epimedium koreanum seeds developed rapidly at a warm stratification temperature (25 °C). After the embryos fully develop, the seeds must undergo cold stratification before germination [39]. Temperature had an effective influence on the seed germination of Halenia elliptica. The effective temperature of cold stratification was less than 8 °C, and the seed germination percentage decreased with the increase in temperature [40]. Compared with other temperatures, 20 °C was the optimal seed germination temperature for Triticum aestivum [41]. Persian walnut seed germination required chilling and heating [42]. In addition, the chilling requirement affected the budbreak date of Persian walnut [43].
MPD can be divided into nine types according to the temperature requirements for embryo development, warm and/or cold stratification for germination, the timing of radicle and plumule emergence, and the responses of seeds to GA3 treatments [17]. The radicles emerged from seeds of H. thibetanus at 15 °C, 20 °C, and 15/20 °C, and the optimal temperature of radicle emergence was 15 °C, with 84.44% of the seeds growing radicles. This indicated that the embryo growth of H. thibetanus required warm stratification before the radicle emerged. No seed radicle emergence was observed at the lower (10 °C) or higher (25 °C and 30 °C) incubation temperatures (Table 1). The embryos of H. thibetanus developed to the torpedo stage at 25 °C and 30 °C, and the torpedo embryos did not develop even if incubation continued. Similarly, the embryos of H. niger grew to the torpedo stage at 20 °C or higher temperatures and further developed to the cotyledon stage only at 15 °C or lower temperatures [19]. In contrast, the seeds of H. foetidu needed treatment at 25 °C for at least 6 weeks before the embryos reached maturity [44]. Stratification could promote the metabolism of seeds, and the contents of stored substances were gradually degraded during stratification [45].
GA3 is also one of the main factors affecting the duration of seed germination. GA3 promotes seed germination in Ricinus communis and Panax notoginseng [46,47]. The GA3 treatment—especially 300 mg/L GA3—significantly increased the radicle emergence rate of H. thibetanus seeds at 10 °C, 15 °C, 20 °C, and 15/20 °C, and it was also effective in shortening the time required for warm stratification, especially at 15 °C (Figure 3), indicating that GA3 could partially replace warm stratification for H. thibetanus. There have been similar findings for other species. GA3 significantly improved the germination rate and promoted the completion of physiological post-ripening in H. foetidus seeds [44]. In Paeonia, GA3 treatment promoted seed embryo development and increased germination percentages [48]. During the development of Jeffersonia dubia seed embryos, warm temperature requirements could be replaced by GA3, while the cold temperature requirement could not be replaced [49]. The content of ABA was higher in the dormant stage of the seeds, whereas the content of GAs increased during embryo development in Epimedium pseudowushanense [50].
Seeds that grew radicles and from which plumules emerged after 3–4 weeks could be classified as having epicotyl MPD [17]. In our study, the epicotyl–plumule of H. thibetanus seeds never germinated after the radicle emerged at 15 °C. Therefore, we concluded that the seeds of H. thibetanus also exhibited epicotyl dormancy. Depending on cold stratification requirements, epicotyl MPD could be classified into two types: deep simple and non-deep simple epicotyl MPD [17]. Seeds from which a radicle emerged germinated epicotyl–plumules after 50 d under cold temperature (5 °C) stratification (Figure 4). This indicated that the seeds of H. thibetanus were likely in deep simple epicotyl MPD. Similar phenomena have occurred in other plants, such as Lilium polyphyllum [51], Allium burdickii, Asarum canadensis, Cimicifuga racemosa, Hydrophyllum appendiculatum, Hydrophyllum macrophyllum, Sanguinaria canadensis [17], and Gymnospermium microrrhynchum [52]. In Helleborus, seeds of H. niger with a fully developed embryo only germinated plumules when they were transferred to 4 °C conditions [19].
Physiological dormancy in the epicotyl required cold temperature stratification, and only the emerged radicle is sensitive to low temperatures [53]. Seeds with a root length of at least 6.0 cm responded to cold stratification and then broke their epicotyl dormancy in Paeonia ludlowii [54]. The epicotyl–plumule emergence percentage of H. thibetanus increased with the duration of cold stratification. When incubated at 5 °C for 70 d, 91.11% of the seeds with 2.5 cm long roots had emerged epicotyl–plumules, and 47.78% of the seeds with 1.5 cm long roots had emerged epicotyl–plumules, while no epicotyl–plumule emerged in seeds with 0.5 cm long roots until 100 d later (Figure 4). Our results showed that the length of the radicle markedly affected the response of the epicotyl physiological dormancy to cold stratification in H. thibetanus.
GA3 treatment could break epicotyl dormancy instead of cold stratification in some species, such as Paeonia ostii [55]. Zhang et al. (2022) found that the epicotyl dormancy of P. ostii might be due to low GA content [56]. In H. thibetanus, the 400 mg/L GA3 treatment partially replaced cold temperature stratification (5 °C). The time needed for cold temperature stratification was significantly shortened, and the epicotyl–plumule emergence rate increased in seeds with roots that were 0.5, 1.5, and 2.5 cm long (Figure 4). These results show that H. thibetanus seeds undergo not only morphophysiological dormancy but also epicotyl physiological dormancy. H. thibetanus seeds fit the characteristics of a deep simple epicotyl morphophysiological dormancy [19]. In addition, embryo development and growth, radicle emergence, and epicotyl–plumule germination can be considered three important steps in the seeds of H. thibetanus.
Seed dispersal in H. thibetanus plants occurs from late May to early June in the Qingling Mountains, and they do not germinate until the following spring. At this point, the soil temperature is >20 °C. Seed embryos develop to a torpedo-shaped embryo stage and stay dormant in summer until autumn when temperatures fall, and the torpedo-shaped embryo continues to develop and grow into a full cotyledon embryo when soil temperatures drop below 20 °C. Seed radicles emerge only when the E/S ratio reaches a certain ratio (ca. 42.6%). Subsequently, the radicle grows continuously. Seeds with roots break their epicotyl dormancy at low winter temperatures, and epicotyl–plumules germinate. The seedlings grow when the temperature increases in spring. In Helleborus, the seeds of H. niger and H. foetidus had morphophysiological dormancy [19,44]. However, the seeds of H. thibetanus had epicotyl morphophysiological dormancy. Seed dormancy is an adaptive trait that can inhibit immediate germination and then waits for the favorable growing season, which is the most advantageous for seedling growth [30,57]. Thus, the seed dormancy characteristics of H. thibetanus may be related to the environmental conditions of its natural distribution area. H. thibetanus is a deciduous perennial plant and has summer dormancy. The shoots emerge in early spring. The epicotyl dormancy characteristic can prevent seedlings from being damaged by frost in winter. In H. thibetanus, embryo development, radicle growth, and the breaking of epicotyl dormancy require a specific temperature and a relatively long time. Under natural conditions, the new germination of seedlings is particularly rare in natural plant populations.

5. Conclusions

The whole process of seed germination was studied in H. thibetanus. The fresh seeds of H. thibetanus had underdeveloped (heart-shaped) embryos at maturity in summer. Before radicle emergence, the E/S ratio increased from 8.58% to 42.6% with warm temperature stratification. The optimal temperature for radicle emergence was 15 °C. The radicle emergence percentage gradually increased with the increase in the warm stratification time, and GA3 shortened the duration of warm temperature stratification. In addition, H. thibetanus seeds also exhibited physiological dormancy in the epicotyl. Cold temperature (5 °C) stratification could release seed epicotyl dormancy, and the root length and cold stratification time affected epicotyl–plumule emergence. GA3 could offset partly the effects of root length and the duration of cold stratification on the release of epicotyl dormancy. The results indicated that H. thibetanus seeds had deep simple epicotyl morphophysiological dormancy. Our study is the first to determine the germination requirements and seed dormancy type of H. thibetanus.

Author Contributions

Conceptualization, methodology, writing and editing, X.Z.; sample collection, F.W. and L.W.; formal analysis, Q.W. and A.L.; writing—review and editing, Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Science and Technology Program of Shaanxi Academy of Sciences (grant number 2021k-17), Science and Technology Planning Project of Xi’an city (grant number 21NYYF0042), Science and Technology Project of Shaanxi Province (grant number 2022ZDLNY03-09), and the project of the first investigation of wild plant resources in Xi’an (grant number [KRDL] K6-2207039).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Acknowledgments

We would like to express our sincere thanks to Ming Yue and Ying Zhang for their assistance in seeds collection.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Wang, W.C. Flora Republicae Popularis Sinicae; Science Press: Beijing, China, 1979; Volume 27, p. 106. [Google Scholar]
  2. Guo, Z.J.; Wang, J.X.; Su, Y.F.; Wei, Y.X.; Zhu, Y.H. Shaanxi Qiyao; Shaanxi Science and Technology Press: Xi’an, China, 2003; p. 35. [Google Scholar]
  3. Meiners, J.; Debener, T.; Schweizer, G.; Winkelmann, T. Analysis of the taxonomic subdivision within the genus Helleborus by nuclear DNA content and genome-wide DNA markers. Sci. Hortic. 2011, 128, 38–47. [Google Scholar] [CrossRef]
  4. Zonneveld, B.J.M. Nuclear DNA contents of all species of Helleborus (Ranunculaceae) discriminate between species and sectional divisions. Plant Syst. Evol. 2001, 229, 125–130. [Google Scholar] [CrossRef]
  5. Yang, F.Y.; Su, Y.F.; Wang, Y.; Chai, X.; Han, X.; Wu, Z.H.; Gao, X.M. Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus (Ranunculaceae). Biochem. Syst. Ecol. 2010, 38, 759–763. [Google Scholar] [CrossRef]
  6. Yang, J.; Zhang, Y.; Miao, F.; Zhou, L.; Sun, W. Two new bufadienolides from the rhizomes of Helleborus thibetanus Franch. Fitoterapia 2010, 81, 636–639. [Google Scholar] [CrossRef] [PubMed]
  7. Zhang, H.; Su, Y.F.; Yang, F.Y.; Zhao, Z.Q.; Gao, X.M. Two new bufadienolides and one new pregnane from Helleborus thibetanus. Phytochem. Lett. 2014, 10, 164–167. [Google Scholar] [CrossRef]
  8. Zhang, H.; Su, Y.F.; Yang, F.Y. Three new steroidal saponins from Helleborus thibetanus. Nat. Prod. Res. 2016, 30, 15. [Google Scholar] [CrossRef] [PubMed]
  9. Zhang, H.; Su, Y.F.; Yang, F.Y. Four new minor spirostanol glycosides from Helleborus thibetanus. Phytochem. Lett. 2016, 18, 213–219. [Google Scholar] [CrossRef]
  10. Liu, Z.; Liu, Y.; Xue, B.; Chen, W.; Xu, W.; Jiang, R. The co-occurrence of bufadienolides and podophyllotoxins from Helleborus thibetanus. Biochem. Syst. Ecolo. 2020, 90, 104042. [Google Scholar] [CrossRef]
  11. Zhao, L.; Liu, P.; Che, X.F.; Wang, W.; Ren, Y. Floral organogenesis of Helleborus thibetanus and Nigella damascena (Ranunculaceae) and its systematic significance. Bot. J. Linn. Soc. 2011, 166, 431–443. [Google Scholar] [CrossRef]
  12. Shi, X.H.; Zou, Q.C.; Jin, L.; An, X.; Zhou, J.H.; Ma, G.Y. Comparative research on pollen fertility and morphology of Helleborus thibetanus. Mol. Plant Breed. 2018, 16, 1690–1697. [Google Scholar] [CrossRef]
  13. Liu, P.; Song, L.; Ren, Y.; Tian, X.H.; Zhang, X.H. Vegetative organ structures and their phylogenic significance of Helleborus thibetanus franch. (Ranunculaceae). Acta Bot. Boreal.-Occident. Sin. 2006, 26, 2208–2213. [Google Scholar]
  14. Shi, X.H.; Ma, G.Y.; Zou, Q.C.; Tian, D.Q. Preliminary analysis of transcriptome information and SSR markers in young leaves of Helleborus thibetanus. Mol. Plant Breed. 2019, 17, 3297–3304. [Google Scholar] [CrossRef]
  15. Nonogaki, H. Seed germination and dormancy: The classic story, new puzzles, and evolution. J. Integr. Plant Biol. 2019, 61, 541–563. [Google Scholar] [CrossRef]
  16. Baskin, J.M.; Baskin, C.C. Some considerations for adoption of Nikolaeva’s formula system into seed dormancy classification. Seed Sci. Res. 2008, 18, 131–137. [Google Scholar] [CrossRef]
  17. Baskin, C.C.; Baskin, J.M. Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination, 2nd ed.; Elsevier/Academic Press: San Diego, CA, USA, 2014; pp. 33–77. [Google Scholar]
  18. Zhao, X.Y.; Wang, L.; Wang, F.Y.; Zhou, Y.F.; Li, Y. Anatomical study of Helleborus thibetanus seed embryo during the morphological post-ripening process. Shaanxi J. Agr. Sci. 2021, 67, 56–58. [Google Scholar]
  19. Niimi, Y.; Han, D.; Abe, S. Temperature affecting embryo development and seed germination of Christmas rose (Helleborus niger) after sowing. Sci. Hortic. 2006, 107, 292–296. [Google Scholar] [CrossRef]
  20. Jang, B.K.; Park, K.; Lee, S.Y.; Lee, H.; Song, S.K.; Kim, J.; Lee, C.H.; Cho, J.S. Comparison of the seed dormancy and germination characteristics of six Clematis species from South Korea. Sci. Hortic. 2023, 307, 11488. [Google Scholar] [CrossRef]
  21. Finch-Savage, W.E.; Leubner-Metzger, G. Seed dormancy and the control of germination. New Phytol. 2006, 171, 501–523. [Google Scholar] [CrossRef] [PubMed]
  22. Yang, L.E.; Peng, D.L.; Li, Z.M.; Huang, L.; Yang, J.; Sun, H. Cold stratification, temperature, light, GA3, and KNO3 effects on seed germination of Primula beesiana from Yunnan, China. Plant Divers. 2020, 42, 168–173. [Google Scholar] [CrossRef] [PubMed]
  23. Xiong, M.; Chu, L.Y.; Li, Q.F.; Yu, J.W.; Yang, Y.H.; Zhou, P.; Zhou, Y.; Zhang, C.Q.; Fan, X.L.; Zhao, D.S.; et al. Brassinosteroid and gibberellin coordinate rice seed germination and embryo growth by regulating glutelin mobilization. Crop J. 2021, 9, 1039–1048. [Google Scholar] [CrossRef]
  24. He, D.; Lv, B.; Wang, X.; Gao, X.; Wang, Z.; Liu, Y.; He, S. Effects of exogenous hormone GA3, 6-BA and root length on breaking of epicotyls dormancy of Paeonia ostii ‘Fengdanbai’ mature embryo. J. Henan Agr. Sci. 2015, 44, 122–126. [Google Scholar]
  25. Liu, A.; Zhang, M.; Yuan, Q.; Wan, Y. Dormancy breaking technologies of Paeonia emodi seeds. Seed 2023, 42, 147–156. [Google Scholar] [CrossRef]
  26. Nonogaki, H. Seed dormancy and germination-emerging mechanisms and new hypotheses. Front. Plant Sci. 2014, 5, 233. [Google Scholar] [CrossRef] [PubMed]
  27. Zhang, K.; Ji, Y.; Song, X.; Yao, L.; Liu, H.; Tao, J. Deep complex morphophysiological dormancy in seeds of Clematis hexapetala Pall. (Ranunculaceae). Sci. Hortic. 2021, 286, 110247. [Google Scholar] [CrossRef]
  28. Liu, Y.; Qiu, Y.P.; Zhang, L.; Chen, J. Dormancy breaking and storage behavior of Garcinia cowa Roxb. (Guttiferae) seeds: Implications for ecological function and germplasm conservation. J. Integr. Plant Biol. 2005, 47, 38–49. [Google Scholar] [CrossRef]
  29. Yang, G.; Yang, L.; Wang, Y.; Shen, S. Physiological epicotyl dormancy and its alleviation in seeds of Yunnanopilia longistaminea: The first report of physiological epicotyl dormancy in China. Peer J. 2017, 5, e3435. [Google Scholar] [CrossRef] [PubMed]
  30. Jaganathan, G.K. Ecological insights into the coexistence of dormancy and desiccation-sensitivity in Arecaceae species. Ann. For. Sci. 2021, 78, 10. [Google Scholar] [CrossRef]
  31. Baskin, C.C.; Baskin, J.M. Deep complex morphophysiological dormancy in seeds of the mesic woodland herb Delphinium tricorne (Ranunculaceae). Int. J. Plant Sci. 1994, 155, 738–743. [Google Scholar] [CrossRef]
  32. Nomizu, T.; Niimi, Y.; Watanabe, E. Embryo development and seed germination of Hepatica nobilis Schreber var. japonica as affected by temperature after sowing. Sci. Hortic. 2004, 99, 345–352. [Google Scholar] [CrossRef]
  33. Vandelook, F.; Van Assche, J.A. Temperature conditions control embryo growth and seeds germination of Corydalis solida (L.) Clairv., a temperate forest spring geophyte. Plant Biol. 2009, 11, 899–906. [Google Scholar] [CrossRef]
  34. Lee, S.Y.; Rhie, Y.H.; Kim, K.S. Non-deep simple morphophysiological dormancy in seeds of Thalictrum rochebrunianum, an endemic perennial herb in the Korean peninsula. Hortic. Environ. Biotechnol. 2015, 56, 366–375. [Google Scholar] [CrossRef]
  35. Zhang, K.; Ji, Y.; Fu, G.; Yao, L.; Liu, H.; Tao, J. Dormancy-breaking and germination requirements of Thalictrum squarrosum Stephan ex Willd. seeds with underdeveloped embryos. J. Appl. Res. Med. Aromat. Plants 2021, 24, 100311. [Google Scholar] [CrossRef]
  36. Lee, S.Y.; Rhie, Y.H.; Kim, K.S. Dormancy breaking and germination requirements of seeds of Thalictrum uchiyamae (Ranunculaceae) with underdeveloped embryos. Sci. Hortic. 2018, 231, 82–88. [Google Scholar] [CrossRef]
  37. Baskin, C.C.; Baskin, J.M. Germination ecophysiology of herbaceous plant species in a temperate region. Am. J. Bot. 1988, 75, 286–305. [Google Scholar] [CrossRef]
  38. Pawłowski, T.A.; Bujarska-Borkowska, B.; Suszka, J.; Tylkowski, T.; Chmielarz, P.; Klupczyńska, E.A.; Staszak, A.M. Temperature Regulation of Primary and Secondary Seed Dormancy in Rosa canina L.: Findings from Proteomic Analysis. Int. J. Mol. Sci. 2020, 21, 7008. [Google Scholar] [CrossRef]
  39. Rhie, Y.H.; Lee, S.Y. Seed dormancy and germination of Epimedium koreanum Nakai. Sci. Hortic. 2020, 272, 109600. [Google Scholar] [CrossRef]
  40. Chen, D.L.; Luo, X.P.; Yuan, Z.; Bai, M.J.; Hu, X.W. Seed dormancy release of Halenia elliptica in response to stratification temperature, duration and soil moisture content. BMC Plant Biol. 2020, 20, 352. [Google Scholar] [CrossRef] [PubMed]
  41. Khaeim, H.; Kende, Z.; Balla, I.; Gyuricza, C.; Eser, A.; Tarnawa, A. Temperature and water stresses on seed germination and seedling growth of wheat (Triticum aestivum L.). Sustainability 2022, 14, 3887. [Google Scholar] [CrossRef]
  42. Vahdati, K.; Aslamarz, A.A.; Rahemi, M.; Hassani, D.; Leslie, C. Mechanism of seed dormancy and its relationship to bud dormancy in Persian walnut. Environ. Exp. Bot. 2012, 75, 74–82. [Google Scholar] [CrossRef]
  43. Hassankhah, A.; Vahdati, K.; Rahemi, M.; Hassani, D.; Khorami, S.S. Persian walnut phenology: Effect of chilling and heat requirements on budbreak and flowering date. Int. J. Hort. Sci. Technol. 2017, 4, 259–271. [Google Scholar] [CrossRef]
  44. Yang, L.; Shi, X.; Du, L. Effects of temperature and gibberellin on seed germination of Helleborus foetidus. Seed 2022, 41, 114–119. [Google Scholar] [CrossRef]
  45. Li, F.H.; Yu, P.; Song, C.H.; Wu, J.J.; Tian, Y.; Wu, X.F.; Zhang, X.W.; Liu, Y.M. Differential protein analysis of Heracleum moellendorffii Hance seeds during Stratification. Plant Physiol. Biochem. 2019, 145, 10–20. [Google Scholar] [CrossRef] [PubMed]
  46. Nurgül, E.; Muhammed, F.K.; Mehmet, D.K. GA3 acts as a germination promoter in wild castor bean (Ricinus communis L.). Seed Sci. Technol. 2022, 50, 295–306. [Google Scholar] [CrossRef]
  47. Ge, N.; Jia, J.S.; Yang, L.; Huang, R.; Wang, Q.; Chen, C.; Meng, Z.; Li, L.; Chen, J. Exogenous gibberellic acid shortening after-ripening process and promoting seed germination in a medicinal plant Panax notoginseng. BMC Plant Biol. 2023, 23, 67. [Google Scholar] [CrossRef]
  48. Zhang, K.; Yao, L.; Zhang, Y.; Baskin, J.M.; Baskin, C.C.; Xiong, Z.; Tao, J. A review of the seed biology of Paeonia species (Paeoniaceae), with particular reference to dormancy and germination. Planta 2019, 249, 291–303. [Google Scholar] [CrossRef] [PubMed]
  49. Rhie, Y.H.; Lee, S.Y.; Kim, K.S. Seed dormancy and germination in Jeffersonia dubia (Berberidaceae) as affected by temperature and gibberellic acid. Plant Biol. 2015, 17, 327–334. [Google Scholar] [CrossRef] [PubMed]
  50. Ma, Y.; Chen, X.; Guo, B. Identification of genes involved in metabolism and signalling of abscisic acid and gibberellins during Epimedium pseudowushanense B.L. Guo seed morphophysiological dormancy. Plant Cell Rep. 2018, 37, 1061–1075. [Google Scholar] [CrossRef] [PubMed]
  51. Dhyani, A.; Phartyal, S.S.; Nautiyal, B.P.; Nautiyal, M.C. Epicotyl morphophysiological dormancy in seeds of Lilium polyphyllum (Liliaceae). J. Biosci. 2013, 38, 13–19. [Google Scholar] [CrossRef]
  52. Rhie, Y.H.; Lee, S.Y.; Kim, K.S. Deep simple epicotyl morphophysiological dormancy in seeds of Gymnospermium microrrhynchum (S. Moore) Takht., a rare species in Korea. Hortic. Sci. Techol. 2019, 37, 669–677. [Google Scholar] [CrossRef]
  53. Copete, E.; Herranz, J.M.; Ferrandis, P.; Baskin, C.C.; Baskin, J.M. Physiology, morphology and phenology of seed dormancy break and germination in the endemic Iberian species Narcissus hispanicus (Amaryllidaceae). Ann. Bot. 2011, 107, 1003–1016. [Google Scholar] [CrossRef]
  54. Hao, H.P.; He, Z.; Li, H.; Shi, L.; Tang, Y.D. Effect of root length on epicotyl dormancy release in seeds of Paeonia ludlowii, Tibetan peony. Ann. Bot. Lond. 2014, 113, 443–452. [Google Scholar] [CrossRef] [PubMed]
  55. Wang, H.; Van Staden, J. Seedling establishment characteristics of Paeonia ostii var. lishizhenii. S Afr. J. Bot. 2002, 68, 382–385. [Google Scholar] [CrossRef]
  56. Zhang, K.; Pan, H.; Baskin, C.C.; Baskin, J.M.; Xiong, Z.; Cao, W.; Yao, L.; Tang, B.; Zhang, C.; Tao, J. Epicotyl morphophysiological dormancy in seeds of Paeonia ostii (Paeoniaceae): Seasonal temperature regulation of germination phenology. Environ. Exp. Bot. 2022, 194, 104742. [Google Scholar] [CrossRef]
  57. Koornneef, M.; Bentsink, L.; Hilhorst, H. Seed dormancy and germination. Curr. Opin. Plant Biol. 2002, 5, 33–36. [Google Scholar] [CrossRef]
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Figure 1. The developmental stages of the embryo and the embryo/seed length ratio of Helleborus thibetanus seeds: (A,C) the heart-shaped embryo of fresh seed at harvest time; (B,D) the cotyledon embryo of seed at the radicle protrusion stage. The arrowheads showed the radicle that was about to emerge. (E) embryo/seed length ratio during seed morphological post-ripening process. Scale bars: (A,B) = 0.5 mm; (C,D) = 0.25 mm. Error bars are ±SD (n = 10).
Figure 1. The developmental stages of the embryo and the embryo/seed length ratio of Helleborus thibetanus seeds: (A,C) the heart-shaped embryo of fresh seed at harvest time; (B,D) the cotyledon embryo of seed at the radicle protrusion stage. The arrowheads showed the radicle that was about to emerge. (E) embryo/seed length ratio during seed morphological post-ripening process. Scale bars: (A,B) = 0.5 mm; (C,D) = 0.25 mm. Error bars are ±SD (n = 10).
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Figure 2. Germination process of Helleborus thibetanus seed: (A) radicle emergence stage; (B) epicotyl-plumule emergence stage; (C) the expansion of the first true leaf.
Figure 2. Germination process of Helleborus thibetanus seed: (A) radicle emergence stage; (B) epicotyl-plumule emergence stage; (C) the expansion of the first true leaf.
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Figure 3. The effects of stratification time, temperature and GA3 on radicle emergence in Helleborus thibetanus: (A) the seeds were incubated at 10 °C; (B) the seeds were incubated at 15 °C; (C) the seeds were incubated at 15/20 °C; (D) the seeds were incubated at 20 °C. Error bars are ±SD (n = 3). Different lowercase letters indicated significant differences among different GA3 concentrations (control, 100, 300, 500 mg/L) at each incubation temperature (p < 0.05) using Tukey test.
Figure 3. The effects of stratification time, temperature and GA3 on radicle emergence in Helleborus thibetanus: (A) the seeds were incubated at 10 °C; (B) the seeds were incubated at 15 °C; (C) the seeds were incubated at 15/20 °C; (D) the seeds were incubated at 20 °C. Error bars are ±SD (n = 3). Different lowercase letters indicated significant differences among different GA3 concentrations (control, 100, 300, 500 mg/L) at each incubation temperature (p < 0.05) using Tukey test.
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Figure 4. The effects of cold stratification time (5 °C) and GA3 on epicotyl dormancy breaking in Helleborus thibetanus seeds. The seeds which emerged radicle were incubated at 5 °C until the epicotyl-plumule emerged: (A) the seeds with a root length of 0.5 cm; (B) the seeds with a root length of 1.5 cm; (C) the seeds with a root length of 2.5 cm. Error bars are ±SD (n = 3). * indicates significant differences between control and GA3 treatment at the same root length (p < 0.05) using Tukey test. ns: indicates no significant differences between control and GA3 treatment at root lengths of 1.5 cm and 2.5 cm using Tukey test.
Figure 4. The effects of cold stratification time (5 °C) and GA3 on epicotyl dormancy breaking in Helleborus thibetanus seeds. The seeds which emerged radicle were incubated at 5 °C until the epicotyl-plumule emerged: (A) the seeds with a root length of 0.5 cm; (B) the seeds with a root length of 1.5 cm; (C) the seeds with a root length of 2.5 cm. Error bars are ±SD (n = 3). * indicates significant differences between control and GA3 treatment at the same root length (p < 0.05) using Tukey test. ns: indicates no significant differences between control and GA3 treatment at root lengths of 1.5 cm and 2.5 cm using Tukey test.
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Table 1. Effects of temperature on Helleborus thibetanus seed radicle emergence. The duration of the seed radicle emergence experiment was 180 days. Data are mean ± standard deviation (n = 3). Means within a column with a different uppercase letter at 15, 15/20, and 20 °C showed a significant difference using Tukey test at p < 0.05.
Table 1. Effects of temperature on Helleborus thibetanus seed radicle emergence. The duration of the seed radicle emergence experiment was 180 days. Data are mean ± standard deviation (n = 3). Means within a column with a different uppercase letter at 15, 15/20, and 20 °C showed a significant difference using Tukey test at p < 0.05.
Temperature (°C)Radicle Emergence Starting Time (d)Radicle Emergence Duration (d)Radicle Emergence Percentage (%)
100.00 ± 0.000.00 ± 0.000.00 ± 0.00
1558.33 ± 1.53 C75.33 ± 0.58 A84.44 ± 1.93 A
2079.33 ± 2.89 A70.67 ± 2.89 A51.11 ± 5.09 B
15/2062.67 ± 1.15 B74.67 ± 4.51 A81.11 ± 1.92 A
250.00 ± 0.000.00 ± 0.000.00 ± 0.00
300.00 ± 0.000.00 ± 0.000.00 ± 0.00
Table 2. Two-way analysis (ANOVA) for the effects of incubation temperature and GA3 on seed radicle emergence in Helleborus thibetanus.
Table 2. Two-way analysis (ANOVA) for the effects of incubation temperature and GA3 on seed radicle emergence in Helleborus thibetanus.
Source of Variations SSdfMSFpR2CV
Model24,073.659151604.911135.89<0.01
Error377.9343211.810
Corrected total 24,451.593
Incubation temperature11,686.69633895.565329.84<0.01
GA35647.90731882.636159.40<0.01
Interactions6739.0569748.78463.40<0.010.9854.833
Note: SS: sum of squares; df: degrees of freedom; MS: mean square; F: ratio. Effects are considered significant or very significant for p < 0.01. R2: the proportion of variation explained by variables; CV: coefficient of variation (%).
Table 3. Two-way analysis (ANOVA) for the effects of GA3 and cold stratification time on epicotyl-plumule emergence of seeds with root lengths 2.5, 1.5, and 0.5 cm in Helleborus thibetanus.
Table 3. Two-way analysis (ANOVA) for the effects of GA3 and cold stratification time on epicotyl-plumule emergence of seeds with root lengths 2.5, 1.5, and 0.5 cm in Helleborus thibetanus.
Source of VariationsSSdfMSFpR2CV
Root length of 0.5 cm
Model46,314.14995146.017217.003<0.01
Error474.2822023.714
Corrected total46,788.43129
GA320,453.530120,453.530862.506<0.01
Cold stratification time18,519.72744629.932195.240<0.01
GA3 × Cold stratification time7340.89341835.22377.390<0.010.99011.267
Root length of 1.5 cm
Model46,742.27695193.586292.157<0.01
Error355.5342017.777
Corrected total47,097.80929
GA33413.12013413.120192.000<0.01
Cold stratification time36,598.09849149.524514.693<0.01
GA3 × Cold stratification time6731.05841682.76494.661<0.010.9926.588
Root length of 2.5 cm
Model45,703.85295078.206274.113<0.01
Error370.5192018.526
Corrected total46,074.37129
GA31920.32011920.320103.656<0.01
Cold stratification time40,614.807410,513.702548.080<0.01
GA3 × Cold stratification time3168.7244792.18142.761<0.010.9926.053
Note: SS: sum of squares; df: degrees of freedom; MS: mean square; F: ratio. Effects are considered significant or very significant for p < 0.01. R2: the proportion of variation explained by variables; CV: coefficient of variation (%).
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Zhao, X.; Wang, F.; Wang, L.; Wang, Q.; Liu, A.; Li, Y. Deep Simple Epicotyl Morphophysiological Dormancy in Seeds of Endemic Chinese Helleborus thibetanus. Agriculture 2024, 14, 1041. https://doi.org/10.3390/agriculture14071041

AMA Style

Zhao X, Wang F, Wang L, Wang Q, Liu A, Li Y. Deep Simple Epicotyl Morphophysiological Dormancy in Seeds of Endemic Chinese Helleborus thibetanus. Agriculture. 2024; 14(7):1041. https://doi.org/10.3390/agriculture14071041

Chicago/Turabian Style

Zhao, Xueyan, Fangyuan Wang, Li Wang, Qing Wang, Ancheng Liu, and Yan Li. 2024. "Deep Simple Epicotyl Morphophysiological Dormancy in Seeds of Endemic Chinese Helleborus thibetanus" Agriculture 14, no. 7: 1041. https://doi.org/10.3390/agriculture14071041

APA Style

Zhao, X., Wang, F., Wang, L., Wang, Q., Liu, A., & Li, Y. (2024). Deep Simple Epicotyl Morphophysiological Dormancy in Seeds of Endemic Chinese Helleborus thibetanus. Agriculture, 14(7), 1041. https://doi.org/10.3390/agriculture14071041

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