Morphological and Seed Germination Behavior of Three Herba Swertiae Species from Hulunbuir, Inner Mongolia: Temperature and Substrate Effects

Abstract

The Mongolian medicinal herb Herba Swertiae is a group of critically endangered medicinal herbs that are difficult to obtain from the wild. This is due to their low percentage of seed germination and difficult domestication. The aim of this study was to investigate the suitable germination conditions for enabling large-scale cultivation and improving the breeding efficiency of these herbs. We used the seeds of Lomatogonium rotatum (L.) Fries ex Nym, Gentianopsis barbata (Froel.) Ma., and Parnassis palustris L. to study their morphological characteristics and the required germination temperature and substrate. Results showed that there were aages outside the seed coats of all three Herba Swertiae collected from Hulunbuir, which had a binding effect on the primary root breaking through the seed coat to form seedlings. In addition, the seeds showed a significantly higher germination rate at 25 °C, and the percentage of seeds germinating in the mixed medium of soil and vermiculite at a 1:1 ratio was significantly higher than on filter paper. We conclude that the morphological characteristics of the seeds have great significance in promoting seed germination. Germination substrate, as an important medium, and temperature, as an important environmental condition, play crucial roles in plant seed germination.

1. Introduction

Herba Swertiae is a genus of widely used medicinal herb in Traditional Mongolian Medicine (TMM), which has a cold nature, and the taste is extremely bitter. Herba Swertiae has a significant importance in clinical therapy, with notable actions of heat-clear and detoxication effects [1,2]. It has been used as the main composition or in combination with other medicinal herbs to prescribe various TMM formulas, such as swertiae-4 decoction, swertiae-8 decoction, and swertiae-10 powder. In TMM clinical practice, these traditional formulae are mainly used to treat jaundice hepatitis, acute enteritis, dysentery, cholecystitis, and pneumonia [3,4,5,6]. Thus, this genus of medicinal herbs has contributed significantly toward TMM medical treatment and the healthy careers of Mongolian nomads. In this study, we mainly focus on the following three TMM medicinal herbs of Herba Swertiae: Lomatogonium rotatum (L.) Fries ex Nym (L. rotatum), Gentianopsis barbata (Froel.) Ma., (G. barbata), and Parnassis palustris L. (P. palustris) to investigate their morphological characteristics and suitable germination conditions. At present, all Herba Swertiae species utilized in manufacturing are collected from wild plants. Because the entire grass is used as medicine, harvesters frequently remove the entire plant during the flowering stage, and the majority of the remaining plants are removed together with the grass when the seeds are immature and the grass is weeded in mid-August on the grassland. There are only a few plant materials surviving on the meadow afterwards. Therefore, artificial domestication has become the primary means of protecting and supplying medicinal resources from the wild.

Temperature is considered the most influential environmental factor for seed germination. Each plant has a unique optimal range for germination temperatures, and it influences the germination process through seed imbibition and biochemical reactions that govern the metabolism [7,8]. In addition, a suitable growing substrate provides appropriate anchoring or support for the plant, a reservoir for nutrients and water, and oxygen and gas exchange between the roots and the atmosphere. Soil and vermiculite are used as a basic medium for planting seeds in nurseries because they are inexpensive and easy to obtain. Vermiculite has been used for years to modify peat-moss potting soils and in horticulture because it promotes aeration, drainage, and water retention [9]. Therefore, the aim of this study was to examine the morphological and germination characteristics of three species of Herba Swertiae seeds in order to find suitable germination conditions for enabling large-scale cultivation and improving the breeding efficiency of these herbs.

2. Materials and Methods

2.1. Materials

The samples used in this study included all wild species collected from Xishan in Hailer, Hulunbuir, Inner Mongolia. All seeds were collected from the wild plants in July 2021, air dried in the laboratory, and stored in kraft paper bags at room temperature until use. The details are shown in

Table 1. Time and location of sample collection.

Plant Name	Geographic Location	Time	Parts
Lomatogonium rotatum (L.) Fries ex Nym	49°11′27″ N 119°44′1″ E	10 August 2021	Mature seeds
Gentianopsis barbata (Froel.) Ma.	49°11′27″ N 119°44′1″ E	10 August 2021	Mature seeds
Parnassis palustris L.	49°11′27″ N 119°44′1″ E	10 August 2021	Mature seeds

.

2.2. Methods

2.2.1. External Characteristic Observation of Seeds

The shape, color, and size of the seeds were observed with a microscope (Leica ZOOM 2000), and width and length of the seeds were measured by Vernier caliper.

2.2.2. Measurement of the Weight of 1000 Seeds

A 100-grain weight determination method was used to measure the weight of 1000 seeds. Six groups of seeds from three wild species were randomly selected, with 100 seeds in each group. Then, the seeds were weighed on a ten-thousandth electronic balance, and the data were recorded. Finally, the weight of 1000 seeds was calculated by the mean value of six repetitions.

2.2.3. Seed Germination Test

According to the “International Rules for Seed Testing” [10] and other germination conditions of Mongolian herbs, germination was carried out on filter paper and a mixed substrate (soil:vermiculite = 1:1). The mold-free and full seeds from three wild species were selected to be dipped in 70% ethanol for 5 min. Then, these seeds were rinsed three to five times with distilled water and placed in a petri dish in rows. Germination was performed in a light incubator at 15 °C, 20 °C, and 25 °C, respectively. Every petri dish had 100 seeds, and each species had three repeats. The number of germinated seeds was counted daily. The germination period was 30 days, after which the germination percentage was calculated.

2.3. Indicator Determination

The germination percentage was determined by the following steps. The primary root grown to half of the seed’s length was considered standard germination. The number of germinated seeds was counted every day. If there were no new germinated seeds for three consecutive days, the seeds in the petri dish were considered dead, and that was the end of germination.

Germination percentage = (total germinated seeds at the end of germination/total seeds) × 100%

2.4. Statistical Analysis

The statistical analyses were performed using GraghPad Prism version 7.04 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com, accessed on 5 March 2016) [11]. Data were analyzed by two-way ANOVA, and the differences among groups were analyzed by the Tukey multiple comparison test. Results were considered significant at p < 0.05.

3. Results

3.1. Morphological Characteristics

L. rotatum is an annual herb with a height of 15–40 cm. Its stem is nearly quadrangular and erect. The leaves on the stem are sessile, elongated lanceolate, and lanceolate to linear, and mid-ribs are obvious on both sides. The flower is 5-merous, terminal, or axillary, and light blue in color with dark veins. Lobes are ellipsoid lanceolate or elliptical, whereas anthers are blue and narrowly rectangular. The capsule is narrowly elliptic or oblanceolate, isometric, or slightly longer than the corolla. Under the microscope, the seed appears spherical, light brown, 0.3–0.4 mm in diameter, and smooth (Figure 1A).

G. barbata is an annual or biennial herb with a height of 8–40 cm. Stems are solitary, erect, and nearly cylindrical. Leaves are cochlear or linear-lanceolate, and mid-ribs are obvious below. Flowers are on the stem or at the top of the branch. The corolla is tubular and funnel-shaped, the tube is yellow-white, and the eave is blue or light blue. The style is short. The capsule has a short stalk, which is as long as the corolla. The seed is brown, oblong, 1 mm in size, and has dense fingerlike projections on the surface (Figure 1B).

P. palustris is a perennial herb of up to 50 cm. Basal leaves are clumped and are oval or heart in shape. There is one leaf in the middle of the flower stem without a petiolate, the base is amplexicaul, and the shape is the same as that of basal leaves. The solitary terminal flower has five sepals and five ovate-round petals, and the flower is white to light yellow. The style is short, with four lobes on the apex. The capsule is oval with four lobes on the top. The seed is brown, oblong, and 1 mm in size, with net-like aages on the surface (Figure 1C).

3.2. Weight of 1000 Seeds

The seeds of the three species were 0.3–1 mm long and 0.2–0.5 mm wide, and the weight of 1000 seeds was 0.02–0.04 g. Variance analysis results showed that the average length of G. barbata seed was the highest among the three species, and L. rotatum seed was the shortest and significantly different from the other two species. G. barbata seed has the largest width, which is significantly different from the other two species (Figure 1). The weight of 1000 seeds of the three wild species was not significantly different (

Table 2. Weights of 1000 seeds of different samples and variance analysis.

Plant Name	Number of Samples	Average Weight of 1000 Seeds	Average Length (mm)	Average Width (mm)
Lomatogonium rotatum (L.) Fries ex Nym	6	0.02 a	0.36 b	0.22 a
Gentianopsis barbata (Froel.) Ma.	6	0.04 a	1.00 a	0.50 b
Parnassis palustris L.	6	0.03 a	0.95 a	0.10 a

^a,b: mean values in the same row with different superscripts differ significantly according to the Tukey multiple comparison (p ≤ 0.05).

).

3.3. Seed Ggermination Characteristics

3.3.1. Effect of Temperature on Seed Germination

Temperature and substrate play crucial roles in seed germination (Figure 2). The germination percentage of seeds from different species was significantly varied at different temperatures. The seed began to germinate at 8 days after culture. Additionally, with the increase in temperature, the germination percentage of three herb seeds showed an upward trend, among which L. rotatum was the most sensitive to temperature changes, with a germination percentage ranging from 45% at 15 °C to 90% at 25 °C, showing a highly significant difference. P. palustris had slightly lower sensitivity to temperature changes than L. rotatum and G. barbata. Its germination percentage was 60% at 25 °C, and the seeds began to germinate at 10 d after culture, which was a little later than the other two species. The germination percentage of G. barbata seeds could reach 88.33% at 25 °C on soil substrate, but smaller germination rates were observed on the filter paper. All three species reached the maximum germination percentage at 25 °C (Figure 3).

3.3.2. Effects of Substrates on Seed Germination

As shown in Figure 4, the germination percentage of G. barbata on the soil substrate was significantly higher than that on filter paper at three temperature gradients, and the highest germination percentage could be up to 90%, while the germination of L. rotatum on these two substrates did not change significantly. In addition, the germination percentage of P. palustris had no significant difference in the two substrates at low temperature (15 °C), but showed a significant difference at 20 °C and 25 °C. In contrast to L. rotatum and P. palustris, a significant interaction effect was found between temperature and substrate effects on the germination percentage of G. barbata (Figure 3 and Figure 4).

4. Discussion

The biological characteristics of seeds, including their external characteristics, the weight of 1000 seeds, and germination percentage, not only fully reflected the plant’s biodiversity but also had a significant contribution in maintaining the balance of the natural world [12]. The studies of the biological characteristics of the seeds have great significance in promoting seed germination. For example, the seed features and seedling characteristics of Picria felterrae Lour were tiny, weighing just 0.0708 g per 1000 seeds, indicating the seeds were more susceptible to environmental changes; hence, water and light must be properly maintained during the seedling phase [13]. In addition, the seed coat of Clematis aethusifolia was hard and thick, and it could be broken by soaking it in concentrated sulfuric acid for 10 s to reach the highest germination percentage [14]. In the present study, microscopic observation showed that all three herb seeds had aages on the surface, especially the fingerlike projections on the surface of G. barbata seeds and the net-like aages on the surface of P. palustris seeds, which resulted in a decrease in germination rate compared to L. ratatum, particularly when the temperature was under 25 °C. Seed aage refers to the pericarp, wings, bracts, crown hairs, and other accessory structures that wrap the seeds, and this trait is essential for the seed to adapt to dispersal [15]. In addition to protecting and dispersing seeds, aages also play a role in regulating seed germination. The previous reports showed that the seed aages of Fagopyrum esculentum and Fagopyrum tataricum could promote seed germination [16], while the seed aages of Angelica sinensis and Ephedra sinica could inhibit seed germination [17,18]. Natural germination rates and yields of wild Herba Swertiae species are very low in the Hulunbuir region of Inner Mongolia. We speculated that the characters of fingertype and net-like aages of seeds may influence the germination of G. barbata and P. palustris and their subsequent yields. Thus, a further research can be suggested for testing the effect of removing the external aages on the seed germination. Moreover, seed weight and size are the key characteristics of the plant and are closely related to seed germination, seed dispersal, and seedling competitiveness [19]. It has been well demonstrated that increasing sowing depth reduces germination and seedling emergence in many weed species [20]. Although the current study did not identify the association between seed weight and sowing depth in the effect on seedling emergence, we found that the seed weight and size are very small among the three seed species, and thus the thinner soil cover is crucial for their germination and seedling emergence. Additionally, regularly maintaining the soil moisture surrounding the seedlings by water management is also required for improving the germination.

Seed germination percentage is an important factor to measure seed viability and has momentous significance in production. Germination substrate, as an important substrate, plays a crucial role in seed germination, and different plants have different seed germination substrates [21]. The present study has applied filter paper and a mix of soil and vermiculite as the substrate to germinate the three Herba Swertiae seed species. It was found that the germination percentage with the mixed substrate was higher than on filter paper, which is consistent with previous studies that soil substrate is superior to filter paper in terms of retaining moisture during the germination process [22]. Moreover, the mixed substrate of soil and vermiculite not only has strong aeration but also has longer moisture retention than filter paper, where the seeds could absorb enough water, which was beneficial for germination. However, an interaction between substrate and temperature effects on the germination of G. barbata was observed; the germination percentage was extremely low (less than 10% at all three temperature gradients) on the filter paper. Therefore, a mixed substrate of soil and vermiculite should be used to germinate these three medicinal plant seeds.

Before field cultivation, seedlings should be grown indoors and then transplanted to the field. Temperature is an important environmental condition for plant seed germination, and seeds usually show the highest germination percentage at an optimized temperature [23,24,25]. Therefore, finding the optimal temperature for seed germination is important for seeding and application. Fluctuating temperatures greatly improve the seed germination of different herbal species, and the optimal temperature of seed germination varies from one plant to another [26,27,28]. In this experiment, the three kinds of Herba Swertiae herb seeds showed the highest germination percentage at 25 °C, significantly higher than at other temperatures. With the increase in temperature, the germination percentage showed an increasing trend, indicating that the three seeds belonged to temperature-sensitive seeds [29]. It can be suggested that the temperature of the greenhouse could be controlled at 25 ± 1 °C during the seedling process, which is consistent with the temperature of the same phenological period of the collection site [30]. Therefore, the temperature range of the habitat could be a reference for studying the germination temperature of other Mongolian herb seeds. Additionally, the three kinds of seeds were all collected from Hulunbuir, Inner Mongolia, and it remains to be verified whether the temperature and substrate concluded in this study are applicable to the species of other regions.

5. Conclusions

The morphological characteristics of the seeds have great significance in influencing seed germination. Germination substrate, as an important medium, and temperature, as an important environmental condition, play crucial roles in plant seed germination. We conclude that the optimum germination conditions for the three Herba Swertiae species are a temperature of 25 °C and a substrate with the mixed medium of soil and vermiculite at a 1:1 ratio. Due to the small size and light weight of seeds, it is recommended that the planting depth and covering soil should be shallow and thin.