Seed Morpho-Anatomy, Dormancy and Germination Requirements in Three Schizanthus Species (Solanaceae) with Ornamental Potential

Abstract

Schizanthus carlomunozii, S. hookeri, and S. porrigens are herbaceous species native to Chile and Argentina and have high ornamental potential. Their propagation through seeds is challenging due to low and uneven germination percentages. This study aimed to determine the morpho-anatomical characteristics, dormancy, and germination requirements of the seeds of these three species. The seeds from all three species have a flattened and reniform shape with a foveolate testa. However, the seeds of S. hookeri are distinguished by their larger size, more pronounced C-shape, seed coat with more marked prominences, and symmetrically arranged areoles. Histological analysis and imbibition tests with methylene blue revealed that the seeds have well-developed embryos and permeable seed coats, ruling out physical and morphological dormancy. Germination tests under various conditions showed that the seeds of the three species exhibit physiological dormancy. Imbibition in gibberellic acid (200 ppm) proved to be an effective treatment to promote germination. When evaluated in S. hookeri seeds, cold stratification and after-ripening also improved germination. The optimal temperatures for seed germination were calculated to be 26 °C for S. carlomunozii, 19 °C for S. hookeri, and 23 °C for S. porrigens.

1. Introduction

The Schizanthus genus, belonging to the Solanaceae family, comprises 14 species of annual or biennial herbaceous plants native to Chile and Argentina [1]. In Chile, these plants are distributed across various habitats, from the Atacama Desert in the Tarapacá region in the north (20°40′ S) to the humid forests in the Los Lagos region in the south (40°30′), and from the Andes Mountain in the east to the Pacific Ocean coast in the west [1,2]. Unlike other solanaceous species, Schizanthus flowers are zygomorphic and bilabiate, with varied and striking colors, making them popular for their ornamental potential. The fruit is a capsule that opens with two valves, containing numerous reniform seeds, with a foveolate surface (i.e., with small gaps) [2].

Schizanthus species are propagated by seeds; however, their low and uneven germination represent significant challenges for commercial nurseries (Mónica Musalem, personal communication). Due to the absence of commercial seed production, wild populations are the primary source of seeds. To compensate for low propagation, larger volumes of seeds need to be collected. Despite high seed production rates [1], it is crucial not to extract more than 20% of the available seeds during collection to avoid negatively impacting the regeneration of wild populations [3]. Given the propagation challenges in commercial nurseries and the importance of preserving Schizanthus species, understanding the causes of poor germination in this genus is essential.

Regarding the difficulty in propagating Schizanthus species by seeds, Jara and colleagues [4] observed that light inhibits germination in S. litoralis Phil. and that scarification, which involves eroding the external seed layers chemically or mechanically, promotes germination. Other researchers [5,6] mention that seeds of species such as S. pinnatus Ruiz & Pav. require stratification—exposure to cold and humid conditions for weeks to months—to germinate. Additionally, some studies indicate that cold stratification for three months can increase the germination of S. hookeri Gillies ex Graham from 0% to 44% but has no effect on related species such as S. grahamii. Gillies ex Hook. Such findings suggest the presence of dormancy in Schizanthus seeds that inhibits germination [7].

Based on these studies, it is hypothesized that seeds of Schizanthus species exhibit physiological dormancy, complicating their propagation under commercial conditions. Given the lack of information on the biology and germination requirements of S. carlomunozii V.Morales & Muñoz-Schick, S. hookeri, and S. porrigens Graham ex Hook., the primary objective of this study was to determine the morpho-anatomical characteristics, dormancy, and optimal germination conditions of these three species to facilitate their identification, manipulation, and use for propagation.

2. Materials and Methods

2.1. Plant Material

Three species of Schizanthus were used in this study: S. carlomunozii, S. porrigens, and S. hookeri. The seeds from S. carlomunozii were collected in the Coquimbo region (31.35° S; 71.59° W; <30 m a.s.l.) in October 2020; S. porrigens seeds were collected in Viña del Mar, Valparaíso region (−33.04° S; −71.55° W; <30 m a.s.l.) in January 2021; S. hookeri seeds were collected in Farellones, Metropolitana region (33.36° S; 70.29° W; ≈2.400 m a.s.l.) in February 2023. The seeds were harvested from dehiscent capsules when they were fully mature (dark brown to black in color, with 7 to 10% water content) and stored in paper bags in a chamber maintained at a constant temperature of 20 °C and 50% relative humidity until evaluation.

2.2. Morpho-Anatomical Characterization of Schizanthus spp. Seeds

The morphological seed characterization was conducted according to the criteria described by Gunn and Gaffney [8], Ramírez and Goyes [9], and Navas [10]. Twenty seeds per species were observed; dimensions (length and width; Figure 1), color, shape, and topography of the seed coat were established through the use of a stereoscopic magnifying glass. Histological sections were made for the characterization of seed anatomy, following the protocol described by [11]. Twelve seeds per species were fixed in an FAA solution (formaldehyde, acetic acid, 70% ethanol; 90:5:5) and dehydrated in a series of alcohols of increasing concentration and then were embedded in paraffin for a period of 14 days. Transverse (Figure 1) and longitudinal sections of the seeds were taken using a 20 µm thick rotating microtome and stained with safranin and fast green to observe the seed anatomy with an optical microscope. For the seeds of S. hookeri, histochemical tests were performed on longitudinal sections of the seeds to identify the presence of starch and lipids in the endosperm using Lugol’s and Sudan III reagents, respectively.

2.3. Characterization of Seed Dormancy and Germination Requirements

For the determination of seed coat permeability, ten seeds of each species were soaked in a solution of methylene blue (1 g/100 mL) for two hours. At the end of the imbibition period, the seeds were washed in running water, their surfaces were dried with paper towel, and they were cut longitudinally for later observation with a stereoscopic magnifying glass.

Germination tests were carried out in glass Petri dishes (90 mm) with two layers of filter paper saturated in distilled water, a gibberellic acid solution (GA₃), or a potassium nitrate solution (KNO₃). In S. hookeri seeds, germination in water was also evaluated after 28, 59, and 94 days of cold stratification (imbibed seeds at 4 °C). For each test and species, four replicates of 30 seeds each were evaluated. The dishes with the seeds were placed in a chamber at 20 °C and constant light (6 µmol m⁻² s⁻¹) or a thermogradient table with a range of 10 temperatures. Germinated seeds (radicle > 2 mm) were counted and removed from each dish three times per week during 28 days. During the evaluation period, distilled water was added to the filter paper whenever necessary, to avoid desiccation of the seeds. When the effect of light was measured, Petri dishes of the dark treatments were wrapped in aluminum foil to prevent light from entering, and only a final count was performed 28 days after sowing.

Germination index. When seeds were evaluated at different temperatures in the thermogradient table, a germination index (GI) was calculated according to the following equation [12]:

$$\mathrm{GI}={\sum }_{i=2}^{28}\left[\left(\mathrm{fraction}\mathrm{of}\mathrm{seeds}\mathrm{germinated}\mathrm{on}\mathrm{day}i\right)/i\right]$$

Estimation of optimal temperature for seed germination. For each species and replication, two trendlines were fitted to the germination indexes (GIs) obtained at the different temperatures in the thermogradient table: one line with the GIs ranging from the lower temperature evaluated to the maximum GI observed, and the other with the values ranging from the maximum temperature evaluated to the maximum GI observed. Then, the optimal temperature for seed germination was estimated as the interception point of these two lines [13].

2.4. Statistical Analysis

For each species, results were analyzed by an analysis of variance (ANOVA), considering a completely randomized design with four replications. When significant differences (p < 0.05) were observed, treatments were compared using Fisher’s LSD multiple comparisons test. To analyze the germination data, an angular transformation corresponding to the arcsine of the root of the proportion number was carried out. InfoStat Software was used for statistical analysis [14].

3. Results

3.1. Seed Morphology and Anatomy

The seeds of the three Schizanthus species studied have a testa ranging from brown to black. The average size was 1.07 ± 0.02 mm long (L) and 0.90 ± 0.02 mm wide (W) in S. porrigens, 1.04 ± 0.02 mm L and 0.92 ± 0.03 mm W in S. carlomunozzi, and 2.10 ± 0.03 mm L and 1.83 ± 0.06 mm W in S. hookeri (Figure 2A–C). The seeds from the three species have a flattened and reniform shape, with a foveolate testa and a poorly defined hilum between lobes. The S. hookeri seeds are distinguished from those of the other two species by their greater size, a more pronounced C-shape, seed coat with more marked prominences, and areoles arranged symmetrically following a horseshoe-shaped curved pattern from the radicular lobe to the cotyledonary lobe (Figure 1). The endosperm responds positively to Sudan III and negatively to Lugol’s reagent, which indicates the presence of lipids and the absence of starch. The embryo is linear and hypocrepiform (Figure 2G–I), which is seen twice when making a transverse cut (Figure 2D–F) with a pair of unexpanded and well-developed cotyledons. The testa is formed by a thin layer of cells with lignified cell walls that do not hinder the entry of water according to the methylene blue test (Figure 3).

3.2. Seed Dormancy and Germination Requirements

3.2.1. Seed Dormancy Characterization

In Figure 3, a longitudinally cut seed of S. hookeri is shown before and after 2 h of imbibition in a methylene blue solution. A similar result occurred in all the seeds of the three Schizanthus species evaluated. These results prove that the seeds of these species are not impermeable and that imbibition occurs in less than 2 h.

The results from the germination tests conducted in February 2023, when the seeds from S. hookeri, S. porrigens, and S. carlomunozii had been stored for 0, 25, and 28 months, respectively, are presented in Figure 4. Because a greater number of S. hookeri seeds were available, more treatments were tested on this species (Figure 4A). In all three species, there was a significant effect of the treatments on germination (p < 0.05). When the seeds were imbibed in water (control), germination reached 21% in S. porrigens (Figure 4C) and no more than 5% in S. carlomunozzi (Figure 4B) and S. hookeri (Figure 4A). Seed imbibition in gibberellic acid improved germination in all three species, while KNO₃ improved germination in S. hookeri and S. carlomunozii (Figure 4). When seed stratification at 4 °C was evaluated in S. hookeri, no effect was observed after 28 days of stratification; however, the seeds stratified for 59 and 94 days reached 87% and 95% germination, higher than in any other treatment evaluated in this test (Figure 4A).

The effect of light or darkness on seed germination was evaluated in seeds soaked in either water or a GA₃ solution. The results are presented in Figure 5. No significant effect of light on seed germination was observed in any of the species or under either germination condition (water or GA₃).

3.2.2. Germination Changes in S. hookeri Seeds during Storage

In the seeds of S. hookeri, germination was evaluated in water and a GA₃ solution from the time of harvest up to 1 year later. The seeds were stored at 20 °C and 50% RH. As shown in Figure 6, the germination of the seeds soaked in GA₃ was consistently higher than those soaked in water; however, in both cases, the germination percentages increased with storage time. For the seeds soaked in GA₃, germination increased from 30% at harvest to 90% after 12 months of storage. Conversely, the seeds soaked in water showed nearly no germination at harvest and up to 4 months of storage, increasing to 5% at 6 months, and reaching 24% at 12 months.

3.2.3. Germination Response to Temperature

When evaluating the germination of seeds treated with GA₃ across a temperature gradient, S. carlomunozii and S. hookeri exhibited high germination percentages between 10 and 32 °C (Figure 7A,B). In contrast, S. porrigens showed maximum germination only within the range of 15 to 25 °C (Figure 7C). Thus, analyzing germination percentages alone did not allow for the determination of the optimal germination temperature for any of the three Shizanthus species. However, when considering germination index values, which account for both the percentage and speed of germination, optimal germination temperatures were estimated to be 26 °C for S. carlomunozii, 23 °C for S. porrigens, and 19 °C for S. hookeri (Figure 7).

4. Discussion

The reniform or C-shape and the presence of a well-developed hypocrepiform embryo is an attribute that is repeated in different Solanaceae genera such as Solanum, Capsicum, Datura, Physalis, Atropa, and Salpiglossis [8], and allow us to rule out the presence of morphological dormancy in these seeds [15]. Additionally, the physical and morphological characteristics reported here for the seeds of three Schizanthus species provide valuable information for their identification, especially in the case of S. hookeri (Figure 1 and Figure 2).

Previous studies on the seeds of S. litoralis reported a positive effect of scarification in promoting germination, and authors have suggested the presence of physical dormancy [4]. However, in the three Shizanthus species included in the present study, seeds possess a testa that does not prevent their rapid imbibition, thus discarding the presence of physical dormancy [15]. A benefit of scarification in promoting the germination of seeds without an impermeable seed coat has been observed in other genera of the Solanaceae family [16] and could be related to the reduction of the growth potential required by the embryo radicle to protrude through the testa [17].

The low germination percentages of Schizanthus seeds when soaked in water suggest the presence of dormancy, which is confirmed by the positive response to the use of GA₃ and KNO₃ (Figure 4), compounds used to promote germination in dormant seeds [18,19,20]. Previously, soaking seeds in a GA₃ solution was suggested to stimulate germination in S. hookeri and S. grahamii seeds [21]. Additionally, newly harvested S. hookeri seeds showed maximum germination values (>90%) after 2 and 3 months of cold stratification (Figure 4A). A positive effect of stratification in stimulating the germination of S. pinnatus seeds has been suggested by other authors [5,6], and a significant increase in germination was observed in S. hookeri seeds stratified in the cold for 3 months, although the germination values did not exceed 50% [7]. In the same study, cold stratification had no effect on the germination of S. grahamii seeds [7].

The presence of light has been reported to affect seed germination in different species of Solanacea [22,23]; however, our results indicate that for the three species of Schizanthus studied, germination was not significantly affected by the presence or absence of light. Differences in the seed germination response to light have been reported for different seed lots of a same species, and could be related to the genotype [24] or the environment in which the seeds developed [25].

In the case of S. hookeri, with a high number of newly harvested seeds available, it was possible to evaluate changes in the germination percentage during the first year of storage (Figure 5). The germination of the seeds imbibed in a gibberellic acid solution was always higher than that of the seeds imbibed in water, indicating that the seeds remained dormant throughout the 12 months of this study. However, the response of the seeds to imbibition in GA₃ increased from the initial evaluations, rising from 30% to 45% in the first 2 months, and reaching 90% after 12 months from harvest. In the case of seeds imbibed in water, although almost no germination was observed during the first 4 months, germination reached 5% and 24% at 5 and 12 months, respectively. These changes indicate a decrease in dormancy depth during seed storage, a process known as after-ripening [18,26], which is characteristic of seeds with physiological dormancy [15].

The temperature is one of the most important environmental factors affecting seed germination, with each species having an optimal temperature characterized by the highest percentage and speed of germination [26]. Conversely, the minimum and maximum temperatures at which a seed lot can germinate depend not only on the species but also on its dormancy and vigor [26,27]. In this study, due to the marked dormancy of the seeds, the determination of the optimal germination temperature was carried out by soaking the seeds in gibberellic acid. This complicates the interpretation of results when determining the minimum and maximum temperatures for seed germination of each species. However, the results indicate that the seeds of S. carlosmunozii and S. hookeri have the potential to germinate over a wide range of temperatures, at least between 10 °C and 30 °C. In the case of S. porrigens, the maximum germination potential would be reached between 15 °C and 25 °C. The results did allow for a good estimation of the optimal germination temperature for each species, at 19 °C, 23 °C, and 26 °C for S. hookeri, S. porrigens, and S. carlosmunozii, respectively (Figure 7). The values for each species can be partly explained by their habitats, as S. carlomunozii is found further north and towards the coast in warmer environments, whereas S. hookeri is found in higher and colder regions, and S. porrigens is found in intermediate areas in terms of latitude, altitude, and temperature [1].

5. Conclusions

The morpho-anatomical characteristics of the seeds and the increase in germination in response to the application of gibberellic acid and potassium nitrate confirm the presence of physiological dormancy in the seeds of S. carlomunosii, S. hookeri, and S. porrigens [15]. Additionally, at least in the case of S. hookeri, the response to cold stratification treatments and the loss of dormancy during seed storage indicate that the physiological dormancy is nondeep [15]. The use of seeds stored for at least 6 months and their imbibition in a gibberellic acid solution (GA₃, 200 to 500 ppm) presents a practical and efficient alternative for propagating these species by seed. Additionally, sowing at temperatures between 20 and 25 °C is recommended to achieve faster and more uniform germination in these species.