Pre-Germinative Treatments and In Vitro Germination of Dianthus caryophyllus and Alstroemeria spp. Seeds

Abstract

Alstroemeria spp. and carnation (Dianthus caryophyllus L.) have considerable and increasing economic importance in the floriculture market, therefore breeders carry out intense breeding programs to select new superior varieties. However, poor germination of hybrid seeds remains a bottleneck. Based on this assumption, seed pre-treatments and in vitro germination protocols, using different germination substrates, were applied in Alstroemeria spp. and carnation to improve germinability. Seed viability was tested using the 2,3,5-triphenyl tetrazolium chloride (TTC) test, and resulted in 91.10% ± 2.33 and 86.66% ± 3.85 in Alstroemeria and carnation, respectively. In Alstroemeria, pre-treatment with potassium nitrate (KNO₃) in combination with modified ½ Murashige and Skoog (MS) medium ensured high germination uniformity combined with high germination percentage, showing significantly higher values than the control. In carnation, a suitable seed sterilization procedure was set up (up to 95.8% sterility); treatments with gibberellic acid (GA₃) and KNO₃ did not influence germination percentage compared to the control. A high multiplication rate of seedling lines was obtained on hormone-free MS medium.

1. Introduction

The genus Dianthus L. belongs to the Caryophyllaceae family and comprises approximately 300 species across Europe, Asia, Africa, and North America. The primary center of diversity for the genus is the Mediterranean region [1]. Within this genus, carnation (D. caryophyllus) is one of the most popular cut flowers with commercial importance in the world floricultural market. The plant has wide color range, long vase life, and versatility in floral arrangements that contribute to its widespread popularity in the floral industry. Another noteworthy flowering species is Alstroemeria L., commonly known as Peruvian lily or Inca lily. Native species of Alstroemeria are mainly found in Chile and Brazil, as well as in other South American countries [2]. Alstroemeria has gained significant recognition for its stunning and long-lasting blooms, making it a sought-after choice for flower growers. Both carnation and Alstroemeria stand out as key players in the ornamental sector, captivating consumers and florists alike with their aesthetic appeal and enduring charm. The two species are among the world’s 10 best-selling cut flowers, together with rose, lily, chrysanthemum, lisianthus, tulip, gerbera, freesia and hydrangea [3]. As a consequence of their dominant position in the global floral market, both species are subject to intense varietal innovation, largely based on the introduction of new or improved traits through hybridization. In a context where each single seed resulting from crossbreeding could be the new candidate to dominate the global market of a species, seeds become a precious resource. Therefore, any technique that increases the germination percentage is significantly beneficial for breeders, particularly in Alstroemeria where germination is generally very poor, as reported by [4] for ten species in which seeds were not subjected to any pretreatment.

Nowadays, biotechnological approaches, like in vitro culture, could be very useful and faster in reaching the goal of an increased germination efficiency. It is reported that many factors could positively influence the germination process: the application of pre-treatments (priming), temperature and light range, and the addition to culture medium of specific plant growth regulators. Gibberellins (GAs) are phytohormones essential for several plant processes (e.g., seed germination, stem elongation, leaf expansion, trichome development, pollen maturation, and flowering induction) and are specifically involved in seed dormancy breakdown in antagonism with abscisic acid (ABA). In several species, high endogenous GAs content in seeds leads to dormancy decreasing [5]. In D. caryophyllus and D. orientalis, the application of gibberellic acid (GA₃) has been shown to be effective in improving germination processes [6,7].

Seed priming is a treatment which consists of hydrating the seeds sufficiently to activate metabolic events without allowing radicle emergence. Hydropriming with hydrogen peroxide (H₂O₂) not only reduces seed colonization by pathogens, but can also improve drought tolerance, growth speed, and germination percentage, and increase the oxygen available to produce energy at the mitochondrial level. In nutripriming, seeds are soaked in a nutrient solution to improve quality germination by increasing their nutrient content. Potassium nitrate (KNO₃) is a compound used for nutripriming, since it provides two crucial elements for plant development: potassium (K) and nitrogen (N). Potassium supports numerous physiological and biochemical processes, while nitrogen is a key component of proteins and nucleic acids [8]. In D. barbatus, soaking seeds with KNO₃ before sowing promoted germination [9]. A stratification period, in which the seeds are subjected to high-low heat regimes for variable periods of time, is considered necessary for a good germination of Alstroemeria seeds [4]. In addition, GAs and KNO₃ have a positive effect on germination rate as well as germination uniformity [10,11]. In vitro germination with suitable substrate composition and seed pre-treatments could enhance the efficiency of germination and guarantee a high number of new plants from which breeders could select élite cultivars. All of the above seed germination-improving methods have shown promising results in some species, improving both germination rate and uniformity. However, the efficacy of these treatments appears to be highly variable and species-specific, making it necessary to further investigate the optimal conditions for each species. Scientific studies focusing on in vitro germination of Alstroemeria spp. and D. caryophyllus and based on priming factors are relatively few, so it is difficult to establish a standardized and effective protocol to improve germination of these species. In addition, very often growth substrate is not analyzed as a factor influencing germination. The following study, by systematically analyzing the effect of different priming treatments and substrates, can contribute to a better understanding of the key factors influencing seed germination in these species. All of this, when applied to breeders’ production systems, can offer practical and applicable solutions, enabling reductions in time and costs associated with propagation and hybridization, and improvements in the quality and uniformity of the seedlings produced. It would also have direct implications for increasing competitiveness in the global market by effectively meeting the diversification and innovation needs required by the floricultural sector.

The aim of this work was to set up an optimal in vitro germination protocol for hybrid seeds of D. caryophyllus and Alstroemeria through the use of specific pre-germination treatments. In particular, the effect of KNO₃ and H₂O₂ priming, the use of gibberellins, and culture media were investigated in order to support the breeders with biotechnological approaches facilitating the possibility of selecting new valuable varieties in the floriculture market.

2. Materials and Methods

2.1. Plant Material

The research was carried out at the Research Centre for Vegetable and Ornamental Crops (CREA) biotechnology platform located in Sanremo (IM), Italy. Hybrid seeds of Alstroemeria spp. and carnation (D. caryophyllus L.) with high potential and ornamental value were provided by HYBRIDA SRL company of Sanremo, Italy.

2.2. Seeds Viability Test

To establish seed viability, 2,3,5-triphenyl tetrazolium chloride (Sigma^®, Kawasaki, Japan), commonly known as tetrazolium salt (TTC), was used as reported by the Food and Agriculture Organization (FAO) [12]. The protocol included three phases: the first one consisted of the elimination or puncturing of the seed coat using a needle; subsequently, seeds were soaked in distilled water for 24 h, in dark conditions at 24 °C; then, seeds were immersed in 1% tetrazolium solution for 24 h, in dark conditions at 24 °C. Viability of the seeds was evaluated under microscope (Leica Wild Heerbrugg M5A) according to embryos and endosperm coloring, which is intense red or pink in viable seeds, while dead seeds appear colorless. The TTC test was performed on three repetitions of 15 seeds each for both species, in comparison with 5 seeds left in distilled water for 2 days as control. The control was included to determine whether the seeds could naturally or for some other reason exhibit a pink or reddish coloration, which would otherwise invalidate the entire TTC treatment.

2.3. Seed Pre-Treatments, Sterilization Protocol and In Vitro Germination

Alstroemeria spp. seeds were initially soaked for 24 h in an aqueous solution of 100 mg L⁻¹ GA₃, or 8 g L⁻¹ KNO₃ or 20 g L⁻¹ H₂O₂, compared to distilled H₂O (control) (

Table 1. Experimental design adopted for Alstroemeria spp.: four pre-treatments combined with two in vitro media.

Pre-Treatment	Substrate
Control (H2O)	H2O + Agar (control)
	½ MS modified
GA3 100 mg L−1	H2O + Agar (control)
	½ MS modified
KNO3 8 g L−1	H2O + Agar (control)
	½ MS modified
H2O2 20 g L−1	H2O + Agar (control)
	½ MS modified

).

After pretreatments, seeds were sterilized for 1 min in a 70% ethanol (EtOH) solution and then in 3% sodium hypochlorite for 20 min. Subsequently, they were rinsed in sterile distilled water three times and then placed in plastic Petri dishes (diameter 9 cm) in the following growth media: H₂O + 8 g L⁻¹ agar (control) or modified ½ MS [13] (half strength micro- and macro-elements, 1 mL L⁻¹ MS vitamins + 10 g L⁻¹ sucrose + 8 g L⁻¹ agar). Afterwards, to break the dormancy, seeds were subjected to the following stratification conditions: 7 days at 24 °C, with photoperiod of 16 h, at photosynthetic photon flux density (PPFD) 30 μE m⁻² s⁻¹ followed by 28 days at 5 °C, in the dark [10]. At the end of this period, seeds were placed in standard cultivation conditions: 24 °C temperature, 16 h of photoperiod and PPFD 30 μE m⁻² s⁻¹ of light intensity, for 60 days (Table 1). Germination data were recorded every two days to evaluate the presence of contamination, the percentage of germination, the average germination time (AGT) and the germination uniformity (GU).

The experimental design adopted for D. caryophyllus, as reported in

Table 2. Experimental design adopted for Dianthus caryophyllus: two pre-treatments combined with three seed sterilization protocols and four in vitro media.

Pre-Treatment	Sterilization	Substrate	GA3 (mg L−1)
Control (H2O)	Control (H2O)	Filter paper	-
		Filter paper	+0.5
		MS	-
		MS	+0.5
	H2O2 10% 13 h + EtOH 96% 2 min	Filter paper	-
		Filter paper	+0.5
		MS	-
		MS	+0.5
	NaOCl 1% 20 min	Filter paper	-
		Filter paper	+0.5
		MS	-
		MS	+0.5
KNO3 250 mg L−1	Control (H2O)	Filter paper	-
		Filter paper	+0.5
		MS	-
		MS	+0.5
	H2O2 10% 13 h + EtOH 96% 2 min	Filter paper	-
		Filter paper	+0.5
		MS	-
		MS	+0.5
	NaOCl 1% 20 min	Filter paper	-
		Filter paper	+0.5
		MS	-
		MS	+0.5

, consisted of one pre-treatment in which seeds were soaked for 6 h in a solution of 250 mg L⁻¹ KNO₃ [9], compared to the control (only distilled water). Subsequently, seeds were sterilized following two different protocols, always compared to a control (H₂O): 10% H₂O₂ solution for 13 h and then in ethanol (96%) for 2 min or solution of 1% NaOCl for 20 min. After the sterilization phase, seeds were rinsed in sterile water 3 times under a horizontal laminar flow hood, and then placed to germinate in a growth chamber at 24 °C, with photoperiod of 16 h, at PPFD 30 μE m⁻² s⁻¹, onto two substrates: filter paper soaked with 5 mL of water, and MS medium (1 mL L⁻¹ MS vitamins + 30 g L⁻¹ sucrose + 8 g L⁻¹ agar). The addition of 0.5 mg L⁻¹ GA₃ to both substrates was analyzed. Germination data were taken every two days, for 60 days, to evaluate the following parameters: presence of contamination, percentage of germination, average germination time (AGT) and germination uniformity (UG). Two months after cultivation, seedlings were multiplied by cutting apical and nodal segments and transferred into MS. The number of new shoots obtained from a single seedling was the multiplication rate (MR). Initially, data analysis was performed using a four-way ANOVA and subsequently simplified by a two-way ANOVA, as reported in the results section.

2.4. Statistical Analysis

In both species, a completely randomized design with 3 replications (15 seeds per replication) for each treatment combination was adopted in all experiments. Germination percentage was calculated as the ratio between the number of germinated seeds and the total number of seeds. Average germination time (AGT) was calculated in accordance with [14]. Germination uniformity (GU) was calculated, according to [15], on the coefficient of variation (CV = (σ/μ) × 100) of AGT indicating the lower the value, the better the uniformity. Collected data were analyzed using the Rstudio computer program (RStudio Team 2020. RStudio: Integrated Development for R. RStudio, PBC, Boston, MA, USA) which includes two-way or multi-way analysis of variance (ANOVA) at p ≤ 0.05, in order to evaluate the presence of interaction between factors and the Student Newman Keuls test (SNK) at p ≤ 0.05. Bars in the graphics represent standard error.

3. Results and Discussion

3.1. Seeds Viability

The triphenyl tetrazolium chloride staining test proved effective for highlighting seed viability in both Alstroemeria and carnation. In fact, in the first species, viable embryos and tissues turned a red color, unlike the unviable ones which did not change color and remained white/colorless (Figure 1A,B). Seed viability was 91.10% ± 2.33. This result suggests that the poor germinability usually observed in Alstroemeria is not due to poor seed viability, opening up the possibility of achieving successful germination rates through the appropriate modulation of factors such as substrate composition, stratification process, light, temperature, humidity, photoperiod, etc., as reported also by [6,7] in Alstroemeria ligtu. Also, in carnation, viable embryos were red, while the unviable seeds remained white (Figure 1C,D). In this species, viability reached 86.66% ± 3.85. A high percentage of viability (98%), revealed by TTC test, was reported by [16] for D. superbus, a critically endangered species in Latvia.

3.2. Pre-Germinative Treatments and In Vitro Seed Germination

In Alstroemeria, two factors were taken into consideration in the germination test: pre-treatments effect and the role of germination substrate. The two-way ANOVA revealed that both factors had a significant effect on germination; however, no significant interaction was found between the two factors, indicating that the influence of each factor on germination is independent from the other. In

Table 3. Alstroemeria spp.: germination percentage, average germination time (AGT) and germination uniformity (GU) related to different seed pre-treatments and germination substrate, compared to the control (different letters, in the columns, indicate values that differ significantly at p ≤ 0.05 according to SNK test). Data are presented as mean ± standard error.

Pre-Treatment	Substrate	Germination (%)	AGT (Days)	GU (%)
Control (H2O)	H2O + Agar (control)	28.89 ± 2.22 bc	2.35 a	40.76
	½ MS modified	48.88 ± 8.01 ab	4.39 a	60.42
GA3 100 mg L−1	H2O + Agar (control)	20 ± 3.85 bc	5.83 a	106.48
	½ MS modified	24.44 ± 14.57 bc	4.25 a	59.70
KNO3 8 g L−1	H2O + Agar (control)	33.33 ± 7.70 bc	4.4 a	78.15
	½ MS modified	64.42 ± 5.88 a	4.1 a	20.23
H2O2 20 g L−1	H2O + Agar (control)	6.67 ± 6.67 c	n.a. 1	n.a. 1
	½ MS modified	15.55 ± 12.37 bc	6.83 a	17.25

¹ n.a. = not available.

, it is possible to appreciate germination percentage, average germination time (AGT), and germination uniformity (GU) values for each combination of pre-treatment and substrate. The presented data represent the mean of three replicates for each experimental combination. The highest germination percentage (64.42%) was achieved when seeds were pre-treated with potassium nitrate and cultured on ½ MS modified medium. Only seeds on the same substrate but without any pre-treatment reached a statistically similar germination percentage (48.88%). The results showed that the use of GA₃ did not improve germination percentage, resulting in a low value compared to the other treatments. This decrease could be attributed to an excessive concentration of the phytohormone used. It is known that too high a dosage of GA₃ can have phytotoxic effects, inhibiting germination rather than stimulating it. Therefore, it is possible that the low values observed are the result of an inhibitory effect caused by overexposure of seeds to GA₃. The authors of [11] used the same pretreatment with potassium nitrate and GA₃ on A. ligtu, ensuring better germination rates compared to the control. The lowest germination percentage (6.67%), achieved on seeds treated with H₂O₂ and cultured on H₂O + agar, was too low to measure AGT and GU percentage, reported as not available (n.a.) in Table 3. No significant differences were observed between the various treatments in the mean germination time (AGT), with values ranging from a minimum of 2.35 to a maximum of 6.83 days. The best combination GU (%) and germination percentage was observed when seeds were pre-treated with potassium nitrate and cultured onto modified ½ MS. In this case, optimal germination uniformity was detected (20.23%), i.e., the seeds germinated very close to 4,1 days (AGT), and this is a very useful condition to ensure homogeneity of the plantlets. Since no interaction between the two factors was detected, a one-way ANOVA was performed separately for each factor to gain a clear understanding of their distinct average effects on germination percentage. Considering only substrates, the medium enriched with micro and macro-elements, vitamins and sucrose (½ MS modified medium) increased the germination percentage by about 16.1% (Δ value) over control, represented by H₂O + Agar (Figure 2). This can be appreciated visually in Figure 3, comparing seeds germinated on H₂O + Agar (Figure 3B) and seeds germinated on ½ MS modified medium (Figure 3A). Considering only pretreatments, hydrogen peroxide had the most negative effect on germination (11.11%) (Figure 4).

For D. caryophyllus, four factors were evaluated in the germination test: pre-treatment with KNO₃, the response to two sterilizing agents (NaOCl or H₂O₂ + EtOH), the role of filter paper or MS, and the addition of GA₃ (Table 2). KNO₃ did not have a significant effect on germination in all treatments, since percentage germination with KNO₃ (34.44%) did not differ statistically from the control (39.25%) (Figure 5A). The same treatment had been tested on D. barbatus by [9] but for this species, KNO₃ (250 mg L⁻¹) had a positive effect on germination, confirming that this pre-treatment could have a species-specific response. The addition of GA₃ did not have a significant effect on germination: germination percentage recorded with GA₃ (36.66%) was comparable to the control (37%) (Figure 5B). The authors of [6] reported that 24-h pre-imbibition with a GA₃ solution at 20 mg L⁻¹ had a considerable effect on D. caryophyllus germination. Therefore, it is possible to hypothesize that the low concentration (0.5 mg L⁻¹) added to the substrate in our study was not sufficient to show its effectiveness and that a higher concentration in the medium or its use via the imbibition method is necessary. Since both KNO₃ and GA₃ did not show significant effects on germination and had no interaction with other factors, the initial multi-way ANOVA statistical model was simplified using the F test [17], not considering the two irrelevant factors. Subsequently, two-way ANOVA was used for data analysis, including growth substrate and sterilizing agent as factors.

Sterilization with H₂O₂ followed by pure ethanol ensured the best sterilization protocol compared to the other tested treatments, reaching the lowest contamination percentage (4.2%. Sodium hypochlorite was not effective in preventing the development of fungi and/or bacteria. In fact, contamination was observed in all substrates/supports in which the seeds were placed to germinate (Figure 6). Even if 100% contamination was observed, at the beginning seeds were able to germinate, although proliferation of contaminant agents was more aggressive on seeds germinated on agarized medium (MS) enriched with sucrose, since MS is a more suitable substrate for the development of pathogens than the filter paper (Figure 6A,B). This resulted in finding an interaction between the substrate and the sterilizing agent.

Sterilizing treatment had a significant effect on germination, depending on the support or growth medium used in the experiments; in fact, the germination percentage obtained on filter paper was not statistically different, neither for the two treatments used nor for the control (Figure 7A). On the contrary, using MS medium, it was possible to appreciate an increasing germination percentage, i.e., 11.67% in the control, 23.80% using NaOCl and 51.11% following treatment with H₂O₂ and ethanol (Figure 7B).

Furthermore, germination percentage observed when seeds were cultured on the support (filter paper) and the substrate (MS) was statistically different, independently from sterilizing agents (Figure 8A); this was caused by contamination, which greatly reduced the germination potential on MS medium. In fact, considering exclusively the seeds treated with hydrogen peroxide and ethanol (less contaminated), no difference was observed between two substrates (Figure 8B).

Since seeds sterilized with NaOCl showed evident contaminations affecting germination, only results related to H₂O₂ + EtOH disinfection treatment were reported and discussed (

Table 4. Dianthus caryophyllus: germination percentage, average germination time (AGT), germination uniformity (GU) and multiplication rate (MR) related to growing media and GA₃ treatment, compared to the control, after H₂O₂ + EtOH sterilization protocol. (Different letters, in the columns, indicate values that differ significantly at p ≤ 0.05 according to SNK test). Data are presented as mean ± standard error.

Growing Media	GA3 (mg L−1)	Germination (%)	AGT (Days)	GU (%)	MR
Filter paper	-	44.44 ± 13.69 a	5.9 ab	17.95	1.1 a
Filter paper	+0.5	44.44 ± 7.95 a	5.6 b	10.45	1.1 a
MS	-	53.33 ± 9.73 a	7.7 a	24.30	5.9 b
MS	+0.5	48.88 ± 6.28 a	6.2 ab	16.20	8.7 b

). Germination did not show significant differences between the various supports/media. The highest germination percentage was 53.33% when the seeds were placed on MS medium, while the lowest percentage was obtained on filter paper (44.44%), but differences were not statistically significant. Seeds treated with GA₃ and placed on filter paper needed less time to germinate (AGT = 5.6) compared to seeds germinate on MS. Furthermore, the GU values remained low in both growing media, confirming a good germination uniformity. Seedlings which germinated on a substrate containing salts, vitamins and sugars can grow more vigorously (Figure 9A) than seedlings arising from filter paper, which appeared shorter and etiolated, and consequently not suitable for massive in vitro propagation (Figure 9B).

In fact, the multiplication rate (MR) was affected by the culture substrate used; the best results were observed on the MS substrate (Table 4 and Figure 10). In Figure 11, it is possible to observe multiplied clones obtained from a single seedling, cultured into a glass vessel (diameter 9 cm) used for in vitro culture.

4. Conclusions

The development of optimized germination protocols is necessary, and crucial, in traditional genetic improvement, especially in an era when varietal innovation is particularly valued by the market. It is also important to address and mitigate the low germination rates that often occur when multiple hybridizations are carried out.

Results obtained on Alstroemeria have shown that external factors can positively affect germination rates. In particular, agarized medium (½ MS) enriched with minerals, sugars and vitamins was better than semisolid medium consisting of water only. In addition, if KNO₃ priming is added to the medium, this can ensure excellent uniformity of germination. This evidence can be an excellent starting point for approaching breeders to implement in vitro germination in their production processes, making genetic improvement efficient and reducing the time to obtain a new variety. Furthermore, remarkable viability of Alstroemeria seeds was recorded, but this is in contrast with the low germination rates observed. This suggests that the difficulty of germination in this species could be attributed to external conditions (i.e., dormancy), partially elucidated in this research but not sufficiently to overcome the germination gap, stimulating future studies in germination rate. In particular, it would be interesting to extend the current research by trying to use an MS medium with a standard concentration of salts to evaluate whether seed germination can further benefit from such conditions. In D. caryophyllus, it was possible to establish that a prolonged seed immersion (13 h) in 10% hydrogen peroxide, followed by 1 min in 70% ethanol, is an excellent method for in vitro sterilization. In all cases, in vitro conditions demonstrated excellent uniformity in germination. Therefore, when it is necessary to obtain starting material as uniform as possible, in vitro methods can provide outstanding results. The high multiplication rate on MS substrate can be an excellent starting point in case it is necessary to micropropagate a high genotype selected by the breeders. As a result, the implementation of in vitro production can guarantee high productivity by reducing the time required for traditional multiplication of selected plants.