Introduction

Cuba is considered the island with the highest level of endemism in the Antilles, where more than half of the plant species are endemic (Méndez et al. 2021). Among the endemic plant formations, those that develop on soils of serpentinized ultrabasic rocks (7% of the island), known as “serpentines”, stand out (Quiala et al. 2004). In this context, serpentine soils have been described to have low fertility due to the combination of factors such as steep slopes, low moisture retention, low organic matter content, high Mg/Ca ratio, and high concentrations of metals such as nickel, chromium, and cobalt (Karatassiou et al. 2021; Saad et al. 2018).

Plant metabolism can be affected by a variety of factors, including environmental conditions, diseases, and pests. Many of these plants growing on serpentine soils have slow growth and low biomass production, pollen and seeds are low (Rodríguez and Andrade 2017; Roebuck et al. 2022). This means that conventional propagation of species growing on this type of soil is limited and very complex, as seed germination is difficult under these conditions (Meindl and Ashman 2017). In addition, once many seeds fall on to the ground, they are eaten by insects and reptiles before they can germinate (Gunarathne et al. 2019; Meindl et al. 2014).

Mosiera bullata (Britton & P. Wilson), is a species of the Myrtaceae family. It is endemic to the central region of Cuba, where it grows on serpentine soils (Goya-Jorge et al. 2022). This species has been classified as threatened with extinction (provisional category) since 2008 (Cruz and Ramos 2008). Humans have caused the alteration, fragmentation, and destruction of its habitat through the development of agricultural activities, civil constructions, mining, and military maneuvers (Méndez-Orozco et al. 2015).

On the other hand, the potential of leaf extracts of M. bullata as antimicrobial agents against plant pathogens has been recently described (Pérez-Gómez et al. 2022). However, a sufficient amount of plant material is first required for possible future use as a biofungicide in agriculture. In addition, its potential for other biotechnological applications such as phytoremediation and phytomining has been described because it is a nickel hyperaccumulator species (Reeves et al. 1999). As a result, this species is a candidate for ex situ conservation through in vitro culture techniques with the dual goal of preserving the wild population and using it on a large scale in biotechnology.

In vitro culture techniques may be an alternative for massive propagation of this species, regardless of environmental conditions. However, to date, there are no published reports of in vitro propagation of this or any other plant in the genus Mosiera. Moreover, in order to develop strategies for the propagation and conservation of a species, it is necessary to know the characteristics of some of the organs involved in propagation.

Therefore, the present study aimed to (1) determine the morpho-physiological characteristics of the fruits and seeds of M. bullata (Britton & P. Wilson) subsp. bullata, (2) determine the suitable conditions for in vitro germination of M. bullata (Britton & P. Wilson) subsp. bullata.

Materials and methods

Plant materials

Physiologically ripe fruits of M. bullata (Britton & P. Wilson) subsp. bullata, were collected in June 2020 near “Albaisa” in Camagüey province, Cuba (21.4300-77.83469). The curator of the “Julián Acuña Galé” herbarium (HIPC) at the University of Camagüey Ignacio Agramonte, identified the plant taxonomically. Seeds were removed by hand from the fruit, washed with a sieve in tap water to remove the pulp, and stored in a tray at a temperature of 25 ± 2 °C for 48 h until completely dry.

Morpho-physiological characterization of fruit and seeds of M. bullata

Twenty ripe fruits were randomly sampled to measure the length and thickness, length of the calyx, and diameter of the calyx using a four-digit digital caliper (Stainless Hardened, Germany). Fruit mass was also determined using a four-tenths analytical balance (Sartorius, Germany), and the number of seeds per fruit was determined. The morphological characteristics of the fruits were determined according to Martin (1946).

Seed morphology

Fifty seeds were taken at random to measure length, width, and thickness using a four-digit digital caliper (Stainless Hardened, Germany) and mass using a four-tenths analytical balance (Sartorius, Germany). Seed morphological characteristics were determined according to Martin (1946).

Embryo morphology

To determine embryo morphology and development, 50 seeds were collected and cut longitudinally with a scalpel (Schreiber, N° 23). The embryos were photographed with a Canon camera (EOS 600D) and a digital caliper (Stainless Hardened, Germany) was used to measure the length of the embryo to calculate the embryo/seed ratio (E/S, %). Embryo morphology and development (E/S) were defined as described by Martin (1946).

Seed viability

One hundred seeds were collected and a viability estimation test was performed with a 1% solution of 2,3,5 triphenyl-2H-tetrazolium chloride (TTC) as described by Altare et al. (2006). Seeds were classified according to their coloration: (1) viable if they were colored deep red or their coloration was light red or with colorless sections, and (2) non-viable if they remained colorless (Maldonado-Peralta et al. 2016).

Seeds germination

Four samples of twenty-five seeds were placed in a Petri dish with filter paper previously moistened with 5 mL of distilled water. The Petri dishes were placed in a germination chamber (Model, RTOP-D series) for 28 days at 30 °C, 80% relative humidity, and a photoperiod of 14 h light / 10 h darkness, under fluorescent light tubes with a photosynthetic photon flux density (PPFD) of 50 μmol m−2 s−1. To describe the type of germination in M. bullata, from the beginning of the germination experiment, photographs were taken daily until all distinctive organs were observed in the new seedling.

In vitro germination of M. bullata seeds

Effect of disinfection time with sodium hypochlorite 2% (w/v) on in vitro germination

Seed germination

Four samples of twenty-five seeds were placed in a Petri dish with filter paper previously moistened with 5 mL of distilled water. The Petri dishes were placed in a germination chamber (model, RTOP-D series) for 28 days at 30 °C, 80% relative humidity, and a photoperiod of 14 h light/10 h dark under fluorescent lights with a photosynthetic photon flux density (PPFD) of 50 μmol m−2 s−1. To describe the nature of M. bullata germination, photographs were taken daily from the beginning of the germination experiment until all characteristic organs were visible on the new seedling.

First, the seeds were washed with plenty of water and commercial powder detergent (1%). After that, the seeds were subjected to three surface disinfection treatments, which were based on 70% (v/v) ethanol/water mixture for 1 min and sodium hypochlorite 2% (w/v) for (1) 10 min, (2) 20 min and (3) 30 min. For each treatment, 40 seeds were used. Seed surface disinfection was done in 50 mL Falcon tubes (Fischer Scientific, USA) keeping constant stirring. Subsequently, the seeds were rinsed three times with sterile distilled water to remove the residues of the disinfectant.

Murashige and Skoog (1962) medium MS was used for the in vitro establishment of seeds, with all its mineral salts and vitamins, and supplemented with myo-inositol (100 mg L−1), and thiamine HCl (1.0 mg L−1), sucrose (30 g L−1), and polyvinylpyrrolidone (250 mg L−1) as an antioxidant. The pH was always adjusted to 5.8 ± 0.05 before adding the solidifying agent. The experiments were conducted in a plant growth chamber, set at a temperature of 25 ± 2 °C, where glass flasks (250 mL capacity) with the seeds of M. bullata on MS medium were exposed to a 16 h photoperiod (12.5–37.5 µmol m−2 s−1) and 8 h of darkness.

The effect of seed disinfection time with sodium hypochlorite 2% (w/v) on the appearance of in vitro contamination by fungi and bacteria was evaluated until 11 days after the explant inoculation. Besides, seed germination percentage was evaluated 28 days after explant inoculation, using the mathematical expression described by Ranal and Santana (2006). It was considered a germinated seed when the emitted radicle reached approximately 2 mm.

Effect of light on in vitro germination

For evaluation of the effect of light, one part (half) of the seeds was kept in the absence of light throughout the entire experiment with black nylon until the radicle emerged (2 mm). The other half was exposed to a 16-h photoperiod of light (12.5–37.5 µmol m−2 s−1) and 8 h of darkness. Seeds were placed in the MS culture medium described above. The percentage of seed germination was determined 28 days after explants were inoculated. Forty seeds were used for each treatment.

Effect of nitrate salts on in vitro germination

Firstly, M. bullata seeds were disinfected taking into account the best results obtained in the previous experiments. Half of the seeds were placed in MS medium with the concentration of nitrate salts reduced by half (MS (NO3 1/2)) and the other half in MS medium without varying the content of the nitrate salts. Subsequently, seeds were placed in culture conditions taking into account the effect of light that showed the best result on in vitro germination in the previous experiment. For each treatment, 40 seeds were used. After 28 days of implantation, the number of germinated seeds in each treatment was quantified.

Data analysis

Quantitative data collected were analyzed using SPSS (version 8.0 for Windows, SPSS Inc, New York, NY). Data were subjected to ANOVA and Tukey tests (p = 0.05) after normality was established (Shapiro–Wilk test), while Levene’s test was performed for homogeneity of variance. Student’s t-test was used to separate the means.

Results

Morpho-physiological characterization of fruit and seeds of M. bullata

M. bullata subsp. bullata has a berry fruit topped by a calyx that is green at physiological maturity and turns red at full maturity (Fig. 1). The fruits had an average length of 8.97 ± 0.30 mm and an average diameter of 7.14 ± 0.21 mm, an average weight of 0.21 ± 0.01 g, and an average amount of seeds/fruit of 18.34 ± 1.96 (Table 1). The calyx had an average length of 4.25 ± 0.10 mm and an average diameter of 3.88 ± 0.07 mm.

Fig. 1
figure 1

External (A and C) and internal (B and D) morphology of fruits of Mosiera bullata. (A and B) Physiologically ripe fruits, (C and D) Fully ripe fruits. Area of calyx (c), length of calyx (cl), diameter of calyx (cd), mesocarp (mc), and seed (sd). The bar indicates 3 mm

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Table 1 Quantitative characteristics of M. bullata fruits
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The seeds of M. bullata were subreniform, light brown on the outside, and had a hard seed coat. The central part of the seed was soft and dark brown (Fig. 2A). Inside, it consisted of an embryo without endosperm (Fig. 2B). Seeds had the following dimensions: length of 3.459 ± 0.094 mm, width of 2.505 ± 0.06 mm, thickness of 1.917 ± 0.057 mm, and weight of 0.006 ± 0.0003 g (Table 2).

Fig. 2
figure 2

Outer (A) and (B) inner M. bullata seeds. Embryo (em) and seed coat (sc). The bar indicates 1 mm

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Table 2 Quantitative characteristics of M. bullata seeds and embryos
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The embryo was curved, had a “C” shape with a length of 3.213 ± 0.06 mm, was whitish in color, and had reflexed cotyledons that were less than 1/4 of the length of the embryo (Fig. 2). The embryo/seed ratio was 93.65 ± 1.46%, so the embryo occupies more than half of the seed length. The viability of the freshly harvested seeds was 86.65 ± 4.41% (Table 2), and the viable embryos had an intense pink color (Fig. 3B), whereas the nonviable embryos retained their natural color (Fig. 3A).

Fig. 3
figure 3

Morpho-physiological characteristics of M. bullata embryos. A nonviable embryo, B viable embryo, and C details of cotyledon. Area of radicle (rr) and cotyledons (co). Each bar indicates 1 mm

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After the seeds were stored under germination conditions for 28 days, their germination percentage was 83.33 ± 5.63% (Table 2), with germination starting on the eleventh day (Fig. 4). After the emergence of the radicle, the hypocotyl elongates, and after 18 days, the cotyledons expand, with epigeal germination observed (Fig. 4).

Fig. 4
figure 4

Germination of M. bullata seeds from 11 to 18 days. Seed (sd), hypocotyl (hp), radicle (rd), and cotyledons (co). The bar indicates 2 mm

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Effect of disinfection time with sodium hypochlorite (2%, w/v) on in vitro germination

The effect of disinfection time of M. bullata seeds with sodium hypochlorite 2% (w/v) is shown in Fig. 5. The lowest percentage of fungal contamination was observed when seeds were disinfected with sodium hypochlorite (2%, w/v) for 20 and 30 min, with no significant differences between them (Fig. 5A). The visible presence of bacteria in the culture medium was not detected. The germination rates determined at the different time points (10, 20 and 30 min) also did not show any statistical differences (Fig. 5B).

Fig. 5
figure 5

Effect of disinfection time of M. bullata seeds with sodium hypochlorite 2% (w/v) in the in vitro establishment phase. Contamination of seeds by fungi (A) and percent germination of seeds (B). Fungal contamination only, as no bacterial contamination was observed. Mean values with the same letters are not statistically different (one-way ANOVA, Tukey, p ≤ 0.05, n = 40)

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Effect of light on in vitro germination

The effect of light on in vitro germination of M. bullata seeds is shown in Fig. 6. Germination of M. bullata seeds occurred in both darkness and photoperiod. However, the percentage of germination was higher in dark conditions, with significant differences in germination in photoperiod. In this sense, germination of M. bullata seeds was increased by about 16% in darkness compared to photoperiod conditions.

Fig. 6
figure 6

Effect of light on in vitro germination of M. bullata seeds after 28 days. Mean values with the same letters are not statistically different (Student’s t, p ≤ 0.05, n = 40)

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Effect of nitrate salts on in vitro germination

The effect of concentration of nitrate salts in the medium MS (Murashige and Skoog 1962) on in vitro germination of M. bullata seeds is shown in Fig. 7. The highest percentage of germination was obtained with the MS nutrient medium with half the content of nitrate salts (MS (NO3 (1/2)), with significant differences compared to the MS nutrient medium without modifications (MS). With this treatment it was possible to increase germination by 10.9%.

Fig. 7
figure 7

Effect of concentration of nitrate salts in medium MS (Murashige and Skoog 1962) on in vitro germination of M. bullata seeds. Mean values with the same letters are not statistically different (Student’s t, p ≤ 0.05, n = 40)

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Discussion

The morphological characteristics observed on the fruits and seeds of M. bullata are consistent with the descriptions of Cruz and Ramos (2008) for the genus Mosiera Small. In this sense, the morphological characteristics are similar to those observed in the fruits of the species Mosiera nipensis (Salywon and Landrum), which belongs to the same genus and is endemic to the eastern region of Cuba (Salywon and Landrum 2014).

The type of embryo (curved or convex) observed in the seeds of M. bullata is consistent with the type of embryo described by Cruz et al. (2011) for this genus. Moreover, the embryo is fully developed at the time of fruit dispersal (embryo/seed ratio: 93.65%). Since this ratio is more than 50%, there is no morphological dormancy in this species according to Baskin and Baskin (2021).

Seed viability was greater than 85% and germination was 80%, indicating that there is no dormancy in this species according to Baskin and Baskin (2021). Although the germination process did not begin until day 11, similar behavior has been described for several species in the Myrtaceae family. Blepharocalyx salicifolius (Kunth) O. Berg, where germination begins between the 6th and 8th day after sowing and ends around the 36th day (Rego et al. 2009). Curitiba prismatica Salywon & Landrum, where germination begins on day 18 and continues until day 65 (Rego et al. 2011) and Myrcianthes pungens O. Berg, which begins on day 14 and ends on day 73 (Armiliato et al. 2018). This is because it exposes the seeds of a population to less environmental stresses that could threaten the survival of the species (Nadarajan et al. 2023).

The results obtained in the present study are consistent with the report of da Silva et al. (2014) on seeds of Eugenia uniflora L., a species of the Myrtaceae family. In this study, disinfection with sodium hypochlorite 2% (w/v) for 10 min caused a higher incidence of visual contamination compared to disinfection for 25 min, with significant differences. In addition, when evaluating the effect of disinfection time with sodium hypochlorite 2% (w/v) on Bucquetia glutinosa (L.f.) DC seeds, Cárdenas et al. (2019) found a significant decrease in contamination by fungi and bacteria for times greater than 10 min.

The efficient disinfection of seeds achieved by using ethanol 70% (v/v) and sodium hypochlorite 2% (w/v) was described by several authors in different species (Barampuram et al. 2014; Salih et al. 2019; Sant’Ana et al. 2018). It has been shown that treatment with ethanol prior to disinfection with sodium hypochlorite is able to remove waxes from cellular tissues and allow the disinfectant to be most effective (Barampuram et al. 2014, Sant’Ana et al. 2018). In many cases, this allows successful disinfection of seeds without the need to apply strong treatments such as chemical fungicides or mercuric chloride (dos Santos et al. 2020; Salih et al. 2019).

Considering the results obtained, disinfection of seeds with sodium hypochlorite 2% (w/v) for 20 min was chosen to continue the experiments.

There are several species in the Myrtaceae family that have shown a stronger response to germination in the absence of light than in the presence of light, such as: E. brasiliensis Lam, E. involucrate DC, E. pyriformis Cambess, E. uniflora L. and Blepharocalyx salicifolius (Lamarca et al. 2011; Rego et al. 2011). This suggests two aspects: 1. these species behave as preferential negative photoblasts, in which seeds germinate preferentially under dark conditions, 2. low concentrations of phytochrome in the active form resulting from exposure to darkness are sufficient to initiate the germination process (Armiliato et al. 2018).

On the other hand, it has been described that the production of phenolic compounds in seeds is stimulated under continuous light conditions. This is due to the activation of the biosynthetic pathways of these compounds or the overexpression of antioxidant compounds resulting from the generation of oxidative stress by the presence of light (Khattak et al. 2007). Świeca et al. (2012) reported that there are some phenolic compounds involved in the inhibition of the germination process. Therefore, the overproduction of phenolic compounds in M. bullata seeds could be one of the reasons that lower germination rates were obtained under light conditions. Considering the results obtained in the present study, the dark environment was chosen for in vitro germination of M. bullata seeds.

There are few reports on in vitro propagation of endemic species of serpentine soils (Alfonso et al. 2018; Bhatia et al. 2002; Marszał-Jagacka and Kromer 2011; Quiala et al. 2004). Endemic species of serpentine soils grow in areas with high concentrations of heavy metals, such as nickel, chromium, and cobalt, but low concentrations of plant nutrients, such as nitrogen, phosphorus, and potassium (Visioli et al. 2013, 2019). In this sense, some studies have hypothesized the presence of evolutionary adaptations, physiological mechanisms, and genetic components in these species that could confer tolerance to the stress conditions under which they evolve, such as low soil nitrogen levels (Brady et al. 2005; Kim and Shim 2008).

In addition to these adaptive mechanisms that may be present in the species M. bullata, there are other factors such as the osmotic potential of the culture medium that may influence the percentage of in vitro seed germination. Martinez-Villegas et al. (2015) reported that the osmotic potential of the culture medium has a direct influence on the germination and growth of explants. In this sense, when the osmotic potential of the culture medium is low, the uptake of water and nutrients is also reduced, which hinders seed germination and shoot growth and multiplication. Silva et al. (2004) reported that the total concentration of salts in a culture medium determines its osmotic potential, so that as the concentration of ions in the solution increases, the osmotic potential decreases. Consequently, in the present study, the MS culture medium with a concentration of nitrate salts (MS (NO3 (1/2)) reduced by half has a higher osmotic potential than the MS culture medium without changes. This could mean that in this medium ((MS (NO3 (1/2))), water uptake by seed tissues was facilitated compared to the MS medium and the germination process was favored. Water uptake is a crucial step for the development of germination (Nadarajan et al. 2023), it allows a proportional increase in respiratory activity and the production of large amounts of energy used mainly for biochemical reactions (Lev and Blahovec 2017).

Taking into account the obtained results, the medium MS with a concentration of nitrate salts reduced by half was selected for in vitro cultivation of M. bullata seeds. In the seeds of this species, the formation of shoots with opposite leaves was observed after 30 days under the conditions established in this study (Fig. 8A). The shoots of M. bullata developed a root system characterized by a long taproot without secondary roots (Fig. 8B). This feature is typical of species growing in serpentine soils. The low water content of these soils forced these species, in order to survive, to develop a deep root system that allowed them to absorb water from the depths of the soil (Quiala et al. 2004).

Fig. 8
figure 8

In vitro establishment of M. bullata seeds. A Seedlings after 30 days in the in vitro establishment phase. B Seedlings after 60 days in the in vitro establishment phase. Each bar indicates 2 cm

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Conclusions

This study reports for the first time the in vitro propagation of M. bullata seeds, an endemic plant of Cuba that is threatened with extinction. This lays the foundation for the future development of a protocol for in vitro propagation of this species that will ensure its conservation and large-scale use in the biotechnology industry.