Plant material and sterilization

The experiment was conducted at the Plant Tissue Culture Laboratory of the National University of Ucayali, located in Pucallpa, Peru. As plant material, seeds of Calycophyllum spruceanum, which were acquired in commercial seed markets, were used. Seed sterilization was performed in a laminar flow chamber. The seeds were immersed in a Benlate® solution (30 g L^-1) for 30 minutes. Subsequently, they were immersed in 70% alcohol and immediately treated with 1% sodium hypochlorite (NaClO), adding three drops of Tween® 20. The solution was kept in constant agitation for 10 minutes. Finally, the seeds were rinsed five times with sterilized distilled water to ensure the elimination of residues.

Culture media, treatments, and variables

For in vitro germination, Murashige and Skoog (MS) and Woody Plant Medium (WPM) at different concentrations were used as base culture medium. Agar alone was used as a control. The experiment consisted of five treatments: T1: 100% MS, T2: 50% MS, T3: 100% WPM, T4: 50% WPM, and T5: control (agar only). Additionally, in each of the treatments, 6.5 g L^-1 of agar (PhytoTech®), 30 g L^-1 of sucrose, and pH was adjusted to 5.8. However, for the control (T5), only 30 g L^-1 sucrose was added, and the pH was adjusted to 5.8, because it already contained agar.

Each treatment consisted of seven replicates, and five flasks (13 cm height and 6 cm mouth) were used in each replicate. Each flask contained 45 mL of medium, which were autoclaved at 121 °C and 1.1 atm for 20 min. Subsequently, the flasks were cooled and three seeds per flask were inoculated, being 105 seeds per treatment, which were placed randomly. The flasks were incubated at a temperature of 23±2 °C, with a relative humidity of 80% and a photoperiod of 16 hours.

The variables related to germination, such as germination percentage (G), germination speed index (GSI), germination initiation day (GID), final day of germination (FDG), germination energy (GE), germination energy period (GEP), germination energy period percentage (% GEP) were evaluated for 21 days after in vitro inoculation (Bewley and Black 1994). Equation 1 was used for the germination percentage G (%):

Where, ∑ni is the total number of germinated seeds and N is the total number of inoculated seeds. The germination speed index (GSI) was calculated using Equation 2:

Where G1, G2, ... Gn refer to the number of germinated seeds and N1, N2, ... Nn, to the number of days after in vitro inoculation. Germination energy (GE) was determined using Equation 3:

Where Gn is the number of seeds germinated in a given day period (seven days), and N is the total number of inoculated seeds. The germination energy period (GEP) was used through Equation 4:

Where PG is the number of seeds where the highest percentage of non-accumulated germination occurs and N is the total number of inoculated seeds.

The morphological development variables, such as plant height (PH), number of nodes (NN), number of leaves (LN), fresh biomass (FB), and dry biomass (DB), were analyzed 70 days after inoculation. To determine the fresh biomass, all seedlings from each treatment were used, cleaned, and weighed on a digital analytical balance accurate to 0.1 mg. Subsequently, the same seedlings were dried in an oven at 70 °C for 72 hours to determine the total dry biomass (Gonzales-Alvarado and Cardoso 2024).

Experimental design and statistical analysis

The experiment was carried out using a completely randomized design. For all variables evaluated, the assumptions of normality were verified using the Shapiro-Wilk test and homogeneity of variances using the Levene test. When these assumptions were not met, the Kruskal-Wallis nonparametric test was applied.

The variables associated with germination and morphological development complied with the assumptions of normality and homoscedasticity, and were therefore subjected to an analysis of variance (ANOVA). In cases where significant differences were found, Tukey's test of means was used with a significance level of 5% (α=0.05). However, the variable number of nodes (NN) did not meet the assumptions, so it was analyzed using the Kruskal-Wallis nonparametric test. To analyze the seed germination percentage as a function of the treatments, a second-degree polynomial regression was applied.

In addition, principal component analysis (PCA) was performed, including all the variables analyzed, using the ggplot2 function and confidence ellipses (level=0.95) in the biplot (Wickham 2016). For the correlation analysis between variables, Spearman's correlation coefficient was used by corrplot, considering -0.6<P >0.6, as strong correlation (Wei et al. 2024). All statistical analyses were performed using R Studio software, version 4.3.2.

Effect of culture media on in vitro germination parameters of Calycophyllum spruceanum

The germination behavior (G) of C. spruceanum shows a trend of progressive increase, stabilizing around 15 to 18 days after in vitro inoculation (Figure 1). The mathematical models fitted to the data correspond to quadratic equations with high coefficients of determination (R²>0.94), indicating an adequate representation of germination behavior in each treatment.

Figure 1: Germination percentage of C. spruceanum during 21 days post-inoculation in different culture media. T1: y = -0.2262x2 + 7.7312x - 15.946. R2 = 0.9474, T2: y = -0.2041x2 + 6.7035x - 12.13. R2 = 0.961, T3: y = -0.33x2 + 10.899x - 15.022. R2 = 0.9598, T4: y = -0.2668x2 + 9.0709x - 14.946. R2 = 0.9413, and T5: y = -0.3613x2 + 11.339x - 11.335. R2 = 0.9449.

It is observed that the culture medium significantly influences germination, with notable differences in the germination rate and the final percentage reached. The treatment with agar (T5) presents the most negative quadratic coefficient (-0.3613x²), suggesting a faster initial germination, but with an earlier stabilization. In contrast, the WPM and 50% WPM treatments (T3 and T4) show a more sustained germination rate over time, reaching higher values compared to the MS and 50% MS treatments (T1 and T2).

Germination is a fundamental aspect for proper management in plant development (Gonzales-Alvarado et al. 2022). Germination of C. spruceanum was influenced by the type of culture medium and its concentration. The use of agar and Woody Plant Medium (WPM) resulted in the highest germination percentages within 21 days, reaching 75.24 and 73.33%, respectively (Table 1 and Figure 1). Similar results were reported by Gonzales-Alvarado et al. (2024) and Marwein and Vijayan (2024), who observed that 100% agar and WPM enhanced the germination of Guazuma crinita and Adinandra griffithii, respectively. In contrast, Guerra-Arévalo et al. (2024) evaluated organic substrates for the germination of C. spruceanum and achieved 62.3% germination. When compared with these previous results, the application of agar and WPM in the present study increased the germination percentage by 17.7 and 20.77%, respectively. According to Pereira (2023), agar is a polysaccharide derived from algae that does not provide nutrients to plants and facilitates seed hydration, activating metabolic pathways that lead to the synthesis of proteins and enzymes essential for germination. In the present study, no statistically significant differences were found between agar (T5) and WPM (T3) regarding germination performance in C. spruceanum (Table 1). This result can be attributed to the early developmental stages of the embryo, during which external nutrients are not essential, as the embryo utilizes seed reserves to generate energy and the necessary chemical compounds for development (Meneses et al. 2022). According to Gonzales-Alvarado et al. (2022), they also emphasized the importance of medium composition in seed germination. For instance, Kirillov et al. (2024) reported that MS medium enhanced germination in Aflatunia ulmifolia, achieving over 50% germination compared to other media. However, in the present study, WPM promoted higher germination percentages than MS treatments. This effect may be associated with the lower concentrations of nitrogen, potassium, and ions in WPM, as well as its reduced ammonium content-approximately 25% lower than that of MS medium (Munthali et al. 2022).

Table 1: Effect of culture medium on in vitro germination parameters (mean ± standard error) of C. spruceanum.

Regarding the germination speed index. (GSI) and germination energy (GE), agar (T5) and WPM (T3) produced the highest values, with GSI reaching 2.32 and 2.02 seeds per day, and GE values of 60 and 52.38%, respectively. Germination was initiated at two days post-inoculation in all treatments except T1 (MS). Furthermore, during the Germination Energy Period (GEP), T1 (MS) exhibited the slowest response, reaching GE at six days with only 15.24%, the lowest value recorded (Table 1). In contrast, T5 and T3 presented GEP values of 28.57 and 25.71%, respectively.

On the Final Day of Germination (FDG), WPM completed the germination process in 19 days, while agar did so in 21 days. These results support the hypothesis that WPM enhances germination. The favorable outcomes observed with agar can be attributed to its stable surface, which supports seed germination and seedling development, despite its lack of nutrients. In contrast, WPM, characterized by lower salt concentrations (Delgado-Paredes et al. 2023), did not negatively impact germination parameters such as G, GSI, GID, GE, GEP, and GEP (%), indicating no phytotoxic effects on C. spruceanum. Conversely, the high salt concentration in MS medium appeared to limit the development of these germination parameters in vitro (Cabral-Miramontes et al. 2022).

Effect of culture media on the morphological development and biomass of Calycophyllum spruceanum

The results of morphological development showed significant differences in all the variables analyzed (Figure 2). Initial plant development encompassed key morphological parameters that influenced subsequent growth and adaptation to different cultivation conditions. Understanding these parameters contributed to the efficient production of seedlings and optimized propagation under in vitro conditions the efficient production of seedlings and optimizes their propagation in in vitro systems.

Figure 2: Effect of culture media on in vitro morphological development of C. spruceanum. A. number of leaves (LN). B. number of nodes (NN), and C. plant height (PH). T1: MS; T2: 50% MS; T3: WPM; T4: 50% WPM; and T5: Agar. Different letters indicate significant differences between treatments according to Tukey&apos;s test at 5% significance level.

The application of Woody Plant Medium (WPM) significantly enhanced the morphological development of C. spruceanum, yielding the highest values for number of leaves (LN), number of nodes (NN), and plant height (PH), with averages of 8.18 leaves per plant, 5.25 nodes per plant, and 16.83 cm in height, respectively (Figure 2).

Similar results were reported by Marwein et al. (2024), Lima et al. (2024), and Meneses et al. (2022), in which WPM demonstrated superior efficiency compared to other culture media, promoting the morphological development of in vitro seedlings of Rhododendron inaequale Hutch, Vismia japurensis, and Vaccinium floribundum, respectively. According to Krasnoperova and Bukharina (2020), indicated that WPM enhances both plant survival and development, supporting the hypothesis that this medium optimizes the morphological development of Calycophyllum spruceanum. To date, no previous studies have evaluated this specific aspect, suggesting that the present findings may contribute to the large-scale propagation of the species with improved morphological traits, suitable for meeting market demand in ecological restoration programs and industrial applications.

According to Stachevski et al. (2013), WPM has 50% of the ionic strength of Murashige and Skoog (MS) medium, as well as higher levels of sulfur (S) and lower concentrations of ammonium (NH₄⁺) and nitrate (NO₃⁻), which reduce the risk of ionic toxicity and improve the adaptability of woody species. Additionally, its balanced composition of macro- and micronutrients, including adjusted concentrations of nitrogen, phosphorus, and potassium, was essential for regulating plant metabolic functions (Delgado-Paredes et al. 2023). Previous studies on C. spruceanum under in vitro conditions employed MS medium for bud establishment and callus induction, with promising results (Olivas 2014; Figueiredo et al. 2014), obtaining promising results. The findings of the present study indicated that WPM represents a more effective alternative for enhancing morphological development and could complement previous efforts in tissue culture research on this species.

After 70 days of in vitro cultivation of C. spruceanum, T3 (WPM) exhibited the highest mean number of leaves (8.18 leaves per plant) and the greatest leaf area. These characteristics are associated with increased survival during the post-in vitro acclimatization phase and contribute to more efficient seedling production in a shorter period (Valladares and Niinemets 2008). In contrast, plants under treatment T5 (agar) displayed yellowish leaves with visible signs of stress, likely due to the absence of nutrients in the culture medium (Figure 3). These observations highlight the need for further studies on nutrient accumulation in plant tissues to complement the present findings. Similar results were reported by Gonzales-Alvarado et al. (2022) in Guazuma crinita, where plants cultured in phytogel for four months exhibited leaf yellowing and other stress-related symptoms.

Figure 3: Morphological characteristics of C. spruceanum at 70 days post-inoculation in vitro.

Regarding the production of fresh biomass (FB) and dry biomass (DB), it was identified that WPM allowed obtaining the highest values, with 147.27 and 16.07 mg per plant, respectively (Table 2), significantly surpassing the other treatments. The results support the hypothesis that the use of WPM optimizes germination, morphological development, and accumulation of biomass in C. spruceanum. In contrast, the agar-only treatment showed a marked limitation in growth, which could be attributed to the deficiency of essential nutrients, since agar alone does not provide the necessary compounds for proper plant development. This had a negative impact on the physiology of the seedlings, which showed an evident yellowing, as illustrated in Figure 3, possibly due to nutritional deficiencies and alterations in chlorophyll synthesis.

Table 2: Effect of culture media on biomass of C. spruceanum.

The use of WPM in C. spruceanum not only optimizes production times but also yields plants with enhanced morphological characteristics, which is essential for large-scale propagation. From an applied perspective, the methodology implemented in this study has the potential to substantially support the mass production of seedlings, thereby facilitating supply for reforestation programs and the restoration of degraded ecosystems. However, it is important to consider that establishing seeds under controlled and aseptic conditions-such as in specific in vitro culture media-may entail higher operational and logistical costs compared to direct sowing in field substrates. Nonetheless, this initial investment is justified by the production of healthy, uniform seedlings with a greater likelihood of survival, which represents a significant advantage for projects requiring high-quality plant material. In vitro propagation of C. spruceanum, therefore, is recommended as an innovative strategy to promote environmental sustainability and ensure the availability of this species for conservation and forest development initiatives. Furthermore, the findings regarding the application of WPM provide a robust foundation for future research in the in vitro culture of C. spruceanum, including studies on micropropagation, genetic transformation, and molecular biology. This approach may also prove valuable for research focused on the extraction of bioactive compounds of pharmacological and industrial relevance, enabling the efficient production of high-value secondary metabolites.

Main component analysis and correlation of germination parameters, morphological development, and biomass of Calycophyllum spruceanum

The main component analysis (PCA) and correlation of germination parameters and morphological development yielded relevant insights. In the PCA, treatments with agar (T5) and WPM (T3) were grouped and associated with germination percentage (G), germination speed index (GSI), and germination energy (GE) (Figure 4A). However, in the analysis of morphological development, these treatments showed clear differentiation. The WPM treatment (T3) exhibited stronger associations with leaf number (LN), number of nodes (NN), plant height (PH), fresh biomass (FB), and dry biomass (DB) (Figure 5A).

Figure 4: A: Main component analysis of germination parameters of C. spruceanum, and B: Correlation analysis between germination variables of C. spruceanum. G: germination percentage, GSI: germination speed index, and GE: germination energy.

Figure 5: A: Main component analysis of morphological development and biomass of C. spruceanum, and B: Correlation analysis of morphological development and biomass variables of C. spruceanum. PH: plant height, NN: number of nodes, LN: number of leaves, FB: fresh biomass, and DB: dry biomass.

Correlation analysis revealed positive associations within both germination and morphological development parameters (Figure 4B and Figure 5B). Among the germination parameters, correlation coefficients exceeded 0.87, with the strongest observed between GSI and germination percentage (G), at 0.93 (Figure 4B). To date, this is the first in vitro study to report such correlations in C. spruceanum, emphasizing the importance of further investigations into its germination processes. Additional research should focus on optimizing germination under ex vitro conditions to enhance propagation efficiency. Moreover, the inclusion of other germination-related parameters is recommended to refine and improve alternative propagation protocols for both natural and controlled environments.

Regarding morphological development, the correlations of plant height (PH), number of nodes (NN), and number of leaves (LN) were greater than 0.84, suggesting a strong association between these growth factors. The highest correlations were recorded between fresh biomass (FB) and dry biomass (DB), with a value of 0.92, confirming that better morphological development contributes directly to higher biomass accumulation. Similar results were reported by Gonzales-Alvarado et al. (2024) and Mori-Vásquez et al. (2024) in Guazuma crinita and Simarouba amara, respectively, where strong correlations were evidenced between fresh and dry biomass, as well as between plant height and biomass. These results reinforce the present findings and confirm the interdependence between morphological traits and biomass production.

The outcomes of this study underscore the necessity for further investigations into the responses of C. spruceanum under various growth conditions. It is particularly important to evaluate root system interactions, assess physicochemical parameters such as pH, electrical conductivity (EC), and the accumulation of macro and micronutrients. Additionally, future research should explore the influence of biochemical components in the culture media on chlorophyll content and the expression of growth- and development-related genes under in vitro conditions. Integrating genetic, physiological, and molecular approaches would facilitate more accurate correlations between morphological responses and molecular regulation in this species. Such insights could significantly enhance propagation protocols and improve the efficiency of C. spruceanum seedling production for both commercial use and conservation or ecological restoration programs.