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Article

Optimizing Somatic Embryogenesis and Biomass Proliferation in Narcissus L. ‘Carlton’ Callus Lines Using Solid and Liquid Media

by
Małgorzata Malik
*,
Justyna Mazur
and
Anna Kapczyńska
Department of Ornamental Plants and Garden Art, University of Agriculture in Krakow, al. 29 Listopada 54, 31-425 Kraków, Poland
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(11), 2460; https://doi.org/10.3390/agronomy15112460
Submission received: 14 September 2025 / Revised: 20 October 2025 / Accepted: 21 October 2025 / Published: 23 October 2025
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

Somatic embryogenesis (SE) in Narcissus offers significant potential for both horticultural propagation and pharmaceutical applications. In this study, embryogenic callus lines derived via primary and secondary SE were evaluated under different in vitro conditions to assess the effects of medium type (liquid vs. solid) and composition (proliferation vs. regeneration) on biomass growth and somatic embryo formation. Lines derived from primary SE (LC1–LC4) were less efficient compared to those obtained through secondary SE (LC5–LC7). Cultures cultivated in liquid proliferation medium for eight weeks showed a greater biomass accumulation than those grown on solid medium. Multivariate analyses revealed distinct growth patterns and responses to medium type among the callus lines. The LC5 and LC7 lines formed a separate cluster characterized by superior biomass proliferation and embryogenic competence. An eight-week culture in a liquid proliferation medium followed by a transfer to a solid medium of the same composition resulted in the highest somatic embryo yield in the LC5 line (54.4 embryos per 0.5 g of callus). Under the same conditions, the LC7 line showed the highest biomass growth (a 23.4-fold increase), but its embryogenic response was more effectively stimulated when the callus was initially proliferated on a solid medium and then transferred to a regeneration medium.

1. Introduction

Narcissus L. is a perennial geophyte with a high ornamental value, commonly used in urban landscapes and home gardens as a cut flower and as a pot plant [1,2]. Among the many available cultivars, ‘Carlton’ stands out as one of the few with a very high commercial importance and is the most extensively grown. The strong interest in this bulbous plant within the floriculture industry is due to its long-lasting flowers and early blooming. ‘Carlton’ is a vigorous, long-lived cultivar suitable for naturalization, having been cultivated for over a century [1].
The importance of ‘Carlton’ goes beyond its ornamental value. It is cultivated as a primary source for extracting alkaloids such as galanthamine, haemanthamine, lycorine, and narciclasine [3,4,5], as well as carbohydrate-binding proteins—lectins [6]—all derived from the bulbs and known for their pharmaceutical properties. Alkaloids are used in the treatment of Alzheimer’s disease and cancer [7,8]. Lectins have been reported as effective inhibitors of infections by human (HIV) and feline (FIV) immunodeficiency viruses, with potential applications in preventing viral transmission [6]. Recent studies suggest that an alternative approach to meeting the current demand for sustainable galanthamine production could involve obtaining this alkaloid from ‘Carlton’ daffodil tissues (bulbs, shoots, and calli) produced in vitro [9,10].
Both organogenesis and SE pathways have been described for the in vitro regeneration of Narcissus species [11,12,13,14,15]. In vitro propagation methods have demonstrated a higher efficiency than traditional techniques, including the separation of adventitious bulbs, chipping, and twin-scaling [1]. Thus, in vitro cultures can serve as a valuable source of high-quality elite propagation material.
The largest number of Narcissus plants was obtained through SE [12,13,14,15,16,17,18]. Both primary and secondary (repetitive, recovery) SE offer the potential to scale up somatic embryo production. The high efficiency of SE has been confirmed by propagation protocols developed for numerous plant species, including Iris germanica L. [19], Theobroma cacao L. [20,21], Piper nigrum L. [22], Lilium longiflorum Thunb. [23], Phoenix dactylifera L. [24], and Cocos nucifera L. [25].
Calli obtained, even within the same genotype, may differ in their propagation and embryogenic potential [26,27]. Frequent and significant differences in somatic embryo regeneration ability arise from the complex nature of the callogenesis and its multifactorial dependence on environmental conditions, genotype, source, and type of explant, and medium composition [28]. Cells respond to various stress factors by reprogramming gene expression and altering their developmental program to acquire embryonic competence [21,29]. The development of different callus lines with variable characteristics is considered a significant limitation in using the indirect SE method for commercial propagation [24,30,31].
Propagation efficiency can be enhanced by technologies based on the use of liquid media in rotary shaker cultures, temporary immersion systems, and bioreactors [16,18,32,33,34]. The literature identifies the ease of nutrient and growth regulator uptake as a key advantage of liquid media, due to the direct contact between tissues and the medium [32]. Moreover, the agitation of cultures in liquid media improves aeration, ensuring the optimal growth and faster multiplication of embryogenic callus [24].
The aim of this study was to evaluate the effects of liquid and solid media on biomass growth and somatic embryo production in various embryogenic callus lines of Narcissus L. ‘Carlton’. Additionally, the study aimed to identify the most effective medium combinations for optimizing in vitro propagation and to investigate the associations between medium type, callus line origin, and regenerative performance.

2. Materials and Methods

2.1. Plant Material

The plant material used in this investigation consisted of six Narcissus L. ‘Carlton’ embryogenic callus lines (Figure 1), maintained in proliferation medium (with the same composition as the initial medium) by repetitive subcultures for different time periods. Primary SE lines (LC1, LC3, and LC4) were developed over a period of 3 months, following the procedure described by Malik and Bach [15]. Secondary SE lines (LC5, LC6, and LC7) were established over 12 months, according to the method described by Malik and Bach [17]. The origin and characteristics of the lines are presented in Table 1. The proliferation medium was composed of MS (Murashige and Skoog) basal medium [35], 30 g L−1 sucrose, and growth regulators—Picloram or 2,4-D—in combination with BA (Table 1). The pH was adjusted to 5.5 before autoclaving (for 20 min at 121 °C and 0.1 MPa), and the medium was solidified 0.7% agar (Purified Difco Agar). Plastic Petri dishes (90 × 25 mm) were used for media distribution. Embryogenic cultures were incubated at 20 ± 2 °C in darkness and subcultured every 4–5 weeks.

2.2. Culture Sysrems

To establish liquid cultures, 0.5 g callus samples from each line were collected (with mature embryos removed) and placed in 100 mL Erlenmeyer flasks containing 20 mL of liquid proliferation medium. The composition of the proliferation media was the same as that used at the stage of the embryogenic callus initiation process (Table 1). Plant material in liquid medium was cultivated in rotary shakers at 100 rpm for 8 weeks (first stage of experiment) and then transferred for 24 weeks to solid media in Petri dishes (second stage of experiment). Half of the material was placed on proliferation medium and half on regeneration MS [35] basal medium containing 30 g L−1 sucrose and growth regulators—0.5 µM NAA and 5 µM BA (Table 1). The pH of regeneration medium was adjusted to 5.8 before autoclaving (for 20 min at 121 °C and 0.1 MPa), and the medium was solidified 0.7% agar (Purified Difco Agar). All cultures (liquid and solid) were cultured in darkness at 20 °C. In liquid cultures, half of the medium volume was replaced weekly; solid cultures were subcultured onto fresh medium every 4 weeks. Every 8 weeks (three terms: TI–III), the increase in biomass ([final weight − initial weight]/initial weight) and the number of forming somatic embryos per 0.5 g of inoculum were determined. The scheme of the experiment is presented in Figure 2.
The control consisted of cultures maintained in Petri dishes filled with 25 mL of agar-solidified medium. The composition of the control media and the initial tissue weight were identical to those used in the liquid culture part of the experiment.

2.3. Statistical Analysis

This experiment was designed as a three-factorial (6 LC callus lines × 2 Msm physical states of medium × 2 Mt medium types) layout comprising 24 treatments with 3 replications, each consisting of 6 vessels. All data were analyzed using the Statistica 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA). The experimental data were subjected to analysis of variance (ANOVA), and Tukey’s multiple range test was used to separate mean values at the significance level of p ≤ 0.05.
The Principal Component Analysis (PCA) and the Hierarchical Cluster Analysis (HCA) were conducted to reveal the general structure in relationships among the studied traits related to medium type, callus line, and callus growth parameters, and to classify the observations. The analyses were performed on standardized data. PCA included dichotomous variables indicating the presence of a particular medium type, which were transformed into dummy variables (binary). Eigenvalues and eigenvectors were calculated from the correlation matrices. The number of principal components representing the most relevant data variation was determined using Kaiser rule and percentage of variance criterion [36,37,38]. Hierarchical Cluster Analysis was performed using the Ward agglomeration method and Euclidean distance criterion [38]. To assess the nature and strength of relationships between medium type and callus growth parameters, both the Pearson correlation coefficient (r) and point-biserial correlation coefficient (rpb) were calculated [39,40,41]. Finally, the analysis of variance (ANOVA), followed by Tukey’s honest significance test (at the p ≤ 0.05), was conducted to indicate and compare the effects of different medium combinations on callus biomass growth and embryo production.

3. Results

3.1. Effect of Callus Line on Biomass Growth

The results revealed significant differences among the six evaluated callus lines. The experiment utilized yellow, nodular calli derived via primary SE (PSE, lines LC1, LC3, and LC4), as well as looser, softer, cream-colored nodular calli with a finer granulation obtained through secondary SE (SSE, lines LC5, LC6, and LC7) (Figure 1). The callus lines (LC1–LC7), differing in origin, proliferated in liquid media with varying intensities. The lowest biomass growth was observed in line LC1, while the highest was recorded for line LC7 (4.04 and 14.89, respectively) (Table 2).

3.2. Effect of Proliferation in Liquid Medium on Biomass Growth and Somatic Embryo and Root Formation

An analysis of the effect of an 8-week cultivation period on the liquid proliferation medium (regardless of other factors) demonstrated that liquid media stimulate biomass growth (Table 2). The average biomass increase in cultures grown in a liquid medium was 8.44, compared to 7.12 on a solid medium. A higher biomass increase was also noted in cultures maintained on a proliferation medium throughout the entire experiment (8.72), in contrast to those grown on a regeneration medium (6.84).
Statistical analysis ANOVA comparing the six lines used in the experiment showed that the number of somatic embryos formed depended on the origin of the callus, while neither the physical state of the proliferation medium used during the first stage of the experiment nor the type of medium used in the second stage had a significant effect (Table 2, Figure 3). Lines LC1, LC3, and LC4 (PSE-derived) were the least productive. No somatic embryo formation was observed in LC1 cultures, and only a few embryos were obtained in LC3 and LC4 (0.13 and 2.03 embryos per 0.5 g of callus, respectively). In contrast, LC5 and LC7 callus cultures yielded the highest numbers of somatic embryos (35.98 and 9.92, respectively).
The development of adventitious roots was also observed in the cultures (Table 2, Figure 3). The highest number of adventitious roots was obtained in the LC6 and LC7 lines, with 6.78 and 7.19 roots per 0.5 g of callus, respectively. These roots were short and thin. Slightly fewer roots were observed in the PSE-derived lines—LC1, LC3, and LC4—ranging from 2.56 to 5.34 roots per 0.5 g of callus, but they were longer and thicker. The lowest root formation occurred in the LC5 cultures (1.94 roots per 0.5 g of callus). Cultures proliferated in a liquid medium and subsequently transferred to a solid regeneration medium produced significantly more roots (6.07 roots per 0.5 g of callus) than those proliferated and regenerated entirely on solid media (3.41 roots per 0.5 g of callus).
The Principal Component Analysis (PCA), a multivariate statistical technique to identify and understand latent interrelations among variables, observations, or objects [37,38], was calculated for five variables and six callus lines (LC1–LC7) obtained from the last stage of the experiment. The first two principal components (PCs) explained 59.29% of total variability (Table 3).
On the basis of PCA outcomes and the biplot, two groups of correlated variables can be distinguished (Table 3, Figure 4A). The first one encompasses the variables that are the most important contributors to the first PC and that are strongly negatively correlated, such as Root No. (−0.79) and Mt medium type (−0.79). The location of those variables’ vectors on the biplot graphs shows the association of the Mt medium type with the process of the roots’ formation. The PC1 separates observation points on the biplot into clusters differed by Mt medium type. Their localization in relation to the Root No. vector suggests that the root formation in callus lines is stimulated by the regenerative medium (Mt R) (Figure 4A). The second group consists of variables constituting the main contribution to the second principal component, which are the Msm physical state of the medium, Embryo No., and biomass growth (Table 3, Figure 4A). These variables’ correlation indicates the relationship of the intensity of embryo formation and tissue growth with the Msm type. The PCA biplot shows that PC2 slightly separates observations into groups in terms of the applied Msm. Observation points representing calli cultivated on a liquid medium (Msm L) slightly cluster toward the vectors for Embryo No. and biomass growth. This suggests that the liquid medium favors biomass growth and embryo development (Figure 4).
The projections of the observation points on the PCA biplot revealed their callus line-related clustering (Figure 4A). Observations that represent LC7 and LC5 among other lines are located closest to the vectors for the biomass growth and Embryo No. variables. This indicates that those two lines are distinguished by a higher overall growth and number of produced embryos from other lines (Figure 4A). To determine whether there are similarities between the callus lines within growth parameters and treatment response on the basis of the studied traits (Embryo No., biomass growth, Root No.), Hierarchical Cluster Analysis was employed. This multivariate technique allows us to group objects based on their features [38]. The analysis of the dendrogram allowed us to classify the studied callus lines into two main clusters (Figure 4B). Cluster 1 encompasses lines LC5 and LC7, while cluster 2 includes all the remaining ones. The HCA outcome confirmed the results obtained from PCA concerning the similarities between LC5 and LC7, and that those two callus lines within the examined features differ from the other lines.

3.3. Effect of Medium Type on LC5 and LC7 Callus Line Parameters

Further analysis of this study focused on callus lines LC5 and LC7, which were shown to be associated with more growth and a higher number of embryos. Correlation coefficients were calculated based on the data obtained from the last stage of the experiment, and analysis of variance (ANOVA) was calculated for data from all terms (TI–TIII). The objectives of these analyses were to assess the strength of association between the medium type and growth features of those callus lines, as well as to determine the combination of medium types for the most efficient embryo production.
The point-biserial correlation coefficient (rpb) is a statistical method used to calculate the correlation between quantitative variables and qualitative variables, such as dichotomous ones, that are coded into dummy (binary) variables [39,40,41]. The correlation coefficient results presented in a graph (Figure 5) revealed the different nature of the associations between medium types and callus lines LC5 and LC7.
For the LC5 callus line, the point-biserial correlation coefficient showed a strong positive correlation of the Msm liquid medium with the variable for Embryo No. (0.71), as well as between the Mt proliferation medium and biomass growth (0.83). This indicates that the average number of embryos produced increases when cultivated in a liquid medium, and that a greater callus biomass growth is correlated with the use of a proliferation medium. Biomass proliferation in LC5 was the highest when a proliferation medium was used in the second stage of the experiment, regardless of whether the callus was initially proliferated on a liquid or solid medium (10.3 and 9.4, respectively) (Figure 6).
The LC5 line proliferated for 8 weeks in a liquid medium, and then on a solid one, produced the highest number of embryos (54.4). A lower, but still considerable number of embryos was obtained (35.8) when cultures were transferred to a solid regeneration medium after proliferation in a liquid medium. When LC5 callus was initially proliferated on a solid proliferation medium, the number of embryos was lower: 26.7 on a solid proliferation medium and 27.1 on a solid regeneration medium in the second stage.
In the LC7 callus line, a strong correlation was observed between the Msm solid medium and the number of embryos (−0.84). Biomass growth showed a moderately positive correlation with the Mt proliferation medium (0.60). The highest biomass increase in LC7 cultures was observed when calli were first proliferated in a liquid proliferation medium and then transferred to a solid medium of the same composition (23.4-fold). Proliferation in a liquid medium clearly enhanced the biomass accumulation. When a solid proliferation medium was used in the first stage, biomass growth decreased in the second stage regardless of whether a regeneration or proliferation medium was used (14.1-fold and 12.9-fold, respectively). The number of roots correlated both with the Mt regeneration medium (−0.55) and with the Msm liquid medium (0.52). In the LC7 callus line, a moderate negative correlation between callus growth and the root number was also observed (−0.58). The LC7 line, characterized by high biomass growth, exhibited a less intense somatic embryo formation. The highest number of embryos—16.4 per 0.5 g of callus—was obtained when the calli were initially cultured on a solid medium and then transferred to a regeneration medium. LC7 calli proliferated in a liquid medium and subsequently transferred to either a solid proliferation or solid regeneration medium did not express significant embryogenic potential (6.1 and 6.2 embryos, respectively).

4. Discussion

The evaluation of the different in vitro culture systems (liquid/solid) demonstrated the usefulness of rotary shaker cultures during proliferation to enhance biomass growth and somatic embryo formation compared to conventional micropropagation systems based on a solid medium. The study on the use of a liquid medium during the proliferation stage was conducted on six callus lines of Narcissus L. ‘Carlton’ obtained through primary and secondary SE. These lines differed in both morphology and regeneration capacity. In many plant species, callus cultures exhibit diverse morphological and developmental characteristics [27]. A high variability in the regeneration potential—ranging from non-embryogenic lines to highly productive ones—among callus lines derived from cultures of the same species has been reported by many researchers, including Bennici et al. [42] in Amaranthus L., Fitch and Moore [43] in sugarcane, Hoenemann et al. [27] in Cyclamen persicum Mill., Šavikin-Fodulović et al. [44] in Dioscorea balcanica Košanin, Li et al. [45] in Rosa hybrid L., Xu et al. [19] in Iris germanica L., Chen and Chang [46] in Oncidium Sw. ‘Gower Ramsey’, and Peng et al. [47] in Pinus koraiensis Siebold&Zucc.
The regeneration potential and embryogenic competence are often correlated with the callus appearance, particularly its texture, which depends on explant type, age, genotype, and medium composition—especially on the type and concentration of growth regulators [31,42,48]. Callus formation is a nonlinear and complex process, influenced by numerous internal and external factors [27,28]. Downey et al. [49] noted that, in many monocot species, the embryogenic callus is typically nodular with a green or yellow color, or appears as a compact white callus, whereas the non-embryogenic callus is usually friable, with a soft, watery appearance or browning tissue.
All callus lines of Narcissus ‘Carlton’ examined in this study were nodular, yellow in the case of PSE-derived lines, and creamy in SSE-derived lines. Calli derived through SSE were characterized by a finer granularity, making them easier to manipulate. The highest biomass proliferation and somatic embryo production were observed in two ‘Carlton’ callus lines—LC5 and LC7—originating from repeated cycles of SE. In contrast, the callus lines LC1, LC3, and LC4, derived from primary explant cultures, showed the lowest performance in terms of the studied parameters. Not only in Narcissus, but also in many other plant species, the efficiency of explants in secondary SE is higher than in primary SE [22,50]. In Theobroma cacao L. cultures, the production efficiency of secondary somatic embryos was up to thirty times higher than that achieved through primary SE. Additionally, secondary embryos were more uniform and developed faster compared to primary ones [20]. Compared to PSE, SSE is characterized by a very high multiplication rate, independence from the explant source, high repeatability, and the ability to maintain embryogenic potential over extended periods [21,51]. It is also considered a time- and cost-effective approach [45]. SSE enables the continuous multiplication of embryos (repetitive SE) [50,52], leading to the development of long-term callus lines that retain embryogenic competence and significantly enhance the potential regeneration rate—by several thousand-fold in some cases, as observed in coconut [53]. Thus, SSE provides opportunities for production scale-up and, due to its greater repeatability, facilitates standardization and mechanization. In contrast, calli derived from PSE rapidly lose embryogenic potential—as in peach palm, where this occurs after only a few subcultures [33].
Auxins and auxin-like plant growth regulators are key factors in callus growth and development and are commonly essential for the induction of SE in many plant species [27,54,55]. Auxins and their analogues promote SE by regulating auxin signaling pathways and inducing stress responses that alter endogenous auxin levels [54,55,56].
The callus lines used in this study were derived from shoot or ovary cultures of Narcissus exposed to Picloram or 2,4-D—two of the most widely used auxins for SE induction. Previous studies have demonstrated the high effectiveness of these compounds. In the re-search conducted by Malik and Bach [15], somatic embryos in primary explant cultures grown on solid media—where calli were not separated from the mother tissue—formed most abundantly on media containing 25 µM 2,4-D or Picloram combined with at least 5 µM BA. Similarly, in subsequent research, the highest number of embryos in repetitive somatic embryogenesis (RSE) cultures was also observed for the same media compositions, when calli were proliferated independently of the original explant [17]. Auxin concentrations either lower or higher than 25 µM, as well as cytokinin concentrations below 5 µM, were found to reduce the efficiency of somatic embryogenesis in Narcissus. Similarly, using flower stem explants instead of ovary explants as the initial material negatively affected the embryogenesis efficiency in both primary SE and secondary SE cultures [15,17]. Many studies have emphasized that, not only the absolute concentrations of plant growth regulators, but also the ratios between auxins and cytokinins play a crucial role in somatic embryogenesis. Auxins influence cytokinin biosynthesis and signaling [57], and the balance between these two hormone groups has proven essential for the successful induction and development of somatic embryos in species such as Coffea canephora Pierre ex A. Froehner [57], the peach rootstock ‘Guardian®’ [58], and Picea spp. [59]. However, the prolonged exposure of cultures to media containing 2,4-D may lead to a decline in embryogenic capacity, despite promoting cell proliferation. 2,4-D has been shown to cause genetic, epigenetic, physiological, and morphological abnormalities, which can impair regeneration or lead to the formation of abnormal somatic embryos [60,61]. It is likely that the LC7 line (induced and proliferated with 2,4-D), which initially showed a high somatic embryogenesis potential similar to that of the LC5 line (induced and proliferated with Picloram) [15,17], gradually lost its embryogenic competence over time, while maintaining its capacity for proliferation. This may explain the observed differences between the two most efficient callus lines.
The alternation between liquid and solid media during the initiation of SE in Narcissus ‘Carlton’, as shown by Malik [16], improved the efficiency of the process. The most favorable SE response was observed when primary explants (ovaries) were shaken in a liquid medium with 25 µM 2.4-D and 5 µM BA for 8 weeks, followed by 4 weeks on a solid medium. The aim of the present study was to assess whether an 8-week proliferation phase in a liquid medium—applied to both primary callus cultures (after separation from the explant) and secondary callus cultures (derived from callus and primary somatic embryos)—could enhance SE efficiency. ANOVA, PCA, and point-biserial correlation analyses demonstrated that an 8-week treatment in a liquid medium promoted either biomass accumulation or somatic embryo formation in embryogenic callus cultures of ‘Carlton’. The liquid culture stimulated biomass growth across all lines. The main reasons for the improved biomass proliferation in liquid media, as indicated in the literature, include the enhanced diffusion and therefore greater availability of nutrients (such as mineral salts, vitamins, and growth regulators) [62]. In a liquid medium, the accessibility of these components is not limited by the physical constraints of solidified media, and they tend to be absorbed more rapidly and dynamically [63]. Additionally, continuous mixing ensures an even distribution of nutrients throughout the culture volume, preventing the localized depletion of essential compounds [64]. Moreover, cells in liquid culture (in a rotary shaker and bioreactor) have a better access to oxygen and can more efficiently release metabolic products compared to cultures grown on solid media [62,64]. However, the somatic embryo formation in response to a liquid medium was particularly enhanced in line LC5. The results also revealed the distinct characteristics of the two most productive callus lines: LC5 showed a high embryogenic capacity, while LC7 exhibited a superior biomass proliferation. Both responses were positively influenced by an 8-week passage in a liquid medium.
The asynchronous development of somatic embryos is considered one of the main limitations hindering the automation of plant production via somatic embryogenesis [19,33,65,66]. Alternating liquid and solid media may help to improve synchronization. Proliferation in liquid media typically delays embryo maturation [18]. In the present study, the advancement of somatic embryos beyond the globular stage and their successful conversion into plants—particularly in embryogenic callus lines derived via SSE—was primarily observed after the transfer from a liquid proliferation medium to a solid regeneration medium with reduced auxin levels (5 µM BA and 0.5 µM NAA). An improved SE through the use of liquid media during the proliferation phase has also been reported in Lilium longiflorum Thunb. [23], Phoenix dactylifera L. [24], and Catharanthus roseus (L.) G. Dom [65], where liquid media enhanced embryo growth and development. Moreover, liquid systems significantly increase SSE yield per unit volume and shorten the overall production cycle [22].
Understanding the factors influencing SE efficiency in shaken cultures is an important step toward the automation of Narcissus production. In addition to improving embryo yield, future studies should focus on the synchronization of somatic embryo development. The medium composition may play a crucial role in ensuring uniform and timely embryo maturation, which is essential for scalable and efficient propagation systems.

5. Conclusions

The study demonstrated that the use of a liquid medium during the proliferation phase significantly enhanced biomass growth and somatic embryo formation in selected callus lines of Narcissus L. ‘Carlton’. Lines LC5 and LC7, derived via SSE, proved particularly effective, highlighting their potential for optimizing in vitro propagation. The LC5 line showed the highest embryogenic efficiency when cultured for 8 weeks in a liquid proliferation medium, followed by a transfer to a solid proliferation medium for 24 weeks, while the LC7 line, under the same conditions, achieved the greatest biomass proliferation.

Author Contributions

Conceptualization, M.M.; methodology, M.M. and J.M.; software, M.M. and J.M.; validation, M.M., J.M., and A.K.; formal analysis, M.M., J.M., and A.K.; investigation, M.M.; resources, M.M.; data curation, M.M.; writing—original draft preparation, M.M. and J.M.; writing—review and editing, M.M., J.M., and A.K.; visualization, M.M. and J.M.; supervision, M.M.; project administration, M.M.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Ministry of Science and Higher Education of the Republic of Poland from subvention funds for the University of Agriculture in Krakow.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Types of Narcissus L. ‘Carlton’ calli used in the experiment: (A)—yellow nodular callus of the LC1 line obtained through primary somatic embryogenesis; (B)—cream-colored nodular callus of the LC5 line obtained through secondary somatic embryogenesis; (C)—cream-colored nodular callus of LC7 line obtained through secondary somatic embryogenesis. Globular-stage somatic embryos are indicated by arrows; bar = 1 cm.
Figure 1. Types of Narcissus L. ‘Carlton’ calli used in the experiment: (A)—yellow nodular callus of the LC1 line obtained through primary somatic embryogenesis; (B)—cream-colored nodular callus of the LC5 line obtained through secondary somatic embryogenesis; (C)—cream-colored nodular callus of LC7 line obtained through secondary somatic embryogenesis. Globular-stage somatic embryos are indicated by arrows; bar = 1 cm.
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Figure 2. Scheme of the experiment on Narcissus L. ‘Carlton’.
Figure 2. Scheme of the experiment on Narcissus L. ‘Carlton’.
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Figure 3. Embryogenic cultures of Narcissus L. ‘Carlton’ after the second stage of the experiment. The first column shows cultures treated with the proliferation medium, and the second column shows cultures treated with the regeneration medium during 24 weeks of the second stage; bar = 1 cm.
Figure 3. Embryogenic cultures of Narcissus L. ‘Carlton’ after the second stage of the experiment. The first column shows cultures treated with the proliferation medium, and the second column shows cultures treated with the regeneration medium during 24 weeks of the second stage; bar = 1 cm.
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Figure 4. Multivariate analysis for Narcissus ‘Carlton’ callus lines: (A) PCA biplots of observation points and variable vectors projected on the plane of the first two principal components (PCs), showing similarities and relationships between types of callus lines, medium/treatment, and callus growth characteristics. Points represent the observations and are distinguished to callus line by color and to treatment types by symbol, n = 96. (B) Dendrogram of Hierarchical Cluster Analysis. HCA allowed us to identify two groups of callus lines based on the following characteristics: Embryo No., biomass growth, Root No.
Figure 4. Multivariate analysis for Narcissus ‘Carlton’ callus lines: (A) PCA biplots of observation points and variable vectors projected on the plane of the first two principal components (PCs), showing similarities and relationships between types of callus lines, medium/treatment, and callus growth characteristics. Points represent the observations and are distinguished to callus line by color and to treatment types by symbol, n = 96. (B) Dendrogram of Hierarchical Cluster Analysis. HCA allowed us to identify two groups of callus lines based on the following characteristics: Embryo No., biomass growth, Root No.
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Figure 5. Graph representing results of point-biserial correlation coefficient (rpb) and Person correlation coefficient (r) of medium types and callus growth features for Narcissus ‘Carlton’ callus lines LC5 and LC7. The size and color of points in graph represents the strength and direction of correlation between pairs of variables, and the significance at p < 0.05 was marked (*). Correlation strength is expressed by color intensity and point size (strong correlations stands out in dark blue/red and larger size).
Figure 5. Graph representing results of point-biserial correlation coefficient (rpb) and Person correlation coefficient (r) of medium types and callus growth features for Narcissus ‘Carlton’ callus lines LC5 and LC7. The size and color of points in graph represents the strength and direction of correlation between pairs of variables, and the significance at p < 0.05 was marked (*). Correlation strength is expressed by color intensity and point size (strong correlations stands out in dark blue/red and larger size).
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Figure 6. Effect of physical state of medium (Msm) and medium type (Mt) on biomass growth and embryo development in Narcissus L. ‘Carlton’ LC5 and LC7 lines across three experimental terms (TI-TIII) during second stage of experiment. Mean values with different small letters are significantly different according to Tukey’s test at p ≤ 0.05.
Figure 6. Effect of physical state of medium (Msm) and medium type (Mt) on biomass growth and embryo development in Narcissus L. ‘Carlton’ LC5 and LC7 lines across three experimental terms (TI-TIII) during second stage of experiment. Mean values with different small letters are significantly different according to Tukey’s test at p ≤ 0.05.
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Table 1. Characteristics of Narcissus L. ‘Carlton’ callus lines used in the experiment.
Table 1. Characteristics of Narcissus L. ‘Carlton’ callus lines used in the experiment.
Callus LineOrigin of Primary ExplantGrowth Regulators in Initiation Medium (µM)Callus Age (Months)Callus Morphology/SE Type *
LC1Flower stem isolated from bulbs chilled for 6 weeks10 Picloram + 1 BA3Yellow, nodular/PSE
LC3Flower stem isolated from bulbs chilled for 6 weeks25 Picloram + 5 BA3Yellow, nodular/PSE
LC4Ovary isolated from bulbs chilled for 6 weeks25 Picloram + 5 BA3Yellow, nodular/PSE
LC5Ovary isolated from bulbs chilled for 12 weeks25 2,4-D + 5 BA12Cream-colored, nodular/SSE
LC6Ovary isolated from bulbs chilled for 3 weeks10 Picloram + 1 BA12Cream-colored, nodular/SSE
LC7Ovary isolated from bulbs chilled for 3 weeks25 Picloram + 5 BA12Cream-colored, nodular/SSE
* SE type—somatic embryogenesis type; PSE—primary somatic embryogenesis; SSE—secondary somatic embryogenesis.
Table 2. Efficiency of Narcissus L. ‘Carlton’ somatic embryogenesis in cultures of six callus lines after 32 weeks of experiment.
Table 2. Efficiency of Narcissus L. ‘Carlton’ somatic embryogenesis in cultures of six callus lines after 32 weeks of experiment.
EffectsBiomass GrowthNumber of EmbryosNumber of Roots
Effect of callus line (LC)
LC14.04 ± 2.05 d A0.00 ± 0.00 c4.63 ± 2.19 ab
LC35.48 ± 0.60 c0.13 ± 0.34 c5.34 ± 1.55 ab
LC48.94 ± 1.57 b2.03 ± 2.64 c2.56 ± 2.14 ab
LC58.76 ± 1.34 b35.98 ± 15.43 a1.94 ± 0.59 b
LC64.56 ± 1.40 cd0.53 ± 1.02 c6.78 ± 1.91 a
LC714.89 ± 5.54 a9.92 ± 4.66 b7.19 ± 3.38 a
Effect of physical state of medium during proliferation (Msm)
Solid7.12 ± 3.89 b7.61 ± 10.09 a3.41 ± 1.00 b
Liquid8.44 ± 5.03 a8.68 ± 18.02 a6.07 ± 1.40 a
Effect of medium type (Mt)
Proliferation8.72 ± 5.34 a8.47 ± 16.9 a0.19 ± 0.11 b
Regeneration6.84 ± 3.31 b7.82 ± 11.9 a9.29 ± 1.47 a
Main effects B
LC*********
Msm***ns***
Mt***ns***
LC × Msm*********
LC × Mt*********
Msm × Mt********
LC × Msm × Mt********
A—Mean values ± SD followed by different letters are significantly different at p ≤ 0.05 according to Tukey’s multiple range test. B—Main effects: *** at p ≤ 0.01; ** at p ≤ 0.05; ns—not significant.
Table 3. Component loading of five traits, eigenvalues, proportion of total variability represented by the first two principal components (PCs), and cumulative variability in Narcissus L. ‘Carlton’ callus lines LC1–LC7.
Table 3. Component loading of five traits, eigenvalues, proportion of total variability represented by the first two principal components (PCs), and cumulative variability in Narcissus L. ‘Carlton’ callus lines LC1–LC7.
VariablePrincipal Component
PC1PC2
Biomass growth0.573802−0.554174
Embryo No.0.384200−0.520377
Root No.−0.789683−0.376311
Msm0.0242850.678211
Mt−0.785065−0.260205
Eigenvalue1.721.25
Percentage of Variance34.3524.94
Cumulative % of Variance34.3559.29
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Malik, M.; Mazur, J.; Kapczyńska, A. Optimizing Somatic Embryogenesis and Biomass Proliferation in Narcissus L. ‘Carlton’ Callus Lines Using Solid and Liquid Media. Agronomy 2025, 15, 2460. https://doi.org/10.3390/agronomy15112460

AMA Style

Malik M, Mazur J, Kapczyńska A. Optimizing Somatic Embryogenesis and Biomass Proliferation in Narcissus L. ‘Carlton’ Callus Lines Using Solid and Liquid Media. Agronomy. 2025; 15(11):2460. https://doi.org/10.3390/agronomy15112460

Chicago/Turabian Style

Malik, Małgorzata, Justyna Mazur, and Anna Kapczyńska. 2025. "Optimizing Somatic Embryogenesis and Biomass Proliferation in Narcissus L. ‘Carlton’ Callus Lines Using Solid and Liquid Media" Agronomy 15, no. 11: 2460. https://doi.org/10.3390/agronomy15112460

APA Style

Malik, M., Mazur, J., & Kapczyńska, A. (2025). Optimizing Somatic Embryogenesis and Biomass Proliferation in Narcissus L. ‘Carlton’ Callus Lines Using Solid and Liquid Media. Agronomy, 15(11), 2460. https://doi.org/10.3390/agronomy15112460

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