Hormonal and Environmental Factors Influencing Secondary Somatic Embryogenesis

Abstract

Secondary somatic embryogenesis (SSE) represents a powerful tool for clonal propagation, efficient genetic modification, and plant conservation, enabling the continuous production of secondary somatic embryos (SSEs) from previously formed embryogenic tissues. The efficiency of SSE is determined both by external factors such as exogenous hormonal and environmental conditions and internal cues such as explant type and genotype. Auxins, particularly synthetic 2,4-dichlorophenoxyacetic acid (2,4-D), represent key factors in inducing and maintaining embryogenic competence, while cytokinins often modulate the differentiation and proliferation of SSEs. The interplay of plant growth regulators (PGRs) not only affects the frequency of SSE induction, but also the morphology and proper development of the resulting embryos. Here, we provide a comprehensive review on hormonal treatments, especially the role of auxins and cytokinins and environmental factors such as temperature, light, and culture medium composition, that shape the embryogenic potential in SSE, with species-specific responses frequently being observed. The importance of primary explant selection, as well as the liquid phase and potential scale-up with bioreactors, are also discussed. Other challenges related to genotype recalcitrance, limited efficiency, maturation and conversion rates, and the lack of an advanced molecular approach are further addressed, providing a framework for improved regeneration and reliability across diverse species.

1. Introduction

Somatic embryogenesis (SE) is a long-studied developmental process in plant biology, having both fundamental and applied significance [1,2]. SE is characterized by the development of embryos from somatic plant cells under inductive conditions [3]. These somatic embryos (SEs), instead of developing into complete plants, can be used as source material to initiate the formation of more embryos in a cyclic manner (Figure 1). This iterative process, often called secondary, cyclic, recurrent, or repetitive embryogenesis, provides a powerful tool for large-scale plant production and efficient genetic modification, as well as the study of plant development [4,5].

While the research on primary somatic embryogenesis (PSE), i.e., the development of embryos from somatic cells, is often focused on understanding the mechanisms of somatic cell reprogramming, research on secondary somatic embryogenesis (SSE) is mostly focused on optimizing propagation techniques and improving efficiency. Embryos obtained by SSE can share a number of characteristics with their primary counterparts, but can also have unique characteristics and development conditions. Like in PSE, SSE developmental expression can follow an indirect pathway where the dedifferentiation of explant tissue occurs through the formation of embryogenic callus followed by the development of secondary somatic embryos (SSEs), or a direct pathway where SSEs develop directly on the explant without the callus phase [6]. SSE shares a similar multi-stage pathway of development with PSE. While the exact stages described in the literature vary, most authors agree that the SE process comprises induction, embryo development (or expression), maturation, and germination (or conversion) [7,8]. The phases of embryo development often follow the similar pattern as zygotic embryogenesis, including the globular, heart, torpedo, and cotyledonary phases [9,10]. These stages and phases, however, are not easily distinguished in many species because gene expression, hormone signaling, and morphology change gradually rather than in discrete steps, and cultures frequently contain heterogeneous mixtures of cell states [8,11,12]. Different stages are often present at the same time on the same explant (embryos of varying stage, or embryos together with a callus), suggesting asynchronous development and expression of SE program in most species [4,9,13,14]. The clear distinction of phases is also influenced by whether the development pathway is direct or indirect, or simultaneously present on the same explant [6,15]. Additionally, the stages and phases of SE development are often under-reported and under-characterized in the available literature.

The hormonal requirements for PSE often include plant growth regulators (PGRs), mainly auxins and cytokinins, in specific ratios to promote embryogenic cell division and differentiation. In many cases, the same media composition would be adequate for inducing both PSE and SSE. However, SSE may require different PGR combinations or concentrations [16,17], reflecting the need to stimulate the dedifferentiation of existing embryos, which, compared to explants for PSE, represent a different explant type. The type of PGRs and their combinations and concentrations are frequently species- and genotype-dependent. Embryogenic tissues formed in PSE and SSE may exhibit different morphological characteristics like differences in size, shape, and developmental stages present, like in the case of hypocotyl-derived embryogenic callus and secondary callus of Carthamus tinctorius L., which showed a transition from friable and fast-growing in PSE to compact and slow-growing in SSE [18]. In another example, while primary somatic embryos (PSEs) induced in Commiphora wightii (Arn.) Bhandari were 1.00–9.00 mm long with abnormal morphology and asynchronous development, SSEs were smaller (1.00–3.00 mm), morphologically normal, and highly synchronized [19]. SSE is often more efficient in producing multiple embryos from a single primary embryo, leading to higher yields and multiplication efficiency in tissue culture compared to PSE [4,15,20,21], which holds significant value for rare or elite genotypes. A higher multiplication rate can be further boosted by cyclic SE, where embryos from previous cycles can initiate subsequent cycles in an iterative manner, prolonging the embryogenic competence without reliance on the original explant [6]. PSE can often take longer to yield new embryos, since there is a need for initial somatic cellular dedifferentiation and reprogramming, while SSE builds upon pre-existing embryogenic structures. Even in cases where PSEs develop faster, secondary embryos often show better maturation and germination efficiency [18]. Although regeneration potential of SSE usually declines over time or number of cycles due to aging effects [4,22,23], in some cases it exhibits significantly higher embryogenic potential [24], offering promise for recalcitrant species or lines [5]. Indeed, the SSE pathway is characterized by a high multiplication index, repeatability, independence from explant source effects, and a high level of uniformity [25], having in some species a clear advantage over PSE, like in Prunus persica L., where SSEs formed faster, in greater numbers, and were larger in size than PSEs [26]. Even in cases where PSE had better callus proliferation and embryo differentiation, as is reported for Catharanthus roseus (L.) G. Don, maturation and germination efficiency in SSE were better than PSE [27]. This capacity for continuous embryo production in SSE systems provides a readily available and renewable source of plant material for various downstream applications, including mass propagation of valuable crop species, conservation of endangered species, and efficient genetic transformation. Although very few studies analyze the molecular basis of PSE and SSE differences, there are indications that many genes exhibit differential expression resulting in large differences in amino acid and protein content [18], as well as the differential expression of many proteins between PSE and several cycles of SSE [24].

While there are more than 130 review articles indexed in Scopus covering PSE, with focus varying from different plant species or genuses [28,29] to molecular aspects [8,30,31], SSE has been systematically reviewed in only two studies [6,15], primarily from a biotechnological perspective. Despite a continual accumulation of research articles on SSE over the years, there is a clear lack of recent overviews focusing on underlying hormonal and environmental factors influencing SSE. Therefore, this study will explore the current understanding of the methodologies used to induce SSE in different species, the role of various hormones like auxins and cytokinins, factors related to in vitro culture, and the effects of initial explant selection. Furthermore, it will address some of the challenges that remain¸ such as genotype dependence and recalcitrance, and discuss future research directions in this promising field, like improving the efficiency and reliability of SSE systems and integrating advanced molecular tools to elucidate the complex molecular pathways that govern SSE.

2. Methods

To provide a comprehensive overview of the existing literature, an extensive Scopus document search was performed to evaluate the current research trends in the field of SSE. Scopus represents the largest academic database [32] with more than 100 million records and more than 30,000 scientific journals indexed. It was chosen due to its high-quality standards, trusted academic reputation, extensive coverage in the field of natural sciences, and ease of information retrieval [33]. The literature search was performed with several keywords commonly used to describe SSE: “secondary”, “cyclic”, “recurrent”, “repetitive” in combination with “somatic embryogenesis”. The search included journal articles and book chapters with the search terms queried in fields article title, abstract, and keywords. The list was manually curated to exclude articles that contain the search terms, but are in fact not related to the topic. This identified 546 scientific papers related to SSE from years 1983 to 2024, encompassing 40 years of research. This comprehensive list served as the basis for analyzing publication dynamics over the 40-year period, with the aim to quantify the number of SSE related studies published each year.

Among 546 studies, 133 papers contained the search terms in the article title, and were therefore considered narrowly focused on SSE (https://hdl.handle.net/21.15107/rcub_ibiss_7767 (accessed on 25 November 2025)). Namely, when the keywords were present only in the abstract and not in the title, SSE was often not described in detail in the text of the study. The subset list encompassed 1 review article, 1 book section, and 131 research articles. The identification of the most studied plant species in the SSE field was based on their highest frequency of occurrence in the total dataset of 133 publications, appearing in at least 3 analyzed publications. Moreover, these 133 studies served as the basis for further content analysis of the factors influencing the SSE process, discussed in the following sections. Specifically, the analysis was designed to address the following questions: (1) which PGRs are most frequently applied in SSE and how their type, concentration, and combinations shape embryogenic potential; (2) how nutritional factors, including mineral formulations, different sugars, and other medium supplements, affect SSE; and (3) what role physical factors, such as temperature, light conditions, and the use of solid or liquid media, have in SSE outcomes. In addition, this dataset was used to (4) identify and analyze the types of starting explants most commonly employed for in vitro induction of SSE.

Using the free software VOSviewer (Visualization of Similarities, ver. 1.6.20) [34], a bibliographic analysis was performed on a dataset of 133 articles from the Scopus database and published between 1983 and 2024, with the aim to identify 30 most frequently used keywords in the title and abstract sections. A minimum occurrence threshold of 10 was applied, with the binary counting method, meaning that only the presence or absence of a term in a document was considered, rather than the total number of times it appeared. Prior to running the analysis, the thesaurus function was used to standardize full names and abbreviations to the most common form, while generic terms not directly related to the topic (e.g., “use”, “year”, “month”, “day”) were excluded. Based on these criteria, the 30 most frequent keywords were clustered according to their association weight.

Factors influencing SSE (PGRs, mineral formulations, sugar types and media supplements, temperature, light conditions, the use of solid or liquid media, and explant types) were quantified across the final dataset of 133 publications. For these factors, the number of studies reporting its use was recorded. Only publications that provided explicit information for a given factor were included in its count, and all values were presented as absolute counts.

3. Secondary Somatic Embryogenesis: 40 Years of Research

Within the broader dataset of 546 publications, the most prevalent term to describe SSE was “secondary somatic embryogenesis”, with 90 scientific articles containing this specific SSE term in the title. While there is a continuing interest in SSE research, four years (1995, 1997, 2010, and 2013) were especially proliferative, with 23 studies published in each year (Figure 2). The research papers discussed 198 distinct species of plants in the context of SSE. Species that raised the most interest in SSE research are markedly those with significant economic importance, critical to various industries including agriculture, food production, manufacturing, and construction, like Vitis vinifera L., Theobroma cacao L., Manihot esculenta Crantz, Quercus suber L., Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg., Quercus robur L., Juglans regia L., Coffea canephora Pierre ex A. Froehner, and others. Some of these species face challenges in conventional vegetative propagation or breeding due to low efficiency (especially for recalcitrant genotypes), long generation times, or susceptibility to diseases, making SSE an efficient strategy to overcome these limitations. For example, C. canephora possesses significant economic and social importance worldwide, but faces challenges in improving through breeding [35], while oak species, although of significant agricultural and ecological relevance, are difficult to regenerate through tissue culture [36].

Similarly, in the subset group of 133 articles narrowly focused on SSE, a significant diversity of species, predominantly economically important, was present (94 species), with the most studied shown in

Table 1. The most studied plant species in SSE research by their Latin/common name. The listed species represent those that appear in at least 3 of the 133 analyzed publications.

Latin/Common Name	No. of Studies
Manihot esculenta Crantz/cassava	10
Quercus suber L./cork oak	5
Medicago sativa L./alfalfa	4
Brassica napus L./rape	4
Musa spp./banana	4
Theobroma cacao L./cacao	4
Coffea arabica L./Arabian coffee	3
Arachis hypogaea L./peanut	3
Rosa hybrida E.H.L.Krause/hybrid tea rose	3
Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg./rubber tree	3
Vitis vinifera L./grapevine	3

. There were also many species belonging to other important plant groups, like medicinal (Rosmarinus officinalis L., Centaurium erythraea Rafn, Cinnamomum camphora (L.) J. Presl, Panax ginseng C.A. Mey., Coriandrum sativum L., Myrtus communis L., Gentiana lutea L., Eucalyptus globulus Labill.) or ornamental plants (Dianthus caryophyllus L., Chrysanthemum morifolium Ramat., C. roseus, Narcissus L., Cyclamen persicum Mill., Gladiolus × grandiflorus Hort.). The steady accumulation of studies over four decades across different plant groups clearly demonstrates an ongoing interest for this topic, particularly in the context of clonal propagation, conservation, and genetic transformation.

A co-occurrence analysis was performed using VOSviewer to identify the 30 most frequently used keywords within SSE-related research. The resulting network revealed three clusters in this research domain, represented by groups of closely connected nodes distinguished by color (Figure 3). Across the entire dataset, the three most occurring keywords were “embryogenesis” (109 occurrences), followed by “culture” (57) and “secondary embryogenesis” (45), highlighting the central focus of the field on in vitro embryogenic processes.

Both primary and secondary SE are profoundly influenced by PGRs and environmental factors. The successful induction of SSE depends on a complex interplay of factors, including internal and external hormonal regulation, media composition, environmental factors, and explant selection. Optimizing these parameters for each species is crucial for achieving high efficiency and consistency in embryo production. These efforts mark four decades of SSE research, and will remain the focus of future progress in this research area.

4. Hormonal Regulation: The Role of Auxins and Cytokinins

While not always required, PGRs—especially auxins and cytokinins—play a pivotal role in both the induction and progression of secondary SE in most species. Auxins are commonly associated with callus induction and the early stages of SE, while cytokinins often play a role in subsequent embryo development and plantlet regeneration [37,38]. However, the interaction between these hormones is intricate and context-dependent, with the optimal ratio, concentration, and combination varying depending on the species and environmental factors. Although SSE can often be induced using the same combinations and concentrations of auxins and cytokinins as PSE, its hormonal requirements can sometimes differ, either in terms of PGR combination or concentration. For instance, in Hepatica nobilis Schreb., 1-naphthaleneacetic acid (NAA) alone or in combination with 6-benzyladenine (BA) yielded the best results for PSE induction, while secondary and subsequent cyclic SE were best induced on MS (Murashige and Skoog) media without PGRs [39]. Similarly, in Trifolium repens L., BA was required for PSE, while SSEs were induced on media without PGRs [40]. On the other hand, in A. hypogaea, lower concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) were required for SSE than for PSE [41]. In some cases (in 10 studies), no external growth regulators were required for inducing PSE, and in most of those species, no PGRs were needed for SSE as well (9), as previously observed by Raemakers et al. [15].

4.1. Auxins Used in SSE

The choice of auxin and its concentration for successful SSE induction are highly dependent on the plant species and genotype, reflecting the complex interplay between auxin signaling pathways and other regulatory mechanisms during SSE. The most commonly used auxin for SSE induction is 2,4-D, a synthetic auxin, herbicide, and defoliant [12,15]. Indeed, 2,4-D was the auxin of choice to induce both PSE (in 68 articles) and SSE (48 articles), as presented in

Table 2. The most commonly used auxins in SSE research. The table presents the auxin type, the number of studies where it was used, the effective concentration range, and the family and species reported, with corresponding references. The concentrations used in the cited articles of different species fall between the minimum and maximum values of the reported concentration range for a specific PGR.

PGR	No. of Studies	Concentration Range (µM)
2,4-D	48	min. 0.23 [17]–max. 180.96 [42]
	family	species
	Amaryllidaceae	Allium ampeloprasum [58], A. cepa [53], Narcissus [60]
	Apiaceae	Bunium persicum [61], Coriandrum sativum [43]
	Asteraceae	Chrysanthemum cv. Euro [62], C. morifolium [63]
	Brassicaceae	Brassica oleracea var. gongylodes [45]
	Caryophyllaceae	Dianthus caryophyllus [25]
	Convolvulaceae	Ipomoea batatas [59]
	Cyperaceae	Lepidosperma drummondii [64]
	Euphorbiaceae	Hevea brasiliensis [65], Manihot esculenta [20,66,67,68,69,70,71]
	Fabaceae	Arachis hypogaea [41,46], Calliandra tweedii [72], Medicago sativa [14,73], Trifolium repens [42]
	Fagaceae	Quercus brantii [49]
	Gentianateae	Centaurium erythraea [4]
	Iridaceae	Gladiolus × grandiflorus [38]
	Malvaceae	Theobroma cacao [7,74]
	Moraceae	Morus alba [17]
	Musaceae	Musa spp. [75,76,77]
	Myrtaceae	Psidium guajava [78]
	Passifloraceae	Passiflora cincinnata [79]
	Pinaceae	Larix × leptoeuropaea [80], Pseudotsuga menziesii [5,24]
	Poaceae	Cynodon daclylon × Cynodon transvaalensis [81]
	Rosaceae	Prunus persica [26], Rosa hybrida [47,57], R. rugosa [82], Sorbus pohuashanensis [83]
	Rubiaceae	Coffea arabica [84,85]
	Vitaceae	Vitis vinifera [86]
NAA	21	min. 0.06 [83]–max. 53.70 [70]
	Amaryllidaceae	Narcissus [60]
	Apiaceae	Angelica glauca [87]
	Asparagaceae	Asparagus officinalis [88]
	Asteraceae	Catharanthus roseus [27]
	Euphorbiaceae	H. brasiliensis [65], M. esculenta [70]
	Fabaceae	Albizia lebbeck [89], C. tweedii [72]
	Fagaceae	Q. robur [90,91]
	Iridaceae	Crocus vernus [92]
	Lauraceae	Cinnamomum camphora [93]
	Musaceae	Musa spp. [77]
	Myrtaceae	Eucalyptus globulus [94]
	Oleaceae	Olea europaea [95]
	Pinaceae	Abies numidica [96]
	Ranunculaceae	Hepatica nobilis [39]
	Rosaceae	Malus × domestica [9], P. persica [26], S. pohuashanensis [83]
	Rubiaceae	C. arabica [85]
IBA	7	min. 0.15 [97]–max. 19.68 [98]
	Araliaceae	Aralia elata [98], Polyscias filicifolia [99]
	Fagaceae	Q. brantii [49], Q. robur [91]
	Theaceae	Camellia reticulata [100], C. sinensis [97]
	Vitaceae	V. rupestris [101]
IAA	6	min. 1.14 [75]–max. 20 [86]
	Clusiaceae	Garcinia indica [102]
	Musaceae	Musa spp. [75,77]
	Rubiaceae	C. canephora [16]
	Vitaceae	V. rupestris [101], V. vinifera [86]
picloram	7	min. 0.8 [103]–max. 124.4 [104]
	Amaryllidaceae	Narcissus [60]
	Arecaceae	Bactris gasipaes [105]
	Caryophyllaceae	D. caryopyllus [103]
	Euphorbiaceae	M. esculenta [106,107,108]
	Fabaceae	A. hypogaea [104]
NOA	1	4.96 [109]
	Vitaceae	V. rupestris [109]
dicamba	1	83 [104]
	Fabaceae	A. hypogaea [104]
2,4,5-T	1	3.91 [10]
	Malvaceae	T. cacao [10]

. The optimal concentration of 2,4-D varied highly, depending on the species. The minimal reported concentration of 2,4-D effective in inducing SSE was 0.23 µM (0.05 mgL⁻¹) [17], and it varied up to 180.96 µM (40 mgL⁻¹) [42]. In Dianthus caryophyllus L., for example, the optimal concentration of 2,4-D for the production of SSEs was 0.90 µM (0.2 mgL⁻¹) for both tested cultivars. Higher concentrations of 2,4-D showed a dose-dependent reduction in SSE, with 9.05 µM (2 mgL⁻¹) proving to be completely inhibitory to SSE, while only non-embryogenic callus was formed [25]. In another example, 4.52 µM (1 mgL⁻¹) of 2,4-D was optimal for SSE induction in C. sativum, while higher and lower concentrations were less successful. This optimal concentration resulted in an impressive 90.7% of PSE explants responding and producing an average of 20.5 secondary embryos per explant [43]. In some cases, the same media formulation and 2,4-D concentration was used for induction of both PSE, SSE, and further cycles [4,43,44,45]. Reflecting the enormous potency of 2,4-D to induce SE, it was often the sole PGR used either in PSE [19,20,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56], SSE [25,41,42,43,45,46,57,58,59], or both [41,42,43,45,46].

It is significant to indicate that the prolonged use of 2,4-D is sometimes connected to the browning of embryogenic tissue and reduction in embryogenic potential [48], as well as the inhibition of further somatic embryo development [11]. In high concentrations or under prolonged exposure, this auxin can induce epigenetic and genetic changes, such as DNA methylation and mutations [110,111,112], and disrupt endogenous auxin balance and polar transport, both essential for proper embryo patterning [113], leading to abnormal embryo morphology (reviewed by Bogdanović et al. [6]). These toxic effects are particularly pronounced during long-term exposure, probably reflecting the accumulation of 2,4-D inside the tissue in high concentrations. Therefore, for some species such as C. arabica [84], C. wightii [19], Hovenia dulcis Thunb. [55], R. hybrida [13], or two D. caryophyllus cultivars [103], the removal of 2,4-D was necessary for completing SE development after the initial callus induction and proliferation. It is also reported, in some cases, that a reduction in 2,4-D concentration, for example, from 9.05 µM to 0.23 µM (2 to 0.05 mgL⁻¹) [17], from 13.57-18.10 to 4.52 µM (3–4 mgL⁻¹ to 1 mgL⁻¹) [82], or from 11.31 µM (2.5 mgL⁻¹) to 0.45–2.26 µM (0.1–0.5 mgL⁻¹) [59], was needed to induce SSE from PSE. In other instances, 2,4-D was completely removed and replaced with other PGR combinations [10,16,27,87,88,92,93,103,106,109,114,115,116], or SSE could be induced on media without any PGRs after the initial 2,4-D input in PSE [13,19,21,48,51,52,54,55,56,99,117,118,119,120,121,122]. These contrasting effects emphasize the crucial consideration that, although 2,4-D is often the most effective auxin, it may not be the optimal choice for every species, which highlights the need to consider alternative auxin types and improve their application to achieve both efficient SSE induction and normal embryo development.

Other auxins, such as NAA and indole-3-butyric acid (IBA), have also been used in SSE induction, although less frequently than 2,4-D. NAA was used in the induction of PSE and SSE in 18 and 21 studies (Table 2), respectively, with concentrations for SSE varying from 0.06 µM (0.01 mgL⁻¹) [83] to 53.70 µM (10 mgL⁻¹) [70]. In a study on Olea europaea L. cv. Dahbia, the highest rate of SSE (30%) and a high conversion rate (40%) was observed on a medium supplemented solely with 0.54 µM (0.1 mgL⁻¹) NAA [95]. Compared to PSE, concentrations of NAA required for SSE were either the same [27,65,85,94] or lower [83,88]. NAA was also commonly used for SSE maturation, regeneration, and germination [44,95,108], as well as subsequent rooting [47], when PSE and SSE were induced using other PGRs. In certain cases, NAA was a better choice than 2,4-D either in terms of higher SSE embryogenic response [83] or better embryo morphology [9,26]. The difference in effectiveness between NAA and 2,4-D was demonstrated in Sorbus pohuashanensis (Hance) Hedl., where low concentrations of NAA were more superior and improved the number of SSEs per explant compared to the 2,4-D and PGR-free treatment. The most effective NAA concentration was 0.3 µM (0.06 mgL⁻¹), resulting in 61% of explants forming SSEs and more than 30 SSEs per explant. Concentrations between 0.06 µM (0.01 mgL⁻¹) and 0.3 µM (0.06 mgL⁻¹) NAA consistently yielded a high number of SSEs per explant (greater than 30) [83].

IBA was less commonly used for the induction of SSE (7), with effective concentrations ranging from 0.15 µM (0.03 mgL⁻¹) [97] to 19.68 µM (4 mgL⁻¹) [98] (Table 2), usually from PSE induced by other PGRs. A study on Aralia elata (Miq.) Seem. used IBA successfully for the induction of both PSE and SSE in concentrations ranging from 1.48 µM (0.3 mgL⁻¹) to 19.68 or 24.60 µM (4 or 5 mgL⁻¹) for SSE and PSE, respectively. The optimal concentration for SSE was 14.76 µM (3.0 mgL⁻¹), which resulted in 100% induction and 16.3 SSEs per explant [98].

Other auxins that are relevant for SSE induction (Table 2) include the natural auxin indole-3-acetic acid (IAA) [16,75,77,86,101,102], as well as several synthetic auxins such as picloram [60,103,104,105,106,107,108], naphthoxyacetic acid (NOA) [109], dicamba [104], and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) [10]. Picloram, for example, was effective for inducing SSE in two D. caryopyllus cultivars in concentrations ranging from 0.8 to 16 µM (0.19 to 3.86 mgL⁻¹), with 2 and 4 µM (0.48 and 0.97 mgL⁻¹) proving optimal. For the ‘Sagres’ cultivar, 2 µM (0.48 mgL⁻¹) picloram resulted in an impressive 90.2% of responding explants and produced an average of 28.7 SSEs per explant, while 4 µM (0.97 mgL⁻¹) picloram resulted in 85.3% response and 32.0 SSEs per explant [103]. Picloram was also very effective for the induction of SSE in A. hypogaea, where concentrations of 82.83 µM (20 mgL⁻¹) or 124.25 µM (30 mgL⁻¹) reliably induced highly repetitive globular-stage SSEs maintained for more than 5 months [104]. For initial SSE induction in Vitis rupestris Scheele, IAA was used as a sole PGR, while for the subsequent elongation, multiplication, and individualization of SSEs, IAA was replaced with IBA, resulting in a very productive and repetitive culture [101].

4.2. Cytokinins Used in SSE

Cytokinins play an important role in the differentiation of somatic embryos, primarily through the regulation of cell division, differentiation, and tissue organization [123]. They stimulate cell proliferation and initiate the formation and maintenance of the shoot meristem, which is essential for establishing bilateral symmetry and the development of embryonal structures [124]. Moreover, cytokinins often act synergistically with auxins, balancing the hormonal status that is crucial for proper SE [123].

Among cytokinins, BA or benzylaminopurine (BAP), a synthetic cytokinin, is by far the most prevalent in the induction of PSE (46), as well as SSE (56), as presented in

Table 3. The most commonly used cytokinins in SSE research. The table presents the cytokinin type, the number of studies where it was used, the effective concentration range, and the family and species reported, with corresponding references. The concentrations used in the cited articles of different species fall between the minimum and maximum values of the reported concentration range for a specific PGR.

PGR	No. of Studies	Concentration Range (µM)
BAP	56	min. 0.04 [81]–max. 2.22 [125]
	family	species
	Amaryllidaceae	Narcissus [60]
	Apiaceae	Angelica glauca [87]
	Araliaceae	Polyscias filicifolia [99]
	Asteraceae	Catharanthus roseus [27], Chrysanthemum morifolium [63]
	Caryophyllaceae	Dianthus caryophyllus [25]
	Clusiaceae	Garcinia indica [102]
	Euphorbiaceae	Hevea brasiliensis [65,130], Manihot esculenta [20,66,67,68,69,70,71]
	Fabaceae	Calliandra tweedii [72], Trifolium repens [40]
	Fagaceae	Quercus alba [131], Q. rubra [131], Q. brantii [49], Q. robur [90,91]
	Iridaceae	Crocus vernus [92], Gladiolus × grandiflorus [38]
	Malvaceae	Theobroma cacao [7,74]
	Moraceae	Morus alba [17]
	Musaceae	Musa spp. [75,76]
	Myrtaceae	Eucalyptus globulus [94], Psidium guajava [78]
	Orchidacae	Oncidium [132]
	Passifloraceae	Passiflora cincinnata [79]
	Pinaceae	Abies numidica [96,133], Larix × leptoeuropaea [80], Pseudotsuga menziesii [5,24]
	Poaceae	Avena sativa [114], Cynodon daclylon x Cynodon transvaalensis [81], Hordeum vulgare [114], Secale cereale [114], Triticum aestivum [114], T. durum [114], T. monococcum [114], T. urartu [114]
	Ranunculaceae	Hepatica nobilis [39]
	Rhamnaceae	Zizyphus jujuba [115]
	Rosaceae	Malus × domestica [9], Prunus persica [26], Rosa hybrida [47], R. rugosa [82]
	Rubiaceae	Coffea arabica [84,85], C. canephora [16]
	Rutaceae	Citrus [125]
	Sapindaceae	Aesculus carnea [116]
	Theaceae	Camellia reticulata [100], C. sinensis [97]
	Vitaceae	Vitis vinifera [86,126], V. rupestris [109]
KIN	15	min. 0.005 [61]–max. 14 [65]
	Amaryllidaceae	Allium cepa [53]
	Apiaceae	Bunium persicum [61]
	Asparagaceae	Asparagus officinalis [88]
	Asteraceae	Chrysanthemum cv. Euro [62]
	Clusiaceae	G. indica [102]
	Euphorbiaceae	H. brasiliensis [65,130], M. esculenta [20]
	Fabaceae	Medicago sativa [14,73]
	Orchidacae	Oncidium [132]
	Rosaceae	Malus × domestica [9], P. persica [26]
	Rubiaceae	C. arabica [85]
	Sapindaceae	Aesculus carnea [116]
ZEA	3	min. 0.91 [75]–max. 2.28 [75]
	Iridaceae	Gladiolus × grandiflorus [38]
	Musaceae	Musa spp. [75,76]
2iP	5	min. 0.1 [72]–max. 20 [72]
	Iridaceae	C. vernus [92]
	Fabaceae	C. tweedii [72]
	Fagaceae	Q. suber [134]
	Oleaceae	Olea europaea [95]
	Orchidacae	Oncidium [135]
TDZ	5	min. 0.45 [132]–max. 10 [9]
	Cyperaceae	Lepidosperma drummondii [64]
	Orchidacae	Oncidium [132]
	Pinaceae	A. numidica [96]
	Rhamnaceae	Z. jujuba [115]
	Rosaceae	Malus × domestica [9]
CPPU	1	min. 0.81 [4]–max. 2.02 [4]
	Gentianateae	Centaurium erythraea [4]

. The minimal reported concentration of BAP effective for inducing SSE was 0.04 µM (0.01 mgL⁻¹) [81] and up to 2.22 mM (500 mgL⁻¹) [125]. In the cases where it was necessary for PSE induction, BAP, sometimes in combination with other PGRs, was used in the same concentration of SSE as well [7,63,65,68,69,71,85,125,126]. In other cases, either a lower [16,25,109] or higher [5,80,114,115] BAP concentration was optimal in SSE compared to PSE. In some studies, BAP was necessary for PSE, but removed for SSE and either replaced with different PGRs [59,83,95,103] or SSE proceeded on media free of PGRs [99,117,120,122,127,128,129]. For example, if PSE was induced with BAP combined with 2,4-D [99,117,122] or NAA [39,127,128], SSE could proceed without PGRs. When PSE was induced using BAP alone, one study reported SSE occurring without the addition of any PGRs [120]. In this example, a spontaneous direct SSE was observed on a medium containing 0.22 µM (0.05 mgL⁻¹) BAP, used to induce PSE in Brassica campestris L. This treatment led to a proliferation of 30–35 SSEs from each PSE, or more than 150 SSEs from each parent zygotic embryo [120]. This demonstrated the profound impact and effectiveness of BAP on SSE, where a carry-over effect from PSE was enough to induce SSE in this species [15,120].

For SSE, BAP was most often combined with 2,4-D [5,7,17,24,26,47,60,62,71,74,79,80,81,84,85,86], but also with IBA [97,100], NAA [27,87,90,92], or IAA [16,75,102]. For example, a medium supplemented with 9 µM (1.99 mgL⁻¹) 2,4-D and 4.5 µM (1.01 mgL⁻¹) BAP was highly effective for inducing SSE in Larix × leptoeuropaea. This combination achieved a remarkable 98% efficiency (PSEs developing secondary embryonal masses) after 9 weeks of culture for 3-week matured PSEs [80]. In C. roseus, a combination of 6.66 µM (1.5 mgL⁻¹) BAP and 5.37 µM (1.0 mgL⁻¹) NAA was very efficient, achieving a 72.1% rate of secondary callusing with SSEs and producing 6.4 SSEs per PSE in the cotyledonary stage. BAP was rarely used alone to induce SSE [116,125,126,133]. In Aesculus carnea Hayne., for example, SSE was most effective when Class II PSEs (5 mm, at the cotyledonary stage) were subcultured on a medium containing 5 µM (1.13 mgL⁻¹) BAP. This concentration yielded the highest embryo-forming capacity (10.50), which combines the mean number of SSEs per PSE and the percentage of PSEs forming SSEs compared to all other treatments [116]. Rather, for the induction, BAP was quite often used for later SSE stages, such as embryo maturation, plant regeneration, and germination [26,44,66,68,75,84,86,93,108], demonstrating its well-documented role in embryo differentiation and conversion to plants.

Kinetin (KIN, 6-furfurylaminopurine) was successfully used to induce SSE in 15 scientific articles with concentrations from 0.005 μM (0.001 mgL⁻¹) [61] to 14 μM (3.01 mgL⁻¹) [65] (Table 3). Kinetin was also combined most often with 2,4-D [14,53,61,62,65,73,85], then NAA [65,88] or IAA [53]. In studies on Chrysanthemum × morifolium (Ramat.) Hemsl cv. Euro, a combination of 9.29 µM (2.0 mgL⁻¹) KIN with 9.05 µM (2.0 mgL⁻¹) 2,4-D was found to be optimal for PSE, yielding a 100% embryogenic response and a maximum of 42.1 ± 5.97 PSEs per responding explant. This specific combination was then successfully utilized for SSE as well [62]. If PSE was induced with KIN and 2,4-D, IAA, or NAA, in some cases, SSE could proceed without PGRs [21,118,136].

Other natural cytokinins relevant for SSE induction (Table 3) included zeatin (ZEA, 6-[4-hydroxy-3-methylbut-2-enylamino]purine) [38,75,76] and 6-(dimethylallylamino) purine (2iP) [72,92,95,134,135], while synthetic cytokinins included thithazuron (TDZ, N-(1,2,3-thiadiazol-5-yl)-N’-phenylurea) [9,64,96,115,132] and N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) [4] (Table 3). For example, 2iP was the best treatment to induce SSE in Calliandra tweedii Benth., producing the highest frequency and number of SSEs, in comparison to 2,4-D and NAA, which led to the browning of the callus and low SSE response. The concentration of 1 µM (0.20 mgL⁻¹) 2iP proved to be the most effective for recurrent production of SSEs, inducing SSE in 70% of cultures and producing an average of 19.44 ± 0.52 cotyledonary SSEs per explant within 6 weeks [72]. TDZ could effectively substitute for auxin–cytokinin combinations in promoting SSE in Malus × domestica Baumg. cv. Gloster 69. Used alone at 10 µM (2.20 mgL⁻¹), it was highly effective in inducing SSE, achieving a rate of 53.3% and mean number of SSEs per PSE of 4.6 ± 1.6, similar to the best auxin–cytokinin combination (BAP, KIN, NAA, 55.5% and 3.8 ± 1.5) [9]. Interestingly, TDZ proved essential for the conversion of SSEs into plants in Lepidosperma drummondii Benth. In this species, the conversion cannot proceed on media without PGRs, and TDZ at 1.0 µM (0.22 mgL⁻¹) resulted in the best conversion rate of 39% [64]. The role of CPPU in SSE was primarily studied in C. erythraea, where it proved essential for embryogenic callus formation, even at the lowest concentration used, 0.4 µM (0.1 mgL⁻¹). Moreover, the number of explants developing secondary embryos drastically increased in a dose-response manner with the addition of CPPU, with medium containing 1.01 µM (0.25 mgL⁻¹) CPPU in combination with 0.45 µM (0.1 mgL⁻¹) 2,4-D proving optimal for SSE induction. This PGR combination resulted in well-developed bi-cotyledonary secondary embryos that could proceed through three successive cycles of SSE [4].

4.3. Other Plant Hormones in SSE

Although less frequently, other plant hormones such as abscisic acid, gibberellins, and ethylene can also influence the SSE process. Abscisic acid (ABA) is widely recognized for its role in stimulating the maturation and SE conversion into plantlets in various species, while its role in inducing SSE and the proliferation of embryos is sometimes contradictory and highly species-specific. ABA was mostly used in the later stages of SE, involving maturation of SSEs (20 studies), but it was also involved in embryo germination and conversion to plantlets (7 studies). In C. tweedii, the addition of ABA was essential for SSE maturation and preventing precocious germination, where 5 μM (1.32 mgL⁻¹) proved optimal for producing bipolar mature SSEs that were later able to germinate into plantlets [72]. Similarly, in B. napus, ABA has been reported to temporarily inhibit SSEs germination while also reducing the rate of embryo growth and survival [137]. In Q. suber and other Quercus species, ABA reduced SSE, probably by preventing the initiation and growth of new embryonic centers, decreasing SSE length, and stimulating the proper formation and maturation of SSEs [134]. While stimulating desiccation tolerance in SSEs, ABA also decreased the embryogenic capacity of these SSEs to form new embryos in M. sativa [73]. ABA promoted the development and maturation of SSEs while reducing the rate of recurrent SE in C. camphora [93]. There were also cases where ABA was unsuccessful to promote embryo maturation, but reduced SSE capacity, and completely inhibited it at the concentration of 10 μM (2.64 mgL⁻¹), as shown for Morus alba L. [17]. Interestingly, while reducing the total number of SSEs produced, ABA increased the frequency of torpedo-stage embryos in C. persicum, enhancing normal embryo development [122].

On the other hand, in some species, like Asparagus officinalis L., ABA increased SSE as well as the conversion of SEs, producing plantlets with long, thin shoots and short, thin roots, which nevertheless grew well and developed properly [88]. In some cases, ABA added to the callus induction medium, especially in combination with BAP, substantially enhanced PSE and SSE in hybrid Cynodon dactylon × Cynodon transvaalensis cv. Tifgreen, leading to more somatic embryo clusters, as well as promoting embryo maturation [81]. Similarly, in R. hybrida, ABA promoted the growth rate of SSE callus and germination of SSEs by 36-fold and 5-fold compared to other treatments, respectively [13]. Both SSE and tertiary SE were enhanced by ABA in Daucus carota L., with the number of embryos increasing with higher ABA concentrations [51]. In another example, well-developed cotyledonary PSEs matured with ABA were an excellent source for SSE induction, with 3-week maturation being the most optimal while 6-week matured SEs formed significantly less SSEs [80].

The role of gibberellins (GA, most often GA₃) in SSE is complex and sometimes species-specific. In most cases, GA₃ appears to have inhibitory or neutral effects on the induction and proliferation of SSEs. For instance, in Oncidium, GA₃ was reported to inhibit SSEs, resulting in 0% SSE rate and no secondary embryos per explant [132]. Similarly, in D. carota [51] and O. europea [95], GA₃ reduced SSE initiation or had no significant effect, as reported for Q. suber [134]. The application of GA₃ inhibitors, like uniconazole in D. carota [138], or ancymidol in A. officinalis [88] and Oncidium [132], promoted SSE from the primary embryo, indicating that high endogenous GA levels may suppress embryogenic competence, possibly by maintaining cells in a somatic state [51]. In line with this observation, lower levels of GA favored SE initiation in Spinacia oleracea L. [139]. Additionally, in Ziziphus jujuba Mill., GA₃ was found to enhance repetitive SE more effectively than cytokinins. Nevertheless, prolonged culture on a GA₃-containing medium led to abnormal embryo development [115]. More consistently, GA₃ primarily plays a positive role during the later stages of SSE, particularly in the maturation, germination, and conversion of embryos into plantlets, as seen in Q. suber [134], M. esculenta [108], Crocus vernus (L.) Hill [92], and Albizia lebbeck (L.) Benth. [89]. In these cases, low-to-moderate GA₃ concentrations improved shoot/root elongation and bipolar structure formation. However, early introduction of GA₃ could lead to abnormal shoot development in M. esculenta [108].

Ethylene, a plant hormone with a wide variety of effects on the growth and development of plants, has been implicated previously in in vitro morphogenetic responses [16]. In some species, ethylene negatively impacted SE in a genotype-specific manner [16]. Therefore, limiting exposure to ethylene, for example, with the use of ethylene inhibitors, could be a viable approach to enhance SE. Known ethylene inhibitors, like silver nitrate, cobaltous ions, and salicylic acid, were found to enhance SSE in C. canephora, with silver nitrate inducing SSE in up to 90% of explants with around 100–150 embryos obtained from each explant [16]. On the other hand, silver nitrate proved to be inhibitory to SSE in two Oncidium cultivars, while 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor to ethylene, was found to induce higher SSE response, leading to a higher percentage and number of SSEs [132]. In another example, ACC addition, as well as ethylene and ethephon, ethylene releaser, strongly reduced the induction of SSE, reducing the percentages of PSEs developing SSE masses in Larix × leptoeuropaea. In the same species, silver nitrate and 2,5-norbornadiene (NBD), both inhibitors of ethylene action, enhanced SSE, especially from PSEs matured for longer periods that had lost their embryogenic potential [80]. These examples highlight the complex and often contradictory roles of ethylene in regulating SSE in various species.

Jasmonates, including methyl jasmonate and jasmonic acid (JA), known to be involved in stress responses and wounding, interact with the induction of SSE, probably through wound-inducible signaling pathways, similarly to SE. Elevated JA levels repressed MYC2 and induced JAZ1, two key regulators of plant growth and stress, favoring IAA accumulation in explants and subsequent SE production in Arabidopsis [140]. Methyl jasmonate was found to both enhance SSE and inhibit the further development of torpedo-stage SEs in D. carota, disturbing the normal morphological development of SSEs [54].

4.4. Complex Interactions Between Exogenous and Endogenous PGRs in SSE

Among other effects, exogenously added PGRs can modulate embryogenic response by altering endogenous hormone levels either directly through the synthesis of specific enzymes or indirectly through signal molecules [141,142]. An externally applied PGR can influence the internal hormone levels, either increasing or decreasing their concentrations, not only affecting hormones of the same group, but other hormone groups as well [143]. This is associated with the expression of genes involved in hormone biosynthesis and signaling pathways, triggering a cascade of physiological changes that enhance embryogenic competence [142].

Although many PGR combinations were proven successful for the induction of SSE in different species, the effects of exogenous plant hormones were highly species-specific and complex. The choice of particular PGRs relies heavily on the previous research experience with a certain species, but mirroring the same induction conditions without optimization is often unlikely. Therefore, testing different inductive PGR combinations is highly recommended and very valuable for determining the optimal conditions for SSE induction.

In one example, TDZ stimulated SSE induction in two Oncidium cultivars, while BAP and KIN were less effective and GA₃ and 2,4-D proved inhibitory [132]. On the contrary, GA₃ was the most effective treatment to induce SSE in Z. jujuba compared to BAP, ZEA, 2iP and TDZ [115]. Little et al. [104] conducted a comprehensive study on optimizing auxin type and concentration for inducing PSE and SSE in A. hypogaea. Eleven different auxins (including 2,4-D, NAA, picloram, dicamba, and centrophenoxine) and cytokinin CPPU were tested at various concentrations. The authors concluded that picloram and centrophenoxine were drastically more effective for inducing both PSE and SSE, yielding the greatest number of embryos per explant and the highest percentage of responding explants. In contrast, NAA and CPPU were not able to produce SEs, while 2,4-D produced mostly misshapen SEs [104]. In another example, NAA proved to be more efficient for SSE induction in Angelica glauca Edgew. than 2,4-D, both in combination with BAP, regardless of explant type [87]. A similar pattern was observed in C. vernus, where NAA in combination with BAP also achieved the best SSE induction compared to NAA with 2iP combination [92]. Wongtiem et al. [20] investigated the effects of various cytokinins (BAP, KIN, ZEA, 2iP, adenine) in addition to 2,4-D on SSE in M. esculenta, concluding that all tested cytokinins, except adenine, reduced the intensity of SSE. While both were successful in inducing PSE in L. drummondii, 2,4-D was markedly more efficient than TDZ for inducing SSE, producing more than 2000 SSEs in 4 weeks [64]. Similarly, 2,4-D in the concentration of 0.90 µM (0.2 mgL⁻¹) was the most effective treatment for SSE induction, while TDZ was completely unsuccessful, regardless of the concentration applied in Ipomoea batatas (L.) Lam. [59]. Perhaps especially interesting are three-hormone combinations used in the induction of SSE. For example, the combination of NAA with BAP and KIN was more effective than IBA or 2,4-D to induce SSE in Malus × domestica. These hormones in concentrations 5.3 µM (0.99 mgL⁻¹) NAA, 0.9 µM (0.20 mgL⁻¹) BAP, and 0.9 µM (0.19 mgL⁻¹) KIN, achieved the highest SSE rate of 55.5% and SSEs production of 3.81 ± 1.54 per PSE [9]. Similarly, IAA with BAP and KIN in concentrations 5.7 µM (1 mgL⁻¹), 16 µM (3.60 mgL⁻¹), 4.6 µM (0.99 mgL⁻¹), respectively, were optimal for inducing SSE in Garcinia indica (Thouars) Choisy, resulting in high SSE percentage and mean number of embryos, as well as the highest maturation percentage and number of matured embryos [102].

Even though it would be very practical to have unified protocols developed for individual species, there are no absolute trend in species’ responses to certain hormone combinations. This could be due to the lack of data—out of 94 different species, 70 appear in only one article, while 24 species appear in two or more articles. Even for the most studied species—M. esculenta (10), there is no universally effective hormone combination for inducing SSE, although three hormones are especially important for SSE in cassava: 2,4-D (7), BAP—either for induction or maturation of SSE (7)—and picloram (3) [20,66,67,68,69,70,71,106,107,108] (Table 2 and Table 3). In Rosa species, for example, the response varied: in two studies, both 2,4-D and BAP were necessary [47,82], in one study only 2,4-D was used [57], and in another, SSE occurred most effectively without auxins and cytokinins [13]. Similarly, for T. cacao, the combination of 2,4-D and BAP was used in two out of four studies [7,74]. It is valuable to note that other factors like nutritional factors, temperature, etc., could be more important for species-specific responses to PGRs. The complexity of these requirements makes further research of the underlying mechanisms of species-specificity particularly challenging.

While there is a prevalent need to include PGRs to induce SSE from PSE, there are also reports in the literature where no PGRs were required to induce PSE and subsequent SSE [52,134,144,145,146,147,148,149]. In those cases, traditionally used plant hormones were either not required or even showed an inhibitory effect. For example, in five out of nine articles on Quercus, the authors reported SSE without PGRs [49,127,134,144,145]. Namely, using PGR-free media resulted in 100% induction of SSE in Quercus brantii L., while adding IBA and BAP reduced SSE, which was the desired outcome for this species in order to obtain mature plants [49]. Similarly, in Q. suber, all tested cytokinins (BAP, KIN, ZEA, 2iP) had a negative effect on the induction of SSE compared to PGR-free media, regardless of concentration [134]. For Brassica species, five out of eight studies reported SSE without PGRs [45,52,120,148,150]. Ćosić et al. [45] demonstrated that SSE in Brassica oleracea L. var. gongylodes was more efficient on a PGR-free medium than on media supplemented with PGRs. In E. globulus, PGR-free medium was more efficient for forming new globular and cotyledonary SSEs than media supplemented solely with NAA or in combination with BAP and/or KIN [94]. Similarly, PGR-free medium was superior for SSE induction in H. nobilis, in comparison with different combinations of NAA and BAP [39]. For D. carota, PGR-free medium resulted in the formation of both embryogenic callus and embryos, while auxin treatment produced only non-embryogenic callus [51,54].

The successful induction of PSE and SSE without external PGRs is likely the result of a favorable endogenous hormone status, reflected in both the content and balance of specific hormones [51,151]. Embryogenic competence often correlates with endogenous hormone levels in the initial explant tissue, embryogenic callus or the resulting embryos. Of particular importance is the balance between auxin and cytokinin levels, which affects the cell dedifferentiation and redifferentiation and determines cell fate [152]. For example, by comparing two types of initial stipe explants in Cyathea delgadii Sternb., differing in their ability to undergo PSE, it was found that explants capable of SE showed increased IAA and total cytokinin levels, accompanied by reduced ABA content [151]. Similarly, IAA levels were significantly higher and total cytokinins were slightly lower, in embryogenic compared to non-embryogenic callus of Ormosia henryi Prain [153]. Illustrating the importance of auxin-cytokinin ratio, a reduced ZEA–IAA ratio, as well as lower levels of ZEA and preserved level of IAA, were associated with a higher embryogenic capacity in one cotyledon line of P. persica L., cultured on PGR-free medium [154]. In one of the few studies measuring endogenous hormone content during SSE, significantly higher ABA levels were detected in PSEs derived from seed coats of D. carota compared with hypocotyl-derived normal embryos and seed-coat-derived SSEs. In addition, endogenous IAA levels were higher in PSEs than in hypocotyl-derived embryos, suggesting that the IAA concentration was sufficient to induce SSEs formation on PSEs [51].

The molecular mechanisms of auxin involvement in PSE are somewhat revealed (reviewed in [30,123,155]), highlighting its essential function in cell reprogramming, embryo identity maintenance, and cross-talk with other signaling pathways. However, almost nothing is known of underlying genetic and epigenetic mechanisms in SSE, as most of the studies about secondary and cyclic embryogenesis focus on methods to improve regeneration efficiency and proliferation. One could infer that similar mechanisms regarding hormone biosynthesis, transport, and signaling are involved in both PSE and SSE; however, there are simply no available data to support this conclusion. Much more research is needed, and not just in model species, to deconvolute this complex and intricate process of transitioning from PSE to SSE and beyond. Studies employing epigenetic, transcriptomic, and proteomic approaches will be pivotal for this deeper understanding and essential for developing more targeted approaches to enhance SSE efficiency.

5. Culture Conditions: Optimizing Nutritional Factors

The culture media composition, including sugars and other nutrients, plays a significant role in both PSE and SSE. The use of specific sugars like sucrose, glucose, or maltose and the addition of activated charcoal or amino acids can influence both the frequency and quality of secondary embryos, as well as their maturation and regeneration ability.

5.1. Mineral Formulations Commonly Used in SSE

A well-balanced medium is important to prevent stunted growth and physiological disorders. Consequently, with the development of in vitro techniques, specific substrates with different mineral, vitamin, and hormonal composition were developed corresponding to the species of interest and observed in vitro process. The mineral composition of the media used was quite variable among different species undergoing SSE, too. Although MS medium (Murashige and Skoog medium [156]) was the most commonly used mineral formulation in at least one of the SSE stages (106), there were often variations in composition such as using half-strength MS formulation [16,64,84,86,121,122,132,135,157,158], MS salts with B5 vitamins [53,79,104,108,136], or other modifications [20,26,41,45,56,65,100,105]. Other notable mineral compositions used were WPM (woody plant medium [159]) [102,117,126,160], SH (Schenk and Hildebrandt medium [161]) [14,92,96,98,133,147], B5 (Gamborg B5 medium [162]) [120,148,163], or DKW (Driver and Kuniyuki Walnut medium [164]) [7,129,165]. Less prominent media formulations mentioned were MT (Murashige and Tucker medium) [166], modified Litvay medium Glitz [5,24], MH (basal medium for Hevea culture) [130], and SCG (secondary callus growth medium) [74]. While in the majority of cases the same media mineral formulation could be used in PSE and SSE (90), there were also examples of using different media formulations, like in the case of Petiveria alliacea L., where MS medium was applied in PSE and MS/2 in SSE [48], A. lebbeck where WPM media was used for PSE and MS for SSE [89], or in Brassica oleracea var. capitata and Brassica oleracea var. botrytis, where B5 was applied for PSE and MS for SSE [52]. The use of different media for distinct stages of SSE has also been reported. For example, in Abies numidica de Lannoy ex Carrière, SH medium was used for callus induction, while MS/2 was used for somatic embryo differentiation and maturation [96,133]. MS/2 was also often applied for the germination of secondary embryos [25,43,72,89,103]. In one study on A. elata, WPM medium was used for embryo germination following SH medium for both PSE and SSE induction [98]. In a study on Chrysanthemum cv. Euro, full-strength MS medium was observed to be the more effective than half-strength MS medium for the proliferation of SSEs [62]. Similarly, Kim et al. [21] observed that MS basal medium had an 88% efficiency for SSE in P. ginseng, while EM media (a modified MS with reduced mineral content, especially nitrogen) had 57% efficiency. The authors also observed that PSEs cultured on EM media remained at the cotyledonary stage as long as they were not detached from the explants, whereas PSEs on MS media developed leafy shoots while still attached, suggesting early embryo conversion [21]. On the other hand, in an effort to control SSE and favor conversion into plantlets, SH/2 medium proved optimal in Q. brantii, offering the lowest SSE (6.25%) and highest conversion percentage (93.75%) compared to MS medium [49].

5.2. Sugar Types in SSE

Sugar type and concentration in the plant growth media is another factor to consider in relation to SSE. An adequate supply of carbon sources is essential for providing the energy needed for the metabolic processes involved in embryo development. Sucrose was by far the most commonly used carbohydrate energy source for SSE, being present in 102 studies and applied in a wide range of concentrations varying from 10 gL⁻¹ [56] to 80.11 gL⁻¹ [130]. The most commonly used concentrations were 30 gL⁻¹ (44), 20 gL⁻¹ (30), and 60 gL⁻¹ (12). In several studies, concentrations over 60 gL⁻¹ proved to be inhibitory or dramatically less efficient in inducing SSE than lower concentrations [62,98], and in certain cases, even lower concentrations of 15 gL⁻¹ [147] or 10 gL⁻¹ [63] were optimal for SSE induction. In contrast, 60 gL⁻¹ was more effective in C. persicum compared to lower-tested concentrations, probably increasing SSE due to osmotic stress [122]. While in most situations the same concentration of sucrose was appropriate for both PSE and SSE, there were examples where a reduction in concentration was needed for successful SSE induction, for example, from 40 to 30 gL⁻¹ in S. pohuashanensis [83] or from 20.54 to 10.27 gL⁻¹ in Larix × leptoeuropaea [80], or from 30 to 20 gL⁻¹ in B. campestris [120]. Alternatively, an increase from 30 to 60 gL⁻¹ in C. persicum [122] or from 30 to 60 gL⁻¹ in Chrysanthemum [62] was optimal for SSE induction. In H. brasiliensis, a higher sucrose concentration (80.11 gL⁻¹) was needed for SSE callus induction, while a lower concentration (20 gL⁻¹) was optimal for secondary embryo development [130].

Other sugars important for SSE are presented in

Table 4. The most commonly used sugars in SSE research. The table presents the sugar type, the number of studies where it was used, the effective concentration range, and the family and species reported, with corresponding references. The concentrations used in the cited articles of different species fall between the minimum and maximum values of the reported concentration range for a specific sugar type.

Sugars	No. of Studies	Concentration Range (gL−1)
sucrose	102	min. 10 [56]–max. 80.11 [130]
	family	species
	Amaryllidaceae	Allium ampeloprasum [58], A. cepa [53], Narcissus [60]
	Apiaceae	Angelica glauca [87], Coriandrum sativum [43], Daucus carota [54]
	Araliaceae	Aralia elata [98], Eleutherococcus senticosus [56], Panax ginseng [21], Polyscias filicifolia [99]
	Arecaceae	Bactris gasipaes [105]
	Asparagaceae	Asparagus officinalis [88]
	Asteraceae	Chrysanthemum cv. Euro [62] C. morifolium [63,167], Carthamus tinctorius [168], Helianthus maximiliani [128]
	Brassicaceae	Bssica campestris [120], B. napus [137,148,150,163], B. oleracea var. botrytis [52], B oleracea var. capitata [52], B. oleracea var. gongylodes [45]
	Burseraceae	Commiphora wightii [19]
	Caryophyllaceae	Dianthus caryophyllus [25,103]
	Clusiaceae	Garcinia indica [102]
	Convolvulaceae	Ipomoea batatas [59]
	Cyperaceae	Lepidosperma drummondii [64]
	Euphorbiaceae	Hevea brasiliensis [65,130], Manihot esculenta [20,66,67,68,69,70,71,106,107]
	Fabaceae	Albizia lebbeck [89], Arachis hypogaea [41,46,104], Calliandra tweedii [72], Medicago sativa [14,73,136,169], M. truncatula [119], Glycine max [50], Trifolium repens [40,42]
	Fagaceae	Quercus alba [131], Q. robur [90] Q. rubra [131], Q. suber [127,145],
	Gentianateae	Centaurium erythraea [4]
	Iridaceae	Crocus vernus [92], Gladiolus × grandiflorus [38]
	Lamiaceae	Rosmarinus officinalis [117]
	Lardizabalaceae	Akebia trifoliate [149]
	Lauraceae	Cinnamomum camphora [93], Ocotea catharinensis [160]
	Malvaceae	Theobroma cacao [7]
	Moraceae	Morus alba [17]
	Musaceae	Musa spp. [75,77,170]
	Myrtaceae	Psidium guajava [44], Myrtus communis [121], Eucalyptus globulus [94]
	Oleaceae	Olea europaea [95]
	Orchidacae	Oncidium sp. [135]
	Passifloraceae	Passiflora cincinnata [79]
	Pinaceae	Abies numidica [96,133], Larix x leptoeuropaea [80], Pseudotsuga menziesii [5,24]
	Piperaceae	Piper nigrum [147]
	Poaceae	Avena sativa [114], Hordeum vulgare [114], Secale cereal [114], Triticum aestivum [114], T. durum [114], T. monococcum [114], T. urartu [114], Cynodon daclylon x Cynodon transvaalensis [81]
	Primulaceae	Cyclamen persicum [122]
	Ranunculaceae	Hepatica nobilis [39]
	Rhamnaceae	Hovenia dulcis [55], Zizyphus jujube [115]
	Rosaceae	Malus × domestica [9], Prunus persica [26], Rosa hybrida [13,47], Sorbus pohuashanensis [83]
	Rubiaceae	Coffea arabica [158], Coffea canephora [16]
	Rutaceae	Citrus [125]
	Sapindaceae	Aesculus carnea [116], A. hippocastanum [118]
	Theaceae	Camellia reticulata [100], Camellia sinensis [157]
	Vitaceae	Vitis rupestris [109], Vitis vinifera [86,109]
glucose	12	min. 15.79 [136]–max. 72 [148]
	Asteraceae	Catharanthus roseus [27]
	Brassicaceae	Brassica napus [148]
	Fabaceae	Medicago sativa [136]
	Fagaceae	Quercus robur [91], Quercus suber [134,144]
	Malvaceae	Theobroma cacao [7,10]
	Moraceae	Morus alba [17]
	Rosaceae	Malus × domestica [9], Rosa hybrida [47], Rosa rugosa [82]
fructose	4	min. 15.79 [136]–max. 72 [148]
	Brassicaceae	Brassica napus [148]
	Fabaceae	Medicago sativa [136]
	Moraceae	Morus alba [17]
	Rosaceae	Malus × domestica [9]
maltose	14	min. 15 [114]–max. 120 [9]
	Asteraceae	Catharanthus roseus [27]
	Euphorbiaceae	Manihot esculenta [108]
	Fabaceae	Arachis hypogaea [104], Medicago sativa [136], M. truncatula [119], Trifolium repens [42]
	Moraceae	Morus alba [17]
	Passifloraceae	Passiflora cincinnata [79]
	Pinaceae	Abies numidica [96,133], Pseudotsuga menziesii [5,24]
	Poaceae	Avena sativa [114], Hordeum vulgare [114], Secale cereal [114], Triticum aestivum [114], T. durum [114], T. monococcum [114], T. urartu [114]
	Rosaceae	Malus × domestica [9]
lactose	1	30 [136]
	Fabaceae	Medicago sativa [136]
xylose	1	0.35 [114]
	Poaceae	Avena sativa [114], Hordeum vulgare [114], Secale cereal [114], Triticum aestivum [114], T. durum [114], T. monococcum [114], T. urartu [114]
ribose	1	0.35 [114]
	Poaceae	Avena sativa [114], Hordeum vulgare [114], Secale cereal [114], Triticum aestivum [114], T. durum [114], T. monococcum [114], T. urartu [114]

. The most significant were glucose, with concentrations varying from 15.79 gL⁻¹ [136] to 72 gL⁻¹ [148], fructose at 15.79 gL⁻¹ [136] to 72 gL⁻¹ [148], and maltose at 15 gL⁻¹ [114] to 120 gL⁻¹ [9]. Maltose was also commonly used for embryo maturation and germination in concentrations from 30 to 60 gL⁻¹ [5,27,42,79,96,104,133]. As an alternative, sucrose was sometimes used for embryo maturation [24,80,92,118] and germination [86,95] in concentrations from 20 to 60 gL⁻¹. When different carbohydrates were tested for their ability to induce SSE, glucose was more effective than sucrose in R. hybrida [47]. In D. caryophyllus, sucrose combined with mannitol produced the best results, although mannitol alone was insufficient to induce SSE [25].

5.3. Other Media Supplements in SSE

The addition of other supplements to the culture medium can also significantly influence SSE. Activated charcoal (AC) has been shown to enhance embryo induction and development in several species, possibly by adsorbing compounds inhibitory to embryogenesis and creating a more favorable environment for embryo development and maturation. While the optimal concentration depended on the species, successful AC application also depended on the developmental phase and explant source. In Ocotea catharinensis Mez, 1.5 gL⁻¹ of AC was successfully used for the induction and maturation of SSEs [160], while 4 gL⁻¹ improved SSE embryo proliferation and quality in Quercus species [131]. In R. hybrida, 3 gL⁻¹ active charcoal was successfully used for embryo maturation [57], while 10 gL⁻¹ was used for Larix × leptoeuropaea embryo maturation medium [80]. Activated charcoal was often important for SSE germination and development into plantlets [21,26,63,72,75,83,96,105,133], while in V. vinifera, 30 gL⁻¹ AC was beneficial during the SSE initiation, maintenance, and germination phase [109]. However, in some cases, AC had inhibitory effects on SSE, such as in M. alba, where even the lowest AC concentration (0.5 gL⁻¹) reduced both the frequency and number of SSEs compared to media without AC, as well as inhibited the development of cotyledonary SSEs, while higher concentrations inhibited SSE completely [17]. Although AC stimulated the production of secondary embryos originating from the suspension of Aesculus hippocastanum L., it reduced SSE in explants originating from anther culture [118]. These inhibitory effects are probably due to unselective nature of adsorption of AC, reflecting the complexity of its role in SSE.

The inclusion of amino acids such as glutamine [26,44,49,80,89,96,97,105,125,129,133,160,170] or proline [10,73] has also been reported to improve the quality and yield of secondary embryos. In A. lebbeck, 75 μM (10.96 mgL⁻¹) of glutamine was optimal for both primary and secondary SE and crucial for maintaining embryogenic competence as well as the maturation of SEs into cotyledonary stage, compared to the other concentrations tested [89]. Glutamine, as a source of reduced nitrogen, is generally claimed to promote SE [127], as well as the development and size of the embryos [97]. However, while improving maturation progression in Q. brantii, it reduced SSE at 1.71 µM (0.25 mgL⁻¹) and 5.13 µM (0.75 mgL⁻¹) concentrations (compared to media without glutamine), proving to be a good treatment to control repetitive SSE and improve embryo maturation in order to obtain fully formed plants [49]. Additionally, while high total nitrogen promoted SSE and better development of cotyledons in SSEs of Q. suber, the high ratio of reduced nitrogen (the presence of glutamine and arginine) decreased the percentage of explants exhibiting polyembryogenesis [127]. Proline, through endogenous accumulation, often induced by osmotic stress or exogenous supplementation, can have a stimulating effect on SE, primarily linked to its involvement with cell wall glycoproteins with morpho-regulatory functions [7]. In T. cacao, proline supplementation of 8.69 µM (1 mgL⁻¹) resulted in a higher production of well-organized, milky SSEs with a better-defined meristematic structure [10]. When tested in different combinations, proline proved to be superior to glutamine in M. sativa for the induction of SSE [14]. Proline, with the addition of glutamine and arginine, when added to the germination medium, has been associated with improved germination rates and plantlet conversion, as demonstrated in C. tinctorius [168]. Casein hydrolysate, a complex mixture of amino acids and peptides [26,83,96,109,116,122,133], or, similarly, casamino acid [91], were also successfully used. In S. pohuashanensis, casein hydrolysate at concentrations between 100 and 400 mgL⁻¹ were stimulatory to the frequency and number of SSEs, with the concentration of 200 mgL⁻¹ proving optimal for SSE proliferation [83].

Other media additives like polyamines, such as putrescine, spermine, and spermidine [114,144], sugar alcohols like mannitol [7,25,67,146], or sorbitol [7,67,146,148,160], coconut water [75], malt extract [44,93,166,170], or PEG [7,118], also proved important in promoting SSE, especially for the maturation phase [44,96,148]. Polyamines are sometimes considered a new category of PGRs that play a role in regulating cell division, differentiation, and morphogenesis, including embryogenesis. In Q. suber, exogenous polyamines significantly increased the number of SSEs formed, with spermidine being the most effective, notably increasing both the quantity (up to 375%) and quality (size increase of about 38%) of SSEs [144].

Silver and copper compounds were also found to increase the efficiency of SSE. Silver nitrate in concentrations of 20 and 40 µM (3.40 and 6.79 mgL⁻¹), for example, was the most efficient treatment for induction of SSE and improving the average number of secondary embryos in C. canephora, compared to cobalt chloride, salicylic acid, or without additional treatment [16]. Silver thiosulphate in a concentration of 20 µM with 4 gL⁻¹ charcoal was the best combination for secondary embryo proliferation in Quercus alba L. and Quercus rubra L. species [131]. The addition of copper sulfate was important for SE in M. esculenta, where a concentration of 3.13 µM (0.5 mgL⁻¹) was used to induce both PSE and SSE [20,107], and a concentration of 2 µM (0.32 mgL⁻¹) was needed only for PSE [106].

6. Culture Conditions: Optimizing Physical Factors

Physical factors in in vitro cultivation like temperature, light conditions, or the use of solid or liquid media can significantly influence both the efficiency and morphology of secondary somatic embryos.

6.1. The Influence of Temperature as a Factor for SSE

Temperature appeared to be an important factor for SSE induction, at least in some species. The temperature range reported in different experimental setups was highly variable, ranging from 20 to 30 °C in different studies. In most cases, a standard temperature for in vitro conditions of around 25 °C was adequate for PSE and SSE induction (Figure 4). Since temperature was not a factor that was thoroughly tested in most SE experiments, observed differences were probably more-so a reflection of the common laboratory practices than the specific experimental requirements. The effects of different temperatures (20 °C, 25 °C, or 30 °C) on the induction of SSE were tested in H. dulcis, where temperature strongly affected the induction frequency of SSE. Interestingly, the highest-tested temperature of 30 °C was the most efficient for the frequency of the SSE induction and the number of SSEs per explant, while lower temperature (20 °C) was more suitable for further embryo development, plantlet conversion, and survival [55].

In most cases, the temperature was kept constant, irrespective of day/night cycle, but there were reports of day/night variations as well. For example, for PSE and SSE in Q. robur [90], Q. alba, and Q. rubra [131], standard conditions were 25/20 °C (day/night), while for M. communis, day/night temperatures were 24/18 °C [121], 26/20 °C for Camellia reticulata [100], and 28/24 °C for M. esculenta [68]. For PSE and SSE induction in H. nobilis, the optimal temperature was 20/18 °C (day/night), while for the cultivation of fully grown plants regenerated from SSE, 25/18 °C (day/night) was more suitable [39]. On the other hand, the germination of SSEs of S. pohuashanensis was more successful with lower (18 °C) rather than higher (25 °C) temperatures [83]. Interestingly, treatment with high temperature (40 °C) for five days was necessary for the induction of PSE from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. mature seeds and subsequent repeated cycles of SSE [56]. In M. esculenta, the storage of SEs at lower temperatures (between 16 and 20 °C) for four months was optimal for frequency of SSE and subsequent plant regeneration, while 16° C only was optimal for longer storage (8–12 months) [66]. Stratification of immature somatic embryos at 4 °C for more than two weeks reduced SSE in Q. brantii, but promoted embryo maturation and progression from the heart to the torpedo stage [49].

6.2. Light Conditions in SSE

Light regime was sometimes crucial for PSE and SSE induction and progression, although the effect was species-specific. In the medicinal plant P. alliacea, a 16 h photoperiod at mean light intensity of 46 µmol m⁻² s⁻¹ significantly improved both the frequency of embryogenesis and the number of PSEs per explant compared to darkness [48]. In contrast, PSE induction in R. hybrida was performed in darkness, but light conditions (14 h photoperiod) gave rise to SSEs of better morphology (vigorous and compact, golden-yellow coloration, and abundant proliferation), while in the dark, SSEs were soft, hyaline/white, waterlogged and non-proliferating, or moribund and withered [47]. Similarly, in Rosa rugosa Thunb., light conditions significantly influenced the morphology of SSEs, with low light intensity (500–1000 lux) proving optimal by producing vigorous, golden-yellow SSEs. In contrast, continuous darkness resulted in brownish and withered embryos, while higher light intensities (2000–2500 lux, or 27–33.75 µmol m⁻² s⁻¹) led to the formation of green and expanded SSEs [82]. The light intensity also affected the proliferation of SSEs in this species, namely 2000–2500 lux or darkness inhibited the proliferation [82].

Continuous darkness was optimal for the induction of PSE and SSE in 21 studies, while continuous light was rarely optimal for the induction and development of SEs [54,101,120], with light intensity sometimes being very low, for example, 15 µmol m⁻² s⁻¹ in V. rupestris [101]. Most often, a certain photoperiod was applied in light/dark cycles varying in the duration of the light phase from 8, 12, 13, 14, 16, 18, and 23 h, with 16 h being the most prevalent day length (64). Depending on the specific phase of SE (induction or development), different combinations of light and dark conditions were optimal in different cases. Light of a certain photoperiod was needed during all of the PSE and SSE phases in 42 articles. In other cases (41), after the initial induction of SE in the dark, cultures required light in the early phases of callus development [26,64,71], for embryo development [19,75,84,115], or maturation of SEs [61,79,107,118]. Light was commonly introduced for the germination of SEs [5,9,38,47,65,81,84,93,96,98,105,109,114,130,133] as, without it, embryos were developing etiolated shoots [4]. It was also introduced for plant development from germinated SEs [26,43,122]. In SSE of O. europaea, dark conditions enhanced the SSE rate (30%) compared to the 16 h photoperiod (15%); however, culturing mature somatic embryos in the dark resulted in a lower germination rate (35%) than that observed under the 16 h photoperiod (45%) [95]. In E. globulus, dark was inducive to SSE proliferation, but light was needed for development from the cotlydonary phase, as embryos and later germinated embryos formed in the light, were green, and had larger leaves than those germinated in the dark, which were whitish, friable, and etiolated [94].

6.3. The Importance of Solid and Liquid Media in SSE

A solid or liquid state of growth media has also been noted as an important factor for the induction and maintenance of PSE and SSE in some species. While solid media, solidified with gelling agents like Phytagel, Gelrite, agar, or agarose, is more prevalently used in SE setups, liquid or suspension culture was used in at least one of the SE phases in 34 studies. Liquid culture offers several advantages, including increased production efficiency, faster embryo development, longer preservation of embryogenic potential, and better uniformity and synchronization of embryogenesis. The higher production rate reported in some species could be due to more efficient nutrient uptake and gas exchange in liquid media, which supports the faster growth and development of plant tissues [69]. In addition, toxic metabolites accumulated around the tissue are more effectively dispersed by a liquid [157]. The more synchronized development of embryos, due to more homogenous conditions in liquid medium, could offer advantages for large-scale plant production in conservation and transformation experiments [69]. Suspension cultures have also been shown to enhance the maturation competence of embryogenic tissues to form well-developed plantlets with proper roots and shoots [27]. Higher multiplication efficiency, due to a higher rate of secondary embryogenesis, is also an important implication, resulting in a substantial increase in biomass, as well as the production of a greater number of SSEs from each initial embryo [48]. Due to the maintenance of embryogenic potential in liquid culture, this can result in highly repetitive embryogenesis where SEs continuously develop into complete plants with good morphology and development, maintaining a consistent supply of plantlets for research and commercial applications.

In Helianthus maximiliani Schrad., for example, secondary embryos were successfully induced in a liquid hormone-free medium, which was found to be more efficient and rapid than primary embryogenesis on solid media with PGRs, promoting the development of new embryos within 10 days [128]. While PSE in M. esculenta could only be obtained on solid media, SSE could be induced in both liquid and on solid medium, with liquid providing a higher, faster, and more synchronized production of embryos, while maintaining comparable shoot conversion rate [69]. In P. alliacea, a medicinal plant, solid and liquid culture were both highly efficient and repetitive in producing SSEs, but the liquid culture offered a significant increase in biomass through a production of a mean of 35 embryos from each embryo inoculated [48]. Liquid culture for SSE was a better choice than solid culture for A. hypogaea, as it was very efficient (approx. 90% of PSEs produced SSEs), cyclic, and repetitive, with exponential growth and no apparent loss of embryogenic potential in over one year, as well as a much higher conversion frequency [41]. When evaluating different electroporation conditions for C. arabica, SSE tissues developing in liquid media were less darkened after electroporation than the ones cultured in solid media, which was ultimately better for plant regeneration [158]. On the other hand, while being comparable in efficiency of SSE in H. dulcis, SSEs formed on a solid medium had larger cotyledons and a better rate of conversion than those formed in a liquid medium [55]. Even when the proliferation of SEs was more efficient in solid culture, embryos from suspension culture showed faster maturation, conversion rate, and shoot-root growth in C. roseus [27]. Interestingly, the complexity of introducing the liquid phase is reflected in the case of M. sativa where after the initial PSE callus induction on solid media, liquid culture was introduced for callus proliferation; however, simple subculturing led to the rapid reduction in embryogenic capacity, which was not successfully mitigated either by fractionation of cell suspension or by SSE [73].

During SSE cultivation, a change between liquid and solid media was sometimes required to support different developmental phases. Liquid culture of embryogenic callus and PSEs of Bunium persicum (Boiss.) Pimenov & Kljuykov was up to 1.3 times more efficient than PSE production on solid media, could be maintained for over one year without the loss of embryogenic potential, and was undergoing efficient SSE. However, the embryos forming in liquid culture were arrested at the globular stage and a transfer to solid media was required for the development of cotyledonary embryos and the conversion to plants [61]. Similarly, cycling between the liquid and solid phase was optimal for SSE in M. esculenta, where liquid culture induced dedifferentiation of the developed PSEs, allowing for the proliferation of embryogenic callus while simultaneously inhibiting embryo development past the globular stage, and a transfer to a solid medium was necessary for further embryo development [106]. Moreover, in Musa spp., liquid culture was more effective than solid media for the maintenance and proliferation of SSEs for 16 months [75], efficient for multiplication and maturation of SSEs in the same species [76], but for conversion into plants, a solid media phase was needed [76]. Liquid culture combined with cultivation on filter paper was used to enhance PSE callus proliferation in P. menziesii in-between cultivation on solid media in PSE and SSE [5]. Liquid culture was very important in PSE of V. rupestris, for the initiation, multiplication, and maturation of PSEs, as well as in SSEs, where, after a phase of SSE initiation on solid, liquid media was needed for the elongation, multiplication, and individualization of SSEs in embryo clusters [101].

The temporary immersion system (TIS) or temporary immersion reactor (TIR), combines the advantages of constant immersion with those of partial immersion with an inert support, avoiding problems such as vitrification and asphyxia, often linked with constant immersion [157]. Compared to other liquid or semi-solid cultivation methods, TIS achieved the highest rate of multiplication of secondary embryos (24-fold) in Camellia sinensis (L.) Kuntze [157]. Similarly, in Q. robur, TIS promoted the proliferation of embryo biomass, but also the synchronization of SEs, enabling the higher production of cotyledonary embryos [90]. Additionally, in H. brasiliensis, TIS was useful for embryo development and maturation [130]. Bobadilla Landey et al. [84] tested the two proliferation systems in C. arabica—embryogenic suspensions in liquid culture and SSE in TIR—concluding that both systems ensured high proliferation rates, with SSE in TIR providing better genetic and epigenetic fidelity. The benefit of using TIS was somewhat contradictory in Bactris gasipaes Kunth, as it greatly improved the number of SSEs obtained and boosted plantlet growth, but reduced survival rate during acclimatization [105]. In another example, TIS only gave rise to non-embryogenic callus, and a simple liquid culture was the most efficient cultivation method in T. cacao [10].

7. Explant Selection: The Importance of the Starting Material

The selection of appropriate explants is a crucial step in maximizing the success of PSE and SSE induction since the initial explant selection for PSE often predetermines the developmental pathway and productivity of embryogenic tissues generated in further embryogenic cycles. In various species undergoing SSE, a wide variety of somatic tissue explants have been successfully employed to induce PSE, including seeds and zygotic embryos (33), leaves and petioles (32), seedlings, cotyledons, and hypocotyls (17), nodal segments and shoot explants (8), and different flower parts like staminodes, anthers petals, and flower buds (14). The choice of explant often depends on factors such as the species, the ease of obtaining the explant, and the embryogenic potential of the tissue. Cells capable of developing SEs are physiologically similar to those in zygotic embryos, hence the further a tissue is from this state, the more reprogramming it needs to become embryogenic [15]. Tissues closely related in developmental stage or origin to embryogenesis, like flower organs, seeds, or developing embryos, represent efficient explants for PSE and subsequent SSE, probably due to less cell reprogramming needed. In concordance with this, direct SSE is often considered to require less cell reprogramming than indirect SSE, having proembryogenic-competent cells already present in the explant [6,10].

The initial PSE explant type often affects the development and efficiency of SSE. For example, seed-coat-derived PSEs in D. carota were able to produce SSEs much more efficiently than hypocotyl-derived PSEs [51]. Similarly to their efficiency in PSE of C. tinctorius, cotyledon-derived primary explants were more efficient in inducing SSE than leaf-derived primary explants [168]. Additional differences were observed in the rate of SSE development, for example, SSEs originating from epicotyl-derived PSEs developed faster than those from hypocotyl-derived PSEs in A. glauca [87]. The success of SSE induction also depended on the choice of explant in M. communis, where only PSEs from immature seeds were able to induce SSE, while PSEs from hypocotyls and cotyledons developed only plantlets [121]. In M. sativa, the frequency of SSE induction depended on the explant ability to develop SEs in PSE. All callus lines that developed PSEs later underwent SSE, with leaf explants proving the most efficient compared to petiole and internode explants [136].

The explant choice for SSE is usually guided by prior knowledge. Primary embryogenic callus, embryogenic clumps, embryo clusters, or primary SEs in various stages of development are often used as explants for SSE induction. Specifically, whole primary somatic embryos, usually at the cotyledonary or globular stage, were the most commonly used as the initial explants for inducing SSE (88). For instance, whole PSEs at the cotyledonary stage were used to successfully induce SSE in C. erythraea on the entire surface of the embryo [4], while in S. pohuashanensis, PSEs at the cotyledonary stage were cut and inoculated to generate secondary embryos, demonstrating a high rate of SSE [83]. SSEs were formed on the surface of globular structures obtained in PSE of Gladiolus × grandiflorus cv. Peter Pears [38]. Rather than individual embryos, primary embryo clusters, or clumps of embryos with different developmental stages were also used for SSE induction (12). Clumps of PSEs, particularly at the early cotyledonary stage, successfully induced secondary embryos in A. glauca [87], while PSE clusters promoted the development of new pre-embryogenic masses and, later, SSEs in C. dactylon × C. transvaalensis [81]. Occasionally, primary embryogenic callus, with or without embryos present, or cell aggregates served as initial explants (6). When primary differentiated nodular callus and embryos were exposed to PGRs for the prolonged period, embryogenic cream-colored soft callus was formed, kept multiplying and differentiated SSEs in Narcissus L. ‘Carlton’ [60]. Rarely, cotyledon or hypocotyl explants from germinated primary somatic embryos were used as explants for SSE (5), such as in B. oleracea var. gongylodes, where secondary embryos spontaneously appeared on hypocotyls of germinated PSEs after 5 weeks of culturing initial zygotic embryos on PGR-free medium [45].

When primary embryos were used for inducing SSE, the embryo developmental stage was the most important factor determining the success of subsequent SSE. To evaluate suitable explants, different primary embryo stages were tested for SSE, assessing their multiplication rate as well as morphology and conversion into plantlets of the resulting SSEs. In certain cases, the latter stages of embryo development, such as cotyledonary or germinated PSEs, proved to be more suitable explants for induction of SSE than the earlier stages, such as globular or torpedo. For example, cotyledonary embryos were more prolific than pre-cotyledonary shaped embryos as explants for SSE in P. ginseng [21] or torpedo-stage PSEs in V. vinifera [86]. Germinated PSEs were the best explant type showing the highest SSE induction frequency in H. dulcis, compared to cotyledonary and heart-stage PSEs [55]. In other species, earlier developmental stages seemed to be more embryogenic than more advanced stages. For example, globular, early heart-shaped stage and early cotyledonary stage PSEs were more potent than more advanced, bigger cotyledonary embryos and germinating embryos in A. carnea [116], with early cotyledonary PSEs also showing the best SSE response in B. oleracea var. gongylodes [45]. Similarly, precotyledonary PSEs in D. caryopyllus formed SSEs directly and more efficiently than cotyledonary PSEs [103], regardless of the tested media and development pathway [25]. The early developmental stages of embryos (globular and heart) showed significantly higher potential for SSE compared to torpedo and cotyledonary embryo stages in Piper nigrum L. as well [147], while in I. batatas PSEs in the early torpedo stage produced more numerous and faster-growing SSEs than elongated and mature primary embryos [59]. Similarly, in C. vernus the highest induction of SSE was observed when heart-shaped and globular PSEs were used, while torpedo-shaped PSEs showed lower conversion rates [92]. Explants prepared from early-stage small translucent embryonic structures were more embryogenic in SSE than those prepared from more advanced embryonic structures in Q. robur [91]. When bipolar and globular PSEs were used in A. officinalis for the induction of SSE, globular embryos were more productive [88]. Interestingly, in E. globulus, the best results were achieved using both early embryo masses, as well as late/germinated cotyledonary embryos [94].

Primary embryo maturation was deemed important in the case of M. sativa, where the authors concluded that the embryogenic capacity declined with PSE maturation and especially with desiccation, and was highest in immature PSEs showing efficiencies comparable to those of petiole explants in PSE [73]. In two Quercus species, primary nodular structures, considered as anomalous somatic embryos with incomplete development, were more effective explant type for SSE compared to well-formed PSEs of various developmental stages [131]. In addition to the PSE developmental stage, embryo size may also be a relevant factor for SSE. Namely, in C. persicum, large-sized globular embryos and small-sized torpedo-stage embryos were significantly more successful than other embryo sizes and stages [122]. Embryo size was also important in C. wightii, where PSEs of the cotyledonary stage were classified in three categories, and the two smaller categories were more efficient in inducing SSE and producing globular SSEs than the larger category [19]. Similarly, in R. rugosa better results were obtained from bigger somatic embryo clumps at the globular stage compared to smaller clumps or cotyledonary embryos [82].

SSE is often clearly advantageous over PSE, having higher efficiency, multiplication rate, and improved development, as demonstrated for different explant types. For example, SSE utilizing PSE cotyledon explants in T. cacao resulted in up to a 30-fold increase in somatic embryo production compared to PSE from staminode explants and, like in PSE, SSE efficiency was genotype dependent [74]. Similarly, each fragment of mature primary cotyledonary embryos produced significantly more secondary SEs than foliar PSE explants in M. esculenta [20]. In another example, SSE in C. wightii resulted in a 74% induction rate from bi-cotyledonary PSEs compared to a 24% induction of PSE from immature zygotic embryos [19]. Additionally, secondary SEs developing on clusters of PSEs in L. drummondii were more uniform, faster to develop and in much greater numbers per gram of tissue compared to primary SEs developed from zygotic embryos [64]. Moreover, SSEs in I. batatas were, in general, more numerous and better synchronized than primary embryos [59].

It can be concluded that the complexity of explant choice for PSE and SSE remains an important factor for efficient SE. Different explant types for PSE induction determine PSE efficiency, but can also determine the success of subsequent SSE. Similarly, various PSE explants used in SSE can provide different results in SSE. These choices, due to different developmental stage and embryogenic tissue capacity, further interact with factors such as PGR choice and concentration, species, and genotype to provide an efficient and reproducible SE induction.

8. Challenges and Future Directions: Improving Efficiency and Reliability in SSE Systems

Secondary or cyclic SE is seen as one of the best methods for the mass multiplication of plants for various downstream applications, ranging from plant conservation to genetic improvement. While significantly improved over decades of research, there are still challenges and bottlenecks that hinder a more widespread use of SSE in many species. Mainly, future research should focus on overcoming genotype dependence and improving maturation and conversion efficiency, while minimizing somaclonal variation. Optimizing culture conditions for different species remains an important prospect, and efforts should be focused ideally on reducing the number of variable factors and simplifying the protocols where applicable, since many reported protocols involve numerous steps and medium variations which can be intimidating for many researchers.

One of the bottlenecks is genotype dependence and the recalcitrance of certain species or valuable genotypes. A strong genotypic effect on SE production has been evident for some crops like cocoa and grapevine, where certain desirable genotypes may show low initiation frequency or even recalcitrance, and much protocol optimization is needed to overcome these hurdles [10,86]. The underlying reasons for this recalcitrance are not fully understood, but they are likely related to complex hormonal interactions influencing signaling and subsequent gene repression during SE initiation. Bearing in mind that the successful induction of SE often occurs only from juvenile tissues, this limits its use, e.g., for the propagation of mature elite trees, while the quality of SEs and conversion rate into plants are also dependent on the genotype of the original explants [134]. The success of SSE can sometimes depend on using PSEs at a specific developmental stage or from certain explant sources, further limiting flexibility [102]. The development of more robust and genotype-independent protocols will undoubtedly lead to further advancements in SSE, expanding its applications in plant breeding, crop improvement, and conservation efforts, and widening its applicability to new species and varieties.

The formation, maturation, and conversion rate of SSEs are still major bottlenecks for many species. They often require specific culture conditions, like determinate PGR combinations, additives in culture media, temperature, or light conditions. These factors are actively optimized, with treatments like proline addition [10], osmoregulators [7], ABA or PEG [148], carbohydrate increase and replacement [10,133], chilling [49,127], and changing light conditions [127], showing promise in some species. Suboptimal conditions sometimes produce physiological and morphological abnormalities, that can affect SE germination and conversion to plantlets [148], but can sometimes be mitigated by using cyclic SE [143].

Being that the SSE process is influenced by numerous factors, like the type and physiological state of the explant, composition of the culture medium, type and concentration of PGRs, light regime, and other factors, the optimization of culture conditions can be very complex and ultimately often species-specific. Developing PGR-free systems where possible would prove beneficial to reduce somaclonal variation risks or aging effects [117,127]. Adjusting auxin/cytokinin type and ratio to achieve a good hormonal balance can improve specific phases of SSE or the efficiency of SSE in general. For example, adenine improved embryoid size and plantlet survival, while other cytokinins inhibited SSE in cassava [20]. In A. trifoliata, the addition of BAP significantly promoted conversion to whole plants, compared to PGR-free medium [149].

Although SSE is seen as an efficient method for the immense multiplication and maintenance of SEs, initial induction or subsequent proliferation can still be low in many species. Its use for truly large-scale clonal propagation, with the potential for mass scale-up, remains yet to be developed for many species, but automated in vitro culture systems, particularly bioreactors and TIS, hold great promise [90,157].

More detailed genetic and phenotypic analyses, and especially molecular analyses, are needed to study specific environmental triggers and genetic pathways that facilitate SSE, as it is markedly less understood than PSE. Studying highly embryogenic plants and genotypes can help discover the mechanisms behind the increased embryogenic potential [128,171]. Since there are species where SSE can progress without external PGRs, understanding the role of endogenous plant hormones in regulating SE [149] and the interplay between externally and internally present hormones remains a significant challenge. Molecular mechanisms of PSE that govern the transition from somatic to embryogenic cell fate, as well as those that regulate the development and maturation of SEs, are still not completely understood and even less known for SSE. Therefore, the future research will most effectively need to combine advanced molecular techniques, such as single-cell transcriptomics and proteomics, to identify key regulatory genes and signaling pathways. For instance, comparative single-cell RNA sequencing of PSE- and SSE-derived tissues could help identify stage-specific regulators, while whole-genome bisulfite sequencing may reveal epigenetic barriers in recalcitrant genotypes. Combining different omics, studying in parallel SE process at the level of genes, proteins, and metabolites, in synergy with histological methods, will be the future approach to advance SE research. Microarray analysis, expressed sequence tag (EST) profiling, and transcriptional profiling will be vital for screening and identifying differentially expressed genes involved in embryogenic acquisition and expression [56]. The identification of new genes, e.g., EsXTH1 and EsPLT1 in Siberian ginseng (E. senticosus) [56] or CeNA1 in common centaury (C. erythraea) [171], that are essential for SE and could be used as markers for SSE initiation or specific developmental-phase expression will prove as invaluable strategy in the new era of SE research. The development of high-throughput screening methods for identifying genotypes with superior potential for SSE would greatly accelerate progress, and identifying genes with expression patterns that can reliably predict this would be most useful. The CRISPR/Cas system would prove significant either for gene knock-out to quickly elucidate the function and effect that these gene-candidates have on the process of SSE, but also for gene knock-in or overexpression to improve recalcitrant genotypes or species.

9. Conclusions

SSE in plants is a complex, variable, and a largely species-specific process. Given that the research on SSE spans more than four decades and includes over one hundred published studies, accumulating into a vast and diverse dataset, an extensive synthesis and a structured review such as this is therefore highly valuable and a first of its kind in this research field. It provides researchers with a comprehensive map of what is known, a consolidated overview of key findings, methodological differences, sources of variability, major factors which highly influence outcomes, and aspects that deserve special attention. Such a synthesis gives researchers an accessible entry point into a complex and diverse literature on factors influencing SSE.

Exogenous PGRs remain one of the key factors influencing the induction and maintaining of SE potential. While sometimes resembling the hormonal requirements of PSE, SSE may also demand altered PGR concentrations or combinations, and in some cases can even occur on a PGR-free medium. Among PGRs, auxins and cytokinins are the most frequently used, with 2,4-D as the predominant auxin and BAP as the most common cytokinin. Special attention should be given to the often contrasting effects of 2,4-D, which, while being very effective, may not be the optimal choice for every species, and exploring alternative auxin types for both efficient SSE induction and normal embryo development will be imperative. Other hormones, such as ABA, gibberellins, jasmonic acid, and ethylene, although less frequently applied, can sometimes be an important factor for SSE induction, maturation, or conversion into plantlets. Culture media formulations for SSE are also highly variable, with MS medium and its modifications being the most widely used, while alternatives such as WPM, SH, or B5 are needed due to species requirements. Sucrose is the primary carbohydrate source, and changes in its concentration can be critical for the transition from PSE to SSE, or towards embryo germination. Additional supplements, including activated charcoal, amino acids, and silver or copper compounds, may also enhance embryogenic responses in certain cases. Although the media components and PGR selection are largely guided by previous studies on a given species, reproducing published induction conditions without optimization is often ineffective; therefore, evaluating multiple combinations is crucial for defining optimal SSE induction conditions. Physical factors further influence SSE. A standard in vitro temperature of 25 °C is suitable for most species, although day/night fluctuations can alter embryogenic outcomes. Similarly, while a 16 h photoperiod is the most common, some studies reported induction under continuous darkness, while constant light was rarely effective. Although solid media are generally used more in SE systems, liquid or suspension cultures are often required, at least in one phase of the process. The choice of starting material, as one of the most important internal factors, strongly influences the embryogenic response. Whole primary somatic embryos, typically at the cotyledonary or globular stage, are the explants most commonly used for efficient SSE induction.

Future research should focus on improving the efficiency and reliability of SSE systems by overcoming genotype dependence, enhancing maturation and conversion rates, and minimizing somaclonal variation. Optimizing culture conditions across species, ideally by simplifying protocols, remains an important task, even though SSE is undoubtedly influenced by multiple factors. A key aspect is the species-specific response to PGRs during SSE induction and maintenance. Clear trends are difficult to establish because the data is sparse: most species appear in only one study so far. Even for the best-studied species, no universally effective hormone combination reliably induces SSE. Ultimately, progress in this field will depend on understanding the underlying mechanisms that need to be mobilized for SSE to occur. Integrating advanced molecular tools, including various omics approaches, to identify key regulatory genes and signaling pathways that control this complex developmental process will indisputably be the focus of future research in this field.