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Review

Somatic Embryogenesis in Conifers: One Clade to Rule Them All?

by
Hugo Pacheco de Freitas Fraga
1,
Paula Eduarda Cardoso Moraes
1,
Leila do Nascimento Vieira
1 and
Miguel Pedro Guerra
2,3,*
1
Departamento de Botânica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba 81530-000, Brazil
2
Graduate Program in Plant Genetic Resources, Laboratory of Plant Developmental Physiology and Genetics, Federal University of Santa Catarina, Florianópolis 88034-000, Brazil
3
Graduate Program in Agricultural and Natural Ecosystems, Federal University of Santa Catarina, Curitibanos Campus, Ulysses Gaboardi Road, Km 3, Curitibanos 89520-000, Brazil
*
Author to whom correspondence should be addressed.
Plants 2023, 12(14), 2648; https://doi.org/10.3390/plants12142648
Submission received: 24 April 2023 / Revised: 29 June 2023 / Accepted: 11 July 2023 / Published: 14 July 2023
(This article belongs to the Special Issue Application of Biotechnology to Woody Propagation)
Review Reports Versions Notes

Abstract

:
Somatic embryogenesis (SE) in conifers is usually characterized as a multi-step process starting with the development of proembryogenic cell masses and followed by histodifferentiation, somatic embryo development, maturation, desiccation, and plant regeneration. Our current understanding of conifers’ SE is mainly derived from studies using Pinaceae species as a model. However, the evolutionary relationships between conifers are not clear. Some hypotheses consider conifers as a paraphyletic group and Gnetales as a closely related clade. In this review, we used an integrated approach in order to cover the advances in knowledge on SE in conifers and Gnetales, discussing the state-of-the-art and shedding light on similarities and current bottlenecks. With this approach, we expect to be able to better understand the integration of these clades within current studies on SE. Finally, the points discussed raise an intriguing question: are non-Pinaceae conifers less prone to expressing embryogenic competence and generating somatic embryos as compared to Pinaceae species? The development of fundamental studies focused on this morphogenetic route in the coming years could be the key to finding a higher number of points in common between these species, allowing the success of the SE of one species to positively affect the success of another.

1. Introduction

Plant biotechnologies associated with somatic embryogenesis (SE) provide effective tools to both capture genetic gains and conserve endangered species. They allow the production of large amounts of plant material and also have other benefits [1]. Zimmerman [2] defined SE as a process unique to the plant kingdom that produces normal embryos and whole plants from somatic cells in an undifferentiated state. This morphogenetic route involves dedifferentiation and acquisition of embryogenic competence by somatic cells in response to appropriate inductive signals [3].
More specifically, somatic cells induced to redifferentiate into an embryogenic state reenter the cell cycle while dividing and specialize tissues, such as the bipolar root and shoot meristematic regions [4,5]. This specific developmental potential has been acknowledged both as a valuable pathway for plant regeneration from cell culture systems and as a model for studying early regulatory and morphogenetic events in plant embryogenesis [1].
In addition, SE is favored over the methods of vegetative propagation due to the possibilities of scaling up the propagation by using bioreactors [6] and of enabling processes such as cryopreservation in several plant species, making it possible to store the plant material in the long term. Embryogenic cultures are also an attractive target for genetic modification [7].
Following the early reports of in vitro somatic embryo formation in Daucus carota cell suspensions by Steward et al. [8] and Reinert [9], SE has been established in a wide range of plant species. However, plants are very diversified and not all of them are able to undergo such drastic biochemical changes and genetic remodeling, which is referred to as SE recalcitrance [10].
The first report of callus induction in gymnosperms was in 1971 for Welwitschia mirabilis (Welwitschiaceae; [11]), and only in 1985 was a complete SE protocol for Picea abies (Pinaceae) published [12,13]. Since then, many other gymnosperm genera, such as Abies, Larix, Pinus, Pseudotsuga, and Sequoia [14,15], have been shown to be able to produce somatic embryos, all of them Pinaceae conifers.
Nowadays, the majority of the SE studies reported are on gymnosperms belonging to the family Pinaceae (mostly the genera Pinus and Picea), which is widely used as a model system in a great number of SE studies [16,17,18,19,20]. This large number of studies suggests that there might be a preference for working with Pinaceae species in light of the great economic importance of this group in silviculture [21,22,23] and/or that non-Pinaceae conifers might be more recalcitrant to SE and need different stimuli.
With regard to the three main hypotheses for conifers’ evolutionary relationships, two of these hypotheses position conifers as a paraphyletic group and include Gnetales as a closely related clade [24,25,26,27,28,29]. If conifers are paraphyletic, this may reflect important differences in their responses during SE and Gnetales cannot be overlooked.
In this review, we used an integrated approach in order to cover the advances in knowledge on SE in conifers (Pinaceae + non-Pinaceae conifers) and Gnetales, discussing the state-of-the-art and shedding light on similarities and current bottlenecks. With our approach, we expect to be able to better understand the integration of these clades within current studies on SE.

2. Taxonomic Considerations

According to the most recent classification of extant gymnosperms proposed by Yang et al. [30], there are 8 orders (Cycadales, Ginkgoales, Araucariales, Cupressales, Pinales, Ephedrales, Welwitschiales, and Gnetales), 13 families, and 86 genera. Also, these authors indicated that gnetophytes constitute a monophyletic group that is a sister group to the Pinaceae (gnepine hypothesis), which was reinforced by phylogenomic results based on thousands of single-copy nuclear genes [30]. These results also negate the monophyly of conifers.
Among conifers (Araucariales, Cupressales, and Pinales), the family Pinaceae (Pinales) stands out. It has the largest number of species (272) distributed across 11 genera, representing ~22.6% of all species of gymnosperms [30]. This family, which typically occurs in the Northern Hemisphere, also has the most economically relevant species in the world, with this aspect possibly being one of the most relevant for the robust scientific production focused on the study of SE in the species of this family.
Comparatively, the other conifer families (Araucariaceae, Cephalotaxaceae, Cupressaceae, Podocarpaceae, Sciadopityaceae, Taxaceae) together have 430 species distributed across 61 genera, representing ~36% of all gymnosperm species [30]. These non-Pinaceae conifers naturally occur in the Southern Hemisphere, except for some species of Cupressaceae and Taxaceae [31]. Many of these species have economic relevance, as will be detailed in other sections of the review.

3. Zygotic Embryogenesis in Conifers

Gymnosperms are distinguished by their naked seeds originating in exposed ovules [32]. A large number of gymnosperm species have orthodox seeds, which undergo maturation drying and can withstand dehydration at less than 5% moisture. Many of these species belong to the family Pinaceae and naturally occur in regions with well-defined seasons. However, seeds of many non-Pinaceae conifers show very different behavior related to the maintenance of germination viability when exposed to low moisture content. These recalcitrant seeds are common in many conifer families, such as Araucariaceae and Podocarpaceae, and possibly have distinct, specific requirements for their germination.
Conifer embryos arise from a single fertilization event within the ovule, creating a diploid embryo that develops within a haploid female gametophyte [33]. Most conifers are pollinated and fertilized during the same season, but several species (for example, from the genus Pinus) have a period of 12–14 months between ovule pollination and fertilization [34]. In addition, there are differences between species regarding the development and structure of the male gametophyte, the types of pollination mechanisms, the development and behavior of the proembryo, and the occurrence and type of polyembryony [34].
Multiple embryos can commonly be found within the early-stage seeds of conifers [33]. This polyembryony continues until determined embryogeny stages when one embryo within the seed becomes dominant through unknown processes and continues to grow and develop while all others are aborted.
The sequence of embryo development in gymnosperms can be classified into three steps: (i) proembryogeny—all stages before suspensor elongation; (ii) early embryogeny—all stages after suspensor elongation and before the establishment of the root meristem; (iii) late embryogeny—intensive histogenesis including the establishment of the root and shoot meristems [34]. The proembryogeny stage initiates with several rounds of nuclear duplication without cytokinesis and enters a free nuclear phase after fertilization, which is an intriguing and almost exclusive characteristic of gymnosperm embryogeny.
Next, the stages of early and late embryogeny occur. In these stages, the primary body plan is established together with the apical–basal symmetry and the radial symmetry, and then the root and shoot apical meristems are delineated and the plant axis is established [34]. In the late embryogeny stage, the cotyledonary primordia arise in a ring around the shoot apical meristem. Here, some striking differences between Pinaceae and non-Pinaceae conifers can be identified, such as the number of cotyledons, which can range from 2 to 24 [35].
Another stage that demonstrates a series of differences between families, genera, and species of conifers is the process of embryonic maturation. The maturation is responsible for (i) synthesizing large amounts of storage products, (ii) inducing water loss, (iii) preventing premature germination, and (iv) establishing a state of dormancy [34]. Issues associated with seed orthodoxy and recalcitrance can significantly alter the biochemical, physiological, and molecular requirements of this stage, which involves water loss at certain levels. For example, conifers with recalcitrant seeds, such as Araucaria angustifolia, certainly exhibit different behaviors compared to certain species of Picea that have orthodox seeds.

4. Somatic Embryogenesis in Conifers

For several species of Pinaceae, SE is well-characterized as multi-step process starting with the development of proembryogenic masses (PEMs) and followed by early somatic embryo formation, maturation, desiccation, and plant regeneration. To successfully progress along this pathway, several chemical and physical stimuli may be employed [7]. Usually, the following five steps are applied: (i) culturing of the primary explant (in the majority of cases, the megagametophytes and isolated immature or mature zygotic embryos) on a culture medium supplemented with plant growth regulators (PGRs), mainly auxin but often also cytokinin, for initiation of embryogenic cultures; (ii) proliferation of embryogenic cultures on a solidified medium or in a liquid medium supplemented with PGRs, similarly as during initiation; (iii) prematuration of somatic embryos in a culture medium in the absence of PGRs, which inhibits proliferation and stimulates cell differentiation; (iv) maturation of somatic embryos in a high-osmotic-pressure culture medium supplemented with abscisic acid (ABA); and (v) plant development in a PGR-free culture medium [7].
Picea abies has been considered the model species for SE studies on conifers, and the first successful report was from 1985 [13]. Since this first report, a series of studies have been able to considerably advance understanding of this species’ morphogenetic route, which has served as a subsidy for the development of countless other SE studies on Pinaceae and non-Pinaceae conifers. However, based on the already-mentioned phylogenetic issues and the particularities of non-Pinaceae conifers associated with their habitat, such as nutritional requirements, etc., the well-established SE model for P. abies may not sufficiently represent other conifer species.
Based on this notion, in the following sections, we discuss the SE reports for Ephedrales, Gnetales, Welwitschiales, and non-Pinaceae conifers (Araucariales and Cupressales), aiming to compare SE step-by-step in each of these clades. The criteria used to choose the studies consulted for this review were as follows: (i) articles directly related to the study of somatic embryogenesis in non-Pinaceae conifers, excluding those that only used this technique for another purpose; (ii) articles published in indexed journals up to the year 2022; (iii) articles written in English. The Google Scholar platform was used to search for the cited articles.

5. Somatic Embryogenesis in Ephedrales, Gnetales, and Welwitschiales

One of the pioneer studies with gymnosperm SE was performed by Button et al. [11] with Welwitschia mirabilis (Welwitchiaceae, Welwitschiales; Table 1). Welwitschia mirabilis is a keystone endemic plant from the Namib Desert in Africa and belongs to a monotypic genus (Bombi et al. [36] 2021). This species demands 3 to 20 years to reach maturity and is extremely recalcitrant to vegetative propagation methods [37]. There are few reports on W. mirabilis SE, mainly because of the rare availability of experimental material and its slow growth behavior [38]. Bornman [39] successfully performed callus induction from the hypocotyl–root axis of germinating embryos of W. mirabilis using Schenk and Hildebrandt (SH) [40] culture medium supplemented with 10 mg L−1 1-naphthaleneacetic acid (NAA). However, somatic embryos could not be obtained.
Somatic embryogenesis studies have also undertaken with species from the genus Ephedra: Ephedra foliata and Ephedra gerardiana of the order Ephedrales. These species are among the few gymnosperms adapted to arid habitats and they are distributed mainly in semi-arid environments across the Palearctic and Nearctic; nevertheless some species can be found in neotropical countries as well [41,42].
A complete protocol was obtained for SE induction and plant regeneration for E. foliata [43]. These authors used zygotic embryos as explants, which were inoculated in MS culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) plus kinetin (Kin); the best results obtained were at the lower concentrations tested (2 μM 2,4-D and 2 μM Kin), reaching 60% somatic embryo maturation. It was also found that 6-benzylaminopurine (BAP) plus 2,4-D showed decreased somatic embryo development. The combination of NAA and Kin resulted in root formation and callus induction but with the complete absence of somatic embryo induction.
Somatic embryos of E. gerardiana were obtained from internodal segment cultures [44]. These authors found that lower concentrations of thidiazuron (TDZ; 0.5–1.0 µM) in MS culture medium resulted in callus induction in all the cultures initiated after two weeks, and 10–30% of the embryogenic cultures yielded embryoid-like structures. Higher TDZ concentrations (10–20 µM) resulted in embryogenic culture induction and shoot bud formation in 100% of the cultures. These authors showed that the embryoid-like structures obtained from embryogenic cultures turned into somatic embryos two weeks after transfer from a basal medium (PGR-free) and germinated into young plantlets with the same culture medium composition. This indicates that lower TDZ concentrations can induce embryoid-like structures, and higher concentrations of TDZ could be used for indirect organogenesis induction in this species.
The order Gnetales includes one genus: Gnetum. Gnetum ula is a large woody climber native to India and a remarkable medicinal plant used to cure a variety of illnesses [45]. The first and only SE report on G. ula by Augustine and D’Souza [46] utilized immature zygotic embryos as explants and presented 86% embryogenic cultures induction in MS culture medium supplemented with BAP (5.0 mg/L). The 2,4-D supplementation resulted in non-embryogenic callus formation.
Thus, reports of SE protocols for these three orders are incipient and scarce, and it is certainly necessary to develop further studies, which would allow a better understanding of this morphogenetic route in related species.

6. Somatic Embryogenesis in Araucariales

Araucaria angustifolia (Araucariaceae, Araucariales) is the most abundant species with SE studies among non-Pinaceae gymnosperms (Table 1). It is a culturally, economically, and ecologically important species in Brazil [47], mainly due to the production of edible seeds and high-quality wood [48,49]. This species is commonly found in south and southeastern Brazil and northwestern Argentina and Paraguay [48], but the remaining A. angustifolia forests are under severe pressure from exploration [50]. In this context, much effort has been directed towards developing an effective SE protocol for A. angustifolia and expanding the methods available for its mass propagation and conservation.
Astarita and Guerra [51] primarily introduced the application of tissue culture tools with this species. For A. angustifolia, the SE model includes two cycles: A, which includes induction and proliferation steps; and B, which covers the maturation phase.
At the beginning of cycle A, the apical portion of the zygotic embryos induces embryogenic cultures in the absence of PGRs or with both auxin and cytokinin supplementation [51,52,53]. Embryogenic cells and suspensor cells (SC) are organized in PEMs [54,55,56]. In the proliferation phase, the PEM I, PEM II, and PEM III stages form the embryogenic culture [57].
The prematuration step triggers early somatic embryo polarization and individualization from PEM III in A. angustifolia. This process is usually triggered by PGR removal from the culture medium and its replenishment with maltose and PEG [54,58,59]. Early somatic embryos appear when compact clusters of embryogenic cells grow from PEM III with two regions: the dense globular embryonal mass in the apex and the suspensor in the basal part [60].
Cycle B begins after prematuration, starting with the maturation phase. Here, early somatic embryos rarely turn into late somatic embryos, which can be achieved when the early embryos are capable of responding to the new specific signals with osmotic (i.e., higher levels of gelling agents and PEG supplementation) and hormonal adjustments (mainly ABA supplementation) during the maturation step [54,58]. The early somatic embryo development indicates the beginning of structural differentiation, with protoderm formation around the early somatic embryo followed by meristem determination (root and shoot apical meristems). Afterward, the formation of plantlets may be achieved through the conversion of the somatic embryos [61].
A. angustifolia requires complex conditions for SE in comparison to the Pinaceae model systems. This indicates possible biochemical or genetic prerequisites that are not met with those SE model systems. With the large number of biochemical, genetic, and histological studies through the years with these species, some of those prerequisites have been determined [51,52,54,57,58,59,62,63,64,65,66,67,68,69,70].
During the transition from the multiplication to the maturation phase, somatic embryos exhibit rising levels of ARGONAUTE (AaAGO), CUP-SHAPED COTYLEDON1 (AaCUC), WUSCHEL-related WOX (AaWOX), S-LOCUS LECTIN PROTEIN KINASE (AaLecK), and VICILIN 7S (AaVIC). In the proliferation phase, expression of AaAGO and SCARECROW-like (AaSCR) was more common but not REVERSIBLE GLYCOSYLATED POLYPEPTIDE (AaRGP) or AaLEC due to the effect of the presence/absence of both auxin and cytokinin [67,68]. AaSERK1 could be found as preferentially expressed in embryogenic cell cultures [64]. Furthermore, in situ hybridization results indicated that AaSERK1 transcription starts to accumulate in groups of cells at the embryogenic culture borders and then becomes limited to the proper developing embryo [64].
Studies involving SE-related gene expression in Pinaceae species have also been reported. Vestman et al. [71] reported that a member of the ARGONAUTE family from white spruce is required for proper shoot and root meristem differentiation during embryo development. Important genes for normal embryo patterning, such as putative homologs of SERK and WOX2, have also been identified as being expressed during both the proliferation and differentiation of early embryos and late embryo development [71]. Rupps et al. [72], investigating SE in Larix decidua, also reported transcript accumulation for LdLEC1 and LdWOX2 during early embryogenesis, whereas LdSERK revealed increased expression during later development. In this context, the expression control of the mentioned genes seems to be conserved during the SE of Pinaceae and non-Pinaceae species.
In addition to SE-specific genes, biochemical markers are important to understanding the regulatory mechanisms of SE. For example, in A. angustifolia, a high glutathione (GSH)/glutathione disulfide (GSSG) ratio demand at the beginning of embryogenesis and the presence of nitric oxide (NO) in embryogenic cells may be responsible for maintaining the polarization of proembryos [66]. The manipulation of GSH/GSSG levels and NO emissions have a clear relationship with the viability of early somatic embryos in A. angustifolia [59]. These authors reported that low GSH concentrations (0.01 to 0.1 mM) resulted in a scavenger role in the culture medium; nevertheless, they improved the number of early SEs formed in the cell suspension culture medium within a few days of inoculation. In contrast, the development of early precotyledonary embryos was induced by high GSH concentrations (5 mM). The function of NO in those embryo developments was suggested by NO being emitted from their apexes. These findings highlight that the modification of the culture redox state might be a reliable strategy for the development of more efficient embryogenic systems for this species in particular [59].
The effects of PGR supplementation during the SE of this species on global DNA methylation (GDM) and the proteomic profiles of A. angustifolia have also been reported [70]. The results showed that the EC induced on PGR-free culture medium had increased GDM compared to the EC induced on PGR-supplemented medium over this period of time, as well as many subcultures, which can be associated with decreased genomic stability and changes in gene expression [70].
SE in Podocarpus lambertii (Podocarpaceae), also a member of Araucariales, has been reported [73,74]. This is the only other species from the order Araucariales with a developed SE protocol. This species occurs naturally in the Atlantic Forest biome (Brazil), one of the 25 biodiversity hotspots in the world [75,76]. It is a dioecious species that depends on insects for pollination. Its distribution in restricted areas and the selective economy that always exploits the best specimens with no conservation nor renovation of their communities resulted in this species being listed as “near threatened” and as having a declining population by the IUCN in 2012 [77].
Somatic embryogenesis in P. lambertii is correlated with what is usually performed with A. angustifolia since they are taxonomically close conifers. Fraga et al. [73] reported an efficient SE protocol for this species with homogeneous somatic embryo formation, especially in the ABA-supplemented treatment of maturation. These authors also showed that GSH supplementation to the maturation culture medium improved the number and quality of somatic embryos. Also, the GSH supplementation affected the endogenous hormone levels during the maturation step. This study opened the possibility of comparative studies of SE in these closely related species, which may result in improvements in maturation and conversion of somatic embryos in A. angustifolia.

7. Somatic Embryogenesis in Cupressales

Among the studies with non-Pinaceae conifers, more than half concern the order Cupressales (Table 1). From this order, the most representative species is the Japanese cedar (Cryptomeria japonica), a monotypic genus with limited distribution in temperate humid regions [78]. Japanese cedar is considered the most important species in Japan, representing 44% of Japan’s forest area [79]. Following C. japonica, the genera Juniperus and Taxus also have some representativity, as shown by the number of SE reports on Cupressales.
Cryptomeria japonica (Cupressaceae) has a record of exploration in Japan over the last 1000 years. Today, C. japonica is still one of the most valuable afforestation tree species in Japan [80], covering 44% of Japanese artificial forests. In addition, it is a famous object of genetic engineering due to the Japanese cedar pollinosis disease, a result of pollen dispersion from C. japonica forests [81]. The high efficiency of genetic transformation and plant regeneration of embryogenic cells from conifers has made them a proper target for genetic transformation [82]. Hence, since the early 2000s, SE and plant regeneration in C. japonica have been reported in numerous studies [79,83,84,85,86,87,88].
In this species, SE can only be obtained from immature zygotic embryos [84,86]. Igasaki et al. [84] have also shown that 80% of somatic embryos can germinate and synchronously sprout cotyledons, hypocotyls, and roots when the induced culture medium is supplemented with phytosulfokine (PSK), a small, sulfated plant growth factor peptide [89]. However, Maruyama et al. [86] achieved greater efficiency in germination by using polyethylene glycol (PEG) 6000.
Tanigushi et al. [87] adapted an SE protocol to C. japonica from previous reports [83,85]. Up to 80% induction rates from embryogenic cells could be achieved both in open-pollinated seeds of 20 clones of C. japonica plus trees and in immature artificially crossed seeds [90,91,92]. The variations in the numbers of somatic embryos in those reports could indicate that somatic embryo formation capacity in C. japonica is highly genotype-dependent [80].
Further species from the order Cupressales with extensive SE research belong to the genus Juniperus. Species from the genus Juniperus (Cupressaceae) grow in several habitats, and they are hardy and adaptable to stress conditions; therefore, they are valuable for long-term rehabilitation purposes. The natural habitat of several Juniperus species has been greatly reduced due to overexploitation for timber and fuel wood and clearance for cultivation [93], and several studies on SE in this genus have been performed due to the fact that the species are highly endangered. The majority of the species from the genus Juniperus are listed as being of “Least Concern” in the IUCN Red List, and all the species listed in the SE protocols published are also classified in this category. Juniperus trees have a great variety of uses, from the wood, which has high value for construction, to the fruits, which have medicinal characteristics that make them suitable for curing headaches and skin diseases [93].
The first report of embryogenic culture induction in a species of the genus Juniperus was for J. oxycedrus [94]. These authors achieved embryogenic culture induction from leaf explants of adult trees cultured in the presence of 2,4-D or picloram, with 6 to 18% of cultures developing somatic embryos [94].
In J. excelsa, it has been shown that MS full-strength culture medium inhibited active callus production, but lower levels of ammonium nitrate (NH4NO3) or total nitrogen minimized this response [95]. Glutamine supplementation in MS culture medium with reduced nitrogen concentration also increased callus proliferation.
Rapid proliferation and development of somatic embryos were also observed in J. communis in PGR-free culture medium with lower nitrogen and calcium levels [93]. Moreover, high initiation frequency for embryogenic cell lines could be obtained from intact megagametophytes at the time of intensive cleavage polyembryogeny. These authors also showed that ABA promoted maturation of early embryos (over 40%), and the germinating embryos retained embryogenic potential in the basal part, leading to the development of new embryogenic tissues.
Based on the study developed for J. communis, Belaineh et al. [96] reported a protocol for embryogenic culture induction and maturation in J. procera. These authors observed recalcitrance towards embryogenic culture maturation; however, supplementation with ABA slightly stimulated maturation in most of the cultures evaluated [96].
Somatic embryogenesis protocols were also described for four species from the genus Taxus: Taxus brevifolia, Taxus cuspidata, Taxus baccata, and Taxus wallichiana. The genus Taxus is a natural source of powerful bioproducts, such as paclitaxel, a potent anticancer drug; toxoids, important in the development of antibodies; and flavonoids, which assist in regulating cellular activity and fight free radicals that cause oxidative stress in the body [97]. Taxus trees are typically slow-growing and long-lived; as a result, these species have been over-used in past centuries, and this fact, combined with other activities, such as illegal cutting, has contributed to the endangerment of the genus [98].
Considering the obvious importance of this species, several attempts have been made to generate SE protocols. Almost 30 years ago, Ewald et al. [99] aimed to establish an SE protocol for three Taxus species (T. baccata, T. brevifolia, and T cuspidata). These authors evaluated several concentrations/combinations of PGRs in the SPE culture medium [100]. The combination of kinetin, BAP, and 2,4-D showed the best results for embryogenic culture formation for the Taxus species, and the obtained embryogenic cultures were able to form proembryonary structures and subcultures after transfer to a culture medium supplemented with kinetin and indole-3-butyric acid (IBA).
The last species from this genus with a published SE protocol is T. wallichiana [101]. The authors described an efficient procedure for regeneration of T. wallichiana plants from embryogenic cultures derived from zygotic embryos using half-strength WPM culture medium supplemented with BAP and NAA. These authors achieved 10% conversion of the mature somatic embryos into complete plantlets on WPM basal medium supplemented at half strength with activated charcoal, obtaining fully formed plantlets seven to eight months after initiation of culturing. These authors also analyzed the content of paclitaxel and related taxanes and revealed that the accumulation of paclitaxel was higher in the embryogenic cultures than in the non-embryogenic callus.
Several other species of Cupressales also have SE protocols described, despite reports being more specific and scarce. Ahn et al. [102] described an SE protocol for Chamaecyparis thyoides without plant growth regulator supplementation. In this protocol, the authors induced embryogenic cultures from megagametophytes containing the precotyledonary zygotic embryos on the modified half-strength embryo maturation culture medium developed by Maruyama et al. [83]. Mature somatic embryos were obtained on half-strength semisolid EM maturation medium supplemented with 50 g L−1 maltose, 100 g L−1 PEG 4000, and ABA concentrations up to 100 µM [102].
Hu et al. [103] proposed an SE protocol for Cunninghamia lanceolata. These authors obtained 13.86% embryogenic culture induction on DCR medium supplemented with 1.5 mg L−1 2,4-D and 0.3 mg L−1 kinetin. They also investigated the maturation process and obtained the best results with supplementation with 50 μM ABA and 100 g L−1 PEG 6000 [103]. A very efficient protocol has been reported for another Cupressales species, Cupressus sempervirens [104]. Despite the strong genotype-dependence observed for this species, the authors were successful in inducing embryogenic cultures in a DCR culture medium supplemented with 10 µM 2,4-D. Maturation occurred in a PGR-free DCR culture medium supplemented with 75 g L−1 PEG 4000, and somatic embryos were converted into seedlings in half-strength LP medium supplemented with 2 g L−1 activated charcoal.
Liu et al. [105] proposed an SE protocol for Sequoia sempervirens using needles from 30-day-old grown seedlings, an unusual type of explant for SE induction in conifers that has the potential to maintain the genotypic fidelity of the donor plant. In this protocol, direct SE was induced on SH culture medium with a combination of BAP (0.5 mg/L) and IBA (1.0 mg/L).
SE has been frequently applied as a potential tool for the in vitro conservation of critically endangered species. Ahn et al. [106] reported a successful protocol for SE induction in Thuja koraiensis using megagametophytes with zygotic embryos at precotyledonary and late embryogeny stages. The highest SE induction rate occurred on a culture medium supplemented with 2.2 μM BAP and 4.5 μM 2,4-D, with somatic embryo maturation implemented on a culture medium containing 100 μM ABA [106].
An SE protocol has been reported for another highly endangered species, Torreya taxifolia [107]. The authors used a complex PGR combination (0.5 mM 2,4-D, 0.2 mM BAP, 0.2 mM kinetin, 0.1 μM brassinolide, 3.8 μM ABA) and obtained 60 to 100% induction. Somatic embryo maturation occurred on a culture medium supplemented with 37.8 μM ABA and 0.1 μM brassinolide.
Table
Table 1. Somatic embryogenesis (SE) protocols published for non-Pinaceae conifers up to 2022.
Table 1. Somatic embryogenesis (SE) protocols published for non-Pinaceae conifers up to 2022.
OrderSpeciesExplant UsedFormulation of Culture MediumSteps of the ProtocolReference
AraucarialesAraucaria angustifoliaZygotic embryosLP salts + vitamins + 0.5 g L−1 nicotinic acid + 45 µM 2,4-D + 11.0 µM KIN + 11.0 µM BAP + 500 mg L−1 casein + 1% PEG 8000 Embryogenic culture inductionAstarita et al. [51]
Zygotic embryosLP salts + 6.8 µM 2,4-D + 2.3 µM KIN + 2.2 µM BAP + 1% PEG 8000 + 0.9 µM sucrose + 38 µM ABAEmbryogenic culture inductionAstarita et al. [62]
Zygotic embryosBM salts + vitamins + 5 µM 2,4-D + 2 µM BAP + 2 µM KIN
BM salts + vitamins + 6% and 9% PEG 3350 + 6% and 9% BAP + 1 µM KIN		Embryogenic culture induction
Precotyledonary somatic embryo development		dos Santos et al. [52]
Zygotic embryosLP salts + vitamins + 5 µM 2,4-D + 2 µM BAP + 2 µM KIN
LP salts + vitamins + 5.0 µM ABA + 1% PEG 4000		Embryogenic culture induction
Precotyledonary somatic embryo development		Silveira et al. [65]
Zygotic embryosBM salts + vitamins + 5 µM 2,4-D + 2 µM BAP + 2 µM KIN
BM salts + vitamins + 5 µM 2,4-D + 2 µM BAP + 2 µM KIN + 9% PEG + 9% maltose		Embryogenic culture induction
Early precotyledonary somatic embryo development		Steiner et al. [58]
Zygotic embryosBM salts + vitamins + 2.0 µM 2,4-D + 0.5 µM BAP + 0.5 µM KIN + 0.5 g L−1 casein + 1.0 µM Spd + 10. µM SpmInduction of stage-three proembryogenic masses (PEM III)Silveira et al. [66]
Zygotic embryosBM salts + vitamins + 0.5 g L−1 casein + 1 µM Put Embryogenic culture inductionSteiner et al. [62]
Zygotic embryosBM salts + 5 µM 2,4-D + 2 µM BAP + 2 µM KINEmbryogenic culture inductiondos Santos et al. [54]
Zygotic embryosBM salts + 5.5 µM myo-inositol + 0.008 µM nicotinic acid + 0.005 µM pyridoxyn-HCl + 0.05 µM glycine + 0.006 µM thiamine-HCl + 6.8 mM L-glutamine + 0.05% casein hydrolysate + 3% sucroseEmbryogenic culture inductionMaurer et al. [108]
Zygotic embryosMSG salts + 0.005 g L−1 nicotinic acid + 0.005 g L−1 pyridoxine-HCl + 0.001 g L−1 thiamine + 1.46 g L−1 L-glutamine + 30 g L−1 sucrose + 3 g L−1 gelrite
MSG salts + 5 µM 2,4-D + 2 µM BAP + 2 µM KIN
MSG salts + vitamins + 1.46 g L−1 L-glutamine + 3 g L−1 gelrite + 120 µM ABA + 9% maltose + 7% PEG 4000 + 3% sucrose + 0.15% AC		Embryogenic culture induction
Embryogenic proliferation
Proembryo formation		Schlögl et al. [68]
Zygotic embryosBM salts + vitamins + 0.5 g L−1 casein hydrolysate + 1 g L−1 myo-inositol + 0.1 g L−1 L-glutamine + 3 g L−1 sucrose + 5 µM 2,4-D + 2 µM BAP + 2 µM KIN
MSG salts + vitamins + 1.46 g L−1 L-glutamine + 90 g L−1 maltose + 70 g L−1 PEG 3350 + 1 µM GSH + 100 µM GSSG + 30 g L−1 sucrose		Embryogenic culture induction
Proembryo formation		Vieira et al. [59]
Zygotic embryosLP salts + 10 g L−1 sucrose + 0.45 g L−1 L-glutamine
BM salts + 30 g L−1 sucrose + 0.5 g L−1 casein hydrolysate + 1 g L−1 myo-inositol + 1.0 g L−1 L-glutamine
DKM salts + 0.5 g L−1 casein hydrolysate + 0.1 g L−1 myo-inositol + 30 g L−1 sucrose + 30 µM fluridone
DKM salts + 0.5 g L−1 casein hydrolysate + 0.1 g L−1 myo-inositol + 90 g L−1 maltose + 70 g L−1 PEG 3350		Embryogenic culture induction
Embryogenic culture proliferation
Pre-maturation
Proembryo formation		Farias-Soares et al. [109]
Zygotic embryosMSG salts + 1.46 µM L-glutamine + 30 g L−1 sucrose + 3 g L−1 gelrite + 1.0 µM 2,4-D + 0.5 µM BAP
MSG salts + 1.46 g L−1 L-glutamine + 30 g L−1 sucrose + 3 g L−1 AC + 70 g L−1 maltose + 90 g L−1 PEG 4000		Embryogenic culture induction
Cotyledonary somatic embryo development		Jo et al. [110]
Zygotic embryosMSG salts + 120 µM ABA + 90.0 g L−1 PEG 4000 + 3.0 g L−1 maltoseEarly precotyledonary somatic embryo developmentElbl et al. [111]
Zygotic embryosBM salts + 1.0 g L−1 L-glutamine + 1.0 g L−1 myo-inositol + 0.5 g L−1 casein hydrolysate + 30 g L−1 sucrose
BM salts + 1.0 g L−1 L-glutamine + 1.0 g L−1 myo-inositol + 0.5 g L−1 casein hydrolysate + 50 g L−1 maltose + 100 g L−1 PEG 4000 + 100 μM ABA		Embryogenic culture induction
Precotyledonary somatic embryo development		Fraga et al. [69]
Zygotic embryosDKM salts + 0.5 g L−1 casein hydrolysate + 0.1 g L−1 myo-inositol + 30.0 g L−1 sucrose + 30 μM fluridone
DKM salts + 0.5 g L−1 casein hydrolysate + 0.1 g L−1 myo-inositol + 90.0 g L−1 maltose or 90.0 g L−1 lactose + 70.0 g L−1 PEG		Embryogenic culture induction
Proembryo formation		Steiner et al. [57]
Zygotic embryosMSG salts + 1.46 g L−1 L-glutamine + 3% sucrose
MSG salts + 1.46 g L−1 L-glutamine + 3% sucrose + 120 µM ABA + 7% maltose + 9% PEG 4000		Embryogenic culture induction
Early somatic embryo development		Navarro et al. [112]
Podocarpus lambertiZygotic embryosMSG salts + BM vitamins + 1.46 g L−1 L-glutamine + 0.1 g L−1 myo-inositol + 30 g L−1 sucrose
MSG salts + BM vitamins + 1.46 g L−1 L-glutamine + 0.1 g L−1 myo-inositol + 50 g L−1 maltose + 100 g L−1 PEG 4000 + 2 g L−1 AC + 75 µM ABA + GSH 500 µM		Embryogenic culture induction
Cotyledonary somatic embryo development		Fraga et al. [73]
Zygotic embryosMSG salts + BM vitamins + 30 g L−1 sucrose
MSG salts + BM vitamins + 50 g L−1 maltose + 100 g L−1 PEG 4000 + 2 g L−1 AC + 75 µM ABA 		Embryogenic culture induction
Cotyledonary somatic embryo development		Guerra et al. [74]
WelwitschialesWelwitschia mirabilisZygotic embryosMS salts + 0.5 g L−1 glycine + 5.0 g L−1 inositol + 0.003 g L−1 calcium pantothenate + 0.03 g L−1 thiamine hydrolysate + 40 g L−1 sucrose + 100/50 mg L−1 NAACallus inductionButton et al. [11]
Zygotic embryosSH salts + 0.3 mg L−1 NAACallus inductionBornman [39]
LeafMS salts + 9.0/22.5/45.0 µM 2,4-D + 2.2/4.4 µM BAP + 4.6 µM KIN + 5.4 µM NAAEmbryogenic culture inductionMisra et al. [38]
GnetalesGnetum ulaMegagametophyte + immature zygotic embryosMS salts + 20 g L−1 sucrose + 5 mg L−1 BAP
MS/2 salts (PGR-free)		Embryogenic culture induction
Immature somatic embryo formation		Augustine and D’Souza [46]
EphedralesEphedra foliataZygotic embryosMS salts + 2 µM 2,4-D + 10 µM KIN
MS salts PGR-free		Embryogenic culture induction and somatic embryo maturation
Somatic plantlet formation		Dhiman et al. [43]
Ephedra gerardianaInternodal segmentsMS salts + 1.0 µM TDZ
MS salts PGR-free		Embryogenic culture induction and somatic embryo maturation
Somatic plantlet formation 		Sharma et al. [44]
CuppressalesCupressus sempervirensZygotic embryosDCR salts + 0.5 g L−1 casein hydrolysate + 0.2 g L−1 myo-inositol + 0.1 g L−1 L-glutamine + sucrose (30 g/L) + 10 µM 2,4-D
DCR salts + 1 µM ABA + 0.02% AC		Embryogenic culture induction
Somatic embryo formation		Lambardi et al. [113]
Zygotic embryosMS salts + 15 g L−1 fructose + 15 g L−1 glucose + 4 g L−1 AC + 10 mL l−1 coconut water
MS + 15 g L−1 fructose + 15 g L−1 glucose + 4 g L−1 AC + 1 g L−1 BSA		Embryogenic culture induction
Proembryo formation 		Sallandrouze et al. [114]
Megagametophyte + immature zygotic embryosDCR salts + 0.5 g L−1 casein hydrolysate + 0.2 g L−1 myo-inositol + 0.1 g L−1 L-glutamine + 30 g L−1 sucrose + 10 µM 2,4-D
DCR salts + 75 g L−1 PEG 4000		Embryogenic culture induction
Somatic embryo development		Barberini et al. [104]
Megagametophyte + immature zygotic embryosDCR salts + 0.5 g L−1 casein hydrolysate + 0.2 g L−1 myo-inositol + 0.1 g L−1 L-glutamine + sucrose (30 g/L) + 10 µM 2,4-D
DCR salts + 75 g L−1 PEG 4000		Embryogenic culture induction
Somatic embryo development		Lambardi et al. [115]
Cryptomeria japonicaZygotic embryosCDm salts + 0.8 g L−1 ammonium nitrate + 1 μM 2,4-D + 6.0 g L−1 L-glutamine
CDm salts + ABA (0, 0.1, 1, and 10 µM) + BAP (0.1, 1, and 10 µM)		Embryogenic culture induction
Proembryo development		Ogita et al. [116]
Zygotic embryosSM1 salts + 10 g L−1 sucrose + 10 μM 2,4-D + 3 μM BAP + 0.5 g L−1 L-glutamine
LPM salts + 10 g L−1 sucrose + 10 μM 2,4-D + 3 μM BAP + 0.5 g L−1 L-glutamine
SM3 salts + PEG 400 + ABA
LP salts + 0.2 g L−1 sucrose + 5 g L−1 AC 		Embryogenic culture induction
Somatic embryo development
Somatic plantlet formation		Maruyama et al. [83]
Zygotic embryosMSG salts + 0.01% myo-inositol + 0.15% L-glutamine + 3.2 μM 2,4-D + 1.8 μM BAP + 3% sucrose + 0.4% gellan gum
EMM salts + 0.2% AC + 0.3% gellan gum
EMM salts + 0.2% AC + 3/5-strength EMM vitamins + 400 mg L−1 L-glutamine + 260 mg L−1 arginine + 20 mg L−1 proline		Embryogenic culture induction
Somatic embryo development
Somatic plantlet formation		Igasaki et al. [84]
Zygotic embryosMSG salts + 0.01% myo-inositol + 0.15% L-glutamine + 3% sucrose + 3.2 μM 2,4-D + 1.8 μM BAP + 32 nM PSK
EMM salts + 5% PEG 4000 + 3% maltose + 100 μM ABA + 32 nM PSK + 0.2% AC + 0.3% gellan gum
SGM salts + 0.2% AC + 10 μM GA3		Embryogenic culture induction
Somatic embryo development
Somatic plantlet formation		Igasaki et al. [85]
Zygotic embryosmCD salts + 3% sucrose + 4.1 mM L-glutamine + 1 µM 2,4-D
mCD salts + 3% sucrose + 16.4 mM L-glutamine +100 µM ABA + 6% maltose		PEM induction
PEM maturation		Nakagawa et al. [117]
Zygotic embryosMSG salts + 0.01% myo-inositol + 0.15% glutamine + 3.2 µM 2,4-D + 1.8 µM BAP + 3% sucrose + 0.4% gellan gumEmbryogenic culture induction and somatic embryo developmentIgasaki et al. [118]
MegagametophyteEM salts + 10 g L−1 sucrose + 10 μM 2,4-D + 5 μM BAP + 0.5 g L−1 casein + 0.5 g L−1 L-glutamine
EM salts + 30 g L−1 maltose + 2 g L−1 AC + 100 μM ABA + amino acids + 150 g L−1 PEG + 3.3 g L−1 gellan gum
EM salts + 20 g L−1 sucrose + 1.5 g L−1 L-glutamine + 10 g L−1 agar		Embryogenic culture induction
Cotyledonary embryo development
Somatic plantlet formation 		Maruyama et al. [119]
Zygotic embryosMS salts + 30 g L−1 sucrose + 3 μM 2,4-D + 1 μM BAP
MS salts + 30 g L−1 maltose + 175 g L−1 PEG 6000 + 2 g L−1 AC + 100 μM ABA		Embryogenic culture induction
Somatic embryo development		Izuno et al. [88]
SeedEM salts + 10 g L−1 sucrose + 10 μM 2,4-D + 5 μM BAP + 0.5 g L−1 casein + 0.5 g L−1 L-glutamine + 3 g L−1 gelrite
EM salts + 30 g L−1 sucrose + 3 mΜ 2,4-D + 1 μM BAP + 1.5 g L−1 L-glutamine + 3 g L−1 −1 gelrite
EM salts + 175 g L−1 PEG + 100 µM ABA + 2 g L−1 L-glutamine + 1 g L−1 asparagine + 0.5 g L−1 arginine + 3 g L−1 gelrite		Embryogenic culture induction
Embryogenic culture proliferation
Somatic embryo maturation		Maruyama et al. [79]
Chamaecyparis pisiferaImmature seedMS salts + 0.5 g L−1 L-glutamine + 2,4-D + BAP
MS salts + 100 μM ABA + 2 g L−1 AC + 150 g L−1 PEG 4000
LP salts + 30 g L−1 sucrose + 5 g L−1 AC + 12.5 g L−1 Wako agar		Embryogenic culture induction
Somatic embryo maturation
Somatic plantlet formation		Maruyama et al. [120]
Immature seedMS salts + 0.5 g L−1 L-glutamine + 2,4-D + BAP
MS salts + 100 μM ABA + 2 g L−1 AC + 150 g L−1 PEG 4000
LP salts + 30 g L−1 sucrose + 5 g L−1 AC + 12.5 g L−1 Wako agar		Embryogenic culture induction
Somatic embryo maturation
Somatic plantlet formation		Maruyama et al. [121]
Immature seedMS/2 salts + 10 μM 2,4-D + 5 μM BAP + 10 g L−1 sucrose
MS/2 salts + 1–10 μM 2,4-D + 0.3 μM BAP + 0.5 g L−1 L-glutamine + 30 g L−1 sucrose
EM salts + 50 g L−1 maltose + 100 μM ABA + 2 g L−1 AC + 150 g L−1 PEG 4000 + amino acids
EM/2 salts + 2 g L−1 AC + 10 g L−1 agar		Embryogenic culture induction
Embryogenic culture proliferation
Somatic embryo maturation
Somatic plantlet formation		Hosoi and Maruyama [122]
Chamaecyparis obtusaZygotic embryosMS salts + 10 μM 2,4-D + 30 g L−1 sucrose
MS salts + vitamins + 100 μM ABA + 150 g L−1 PEG 4000 + 30 g L−1 maltose
MS salts + 2 g L−1 AC + 20 g L−1 sucrose + 5 g L−1 gelrite 		Embryogenic culture induction
Cotyledonary embryo development
Somatic plantlet formation		Konogaya et al. [123]
Zygotic embryosEM salts + 0.40 g L−1 KCl + 0.5 g L−1 casein + 1 g L−1 L-glutamine + 10 g L−1 sucrose + 10 µM 2,4-D + 5 µM BAP
EM salts + 50 g L−1 maltose + 100 g L−1 PEG 4000 + 2 g L−1 AC + 100 µM ABA + EMM amino acids 		Embryogenic culture induction
Somatic plantlet formation		Maruyama et al. (2005) [124]
Chamaecyparis thyoidesZygotic embryosEM salts + 0/4.5/9 µM 2,4-D + 0/2.2/4.4 µM BAP + 10 g L−1 sucrose + 0.5 g L−1 myo-inositol + 1 g L−1 L-glutamine
EM salts + 50 g L−1 maltose + 100 gL−1 PEG 4000 + 0/10/50/100 µM ABA + 0/2 g L−1 AC
EM salts + 10 g L−1 sucrose + 2 g L−1 AC		Embryogenic culture induction
Somatic embryo development
Somatic plantlet formation		Ahn et al. [102]
Cunninghamia lanceolataZygotic embryosDCR salts + g L−1 sucrose + 0.5 g L−1 casein hydrolysate + 0.4 g L−1 L-glutamine + 5 g L−1 Phytagel + 1 g L−1 AC + 0.5 mg L−1 2,4-D + 0.5 mg L−1 KIN
DCR salts + 50 μM ABA + 100 g L−1 PEG 6000		Embryogenic culture induction
Somatic embryo development		Hu et al. [103]
Zygotic embryosDCR salts + 1–2 mg L−1 2,4-D + 0.5 mg L−1 KIN + 0.5 mg L−1 BAP + 1 mg L−1 vitamin C + 0.45 g L−1 L-glutamine + 20 g L−1 maltose + 2.5 g L−1 AC + 2.3 g L−1 gelrite
DCR salts + 2–8 mg L−1 ABA + 1.5 mg L−1 GA + 0.2 g L−1 proline + 0.45 g L−1 L-glutamine + 1 mg L−1 vitamin C + 0.5 g L−1 casein hydrolysate + 25 g L−1 maltose + 2 g L−1 AC + 0.2 g L−1 aspartic acid + 2.8 g L−1 gelrite + 100–200 g L−1 PEG		Embryogenic culture induction
Somatic embryo development		Wang et al. [125]
Zygotic embryosDCR salts + 2.0–6.0 mg L−1 2,4-D + 0.5 mg L−1 BAP + 500 mg L−1 casein hydrolysate + 450 mg L−1 L-glutamine + 100 mg L−1 myo-inositol + 20 g L−1 maltose + 2.1 g L−1 gellan gum
DCR salts + 3 mg L−1 ABA + 1.0 mg L−1 GA3 + 500 mg L−1 casein hydrolysate + 120–200 g L−1 PEG 8000 + 30 g L−1 maltose		Embryogenic culture induction
Somatic embryo development		Zhou et al. [126]
Torreya taxifoliaZygotic embryos2207 salts + 0.25% AC + 15 g L−1 maltose + 1 g L−1 myo-inositol + 110 mg L−1 + 2,4-D + 45 mg L−1 BAP + 43 mg L−1 KIN + 0.048 mg L−1 brassinolide + 1 mg L−1 ABA + 5 mg L−1 biotin + 50 mg L−1 folic acid + 250 mg L−1 MES + 60.7 mg L−1 pyruvic acid + 450 mg L−1 L-glutamine
2207 salts + 1% AC + 15 g L−1 maltose + 200 mg L−1 ABA + 5 mg L−1 biotin + 0.1000 μM brassinolide + 50 mg L−1 folic acid + 250 mg L−1 MES + 60.7 mg L−1 pyruvic acid + 0.048 mg L−1 brassinolide + 450 mg L−1 L-glutamine		Embryogenic culture induction
Somatic embryo development		Ma et al. [107]
Sequoia sempervirensNeedlesSH salts + 0/0.05/0.1 g L−1 BAP + 0/0.02/0.05 g L−1 KT + 0.02/0.05/0.1 g L−1 IBA + 0.05% casein hydrolysate
SH salts + 0.05 g L−1 IBA + 0.01 g L−1 NAA + 0.1% AC + 1.5% sucrose + 0.05% casein hydrolysate		Embryogenic culture induction
Somatic embryo development		Liu et al. [105]
Juniperus communisZygotic embryosLP salts + 15 mM ammonium nitrate + 30 g L−1 sucrose + 9 µM 2,4-D + 4.4 µM BAP + 0.044 g L−1 L-glutamine
LP salts + 15 mM ammonium nitrate + 30 g L−1 sucrose + 32 mg L−1 ABA + 0.044 g L−1 L-glutamine		Embryogenic culture induction
Proembryo development		Belaineh et al. [96]
MegagametophytesLP salts (PGR-free)
DCR salts + 2.02 mM potassium nitrate + 1.16 mM calcium chloride + 60 µM ABA + 20 or 100 µM GA4/7		Embryogenic culture induction
Early somatic embryo development		Helmerson et al. [93]
Juniperus proceraZygotic embryosLP salts + 15 mM ammonium nitrate + 3.0 g L−1 sucrose + 9.0 µM 2,4-D + 4.4 µM BAP + 0.044 g L−1 L-glutamine + 1.6 g L−1/3.2 g L−1/6.4 g L−1/12.8 g L−1/25.6 g L−1 ABA + 7.5% PEG 4000Non-embryogenic callusBelaineh et al. [96]
Juniperus excelsaShootsMS salts + ammonium nitrate/potassium nitrate + L-glutamineCallus inductionShanjani et al. [95]
Thuja koraiensisZygotic embryosEM salts + SH + Litvay + 2.2 µM BAP + 4.5 µM 2,4-D + 1 g L−1 L-glutamine + 0.5 g L−1 myo-inositol + 1 g L−1 AC + 10 g L−1 sucrose
IM salts + 1% sucrose 		Embryogenic culture induction
Somatic plantlet formation		Ahn et al. [106]
Taxus cuspidataZygotic embryosSPE salts + 0.001 g L−1 KIN + 0.002 g L−1 NAA Embryogenic culture inductionEwald et al. [99]
Taxus brevifoliaZygotic embryosSPE salts + 0.001 g L−1 KIN + 0.002 g L−1 NAA Embryogenic culture inductionEwald et al. [99]
Taxus baccataZygotic embryosSPE salts + 0.001 g L−1 KIN + 0.002 g L−1 NAA Embryogenic culture inductionEwald et al. [99]
Taxus wallichianaZygotic embryosB5 salts + SH vitamins + 3% sucrose + 0.01 g L−1 NAA + 0.005 g L−1 BAP
WPM salts + SH vitamins + 3% sucrose + 0.02 g L−1 NAA + 0.05 g L−1 BAP
WPM salts + 0.01 g L−1 ABA + 1.0% AC		Embryogenic culture induction
Early precotyledonary somatic embryo development
Somatic plantlet formation		Datta and Jha [101]
MS—Murashige and Skoog [127]; WPM—woody plant medium [128]; B5—Gamborg et al. [129]; BM—Gupta and Pullman [130]; MSG—Becwar et al. [131]; LM—Litvay et al. [132]; DKM—von Arnold and Clapham [133]; EM—embryo maturation medium [83]; LP culture medium [96]; DCR—Gupta and Durzan [134]; SPE—Gupta and Durzan [100]; 2207 [135]; Cd culture medium [136]; EMM/SGM—Smith’s germination medium [137]; 2,4-D—2,4 dichlorophenoxyacetic acid; GA3—gibberellic acid; TDZ—thidiazuron; BA—benzyladenine; BAP—6-benzylaminopurine; NAA—1-naphtaleneacetic acid; KIN—kinetin; ABA—abscisic acid; IBA—indole-3-butyric acid; AC—activated charcoal; PGR—plant growth regulator; BSA—buthionine sulfoximine; GSSG—glutathione disulfide; GSH—glutathione reduced; Put—putrescine; Spm—spermine; Spd—spermidine; PEG—polyethylene glycol.

8. Conclusions and Future Directions

Some issues encountered in triggering SE in conifers have not yet been solved despite the assiduous research efforts over time. While most of the conditions required for SE in Pinaceae species are known, somatic embryo formation and development in many non-Pinaceae conifers are hampered. Non-Pinaceae conifers’ recalcitrance towards SE in general—for example, the slow-growing nature of species, such as W. mirabilis, or the complex conditions required for a complete SE protocol, such as with A. angustifolia—may be an obstacle to obtaining satisfactory protocols.
A better understanding of SE in non-Pinaceae conifers should prove fruitful in the future as SE is now one of the most critical methods for the large-scale propagation of elite genotypes and their conservation. This is especially important because most non-Pinaceae conifers have endangered status.
Some level of understanding has indeed been achieved for most non-Pinaceae conifers, but it is also true that more coordinated research on the SE of these species is urgent. Analyzing the main variables tested in the protocols described in this review, it is possible to list some common points and many dissimilarities in SE protocols for non-Pinaceae conifers. Among the main points held in common are: (i) the use of zygotic embryos as the preferable explant; (ii) the use of strong auxins, such as 2,4-D, in the induction step; (iii) the use of ABA and osmotic agents (such as PEG), sometimes combined with higher concentrations of gelling agents, during the maturation stage; and (d) the use of activated carbon in the conversion to somatic seedlings step. Despite these points in common, there are many heterogeneities in the described protocols, such as: (i) the use of different saline formulations, often in protocols for the same species; (ii) the use of various vitamin compositions; (iii) supplementation of the culture medium with different amino acids or polyamines; and (iv) non-standardization in the carbon sources used.
Therefore, we expect to see increased integration of non-Pinaceae species in future SE studies considering the higher phylogenetic proximity that they present. The development of fundamental studies focused on this morphogenetic route in the coming years could be the key to finding a higher number of points in common between the described protocols, allowing the success of the SE of one species to positively affect the success of another.

Author Contributions

All authors contributed to the conceptualization of the study. Data collection was performed by P.E.C.M. The first draft of the manuscript was written by P.E.C.M., L.d.N.V. and H.P.d.F.F. All authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant numbers HPFF [406261/2018-0] and MPG [302798/2018-8 and 407974/2018-0]).

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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MDPI and ACS Style

Fraga, H.P.d.F.; Moraes, P.E.C.; Vieira, L.d.N.; Guerra, M.P. Somatic Embryogenesis in Conifers: One Clade to Rule Them All? Plants 2023, 12, 2648. https://doi.org/10.3390/plants12142648

AMA Style

Fraga HPdF, Moraes PEC, Vieira LdN, Guerra MP. Somatic Embryogenesis in Conifers: One Clade to Rule Them All? Plants. 2023; 12(14):2648. https://doi.org/10.3390/plants12142648

Chicago/Turabian Style

Fraga, Hugo Pacheco de Freitas, Paula Eduarda Cardoso Moraes, Leila do Nascimento Vieira, and Miguel Pedro Guerra. 2023. "Somatic Embryogenesis in Conifers: One Clade to Rule Them All?" Plants 12, no. 14: 2648. https://doi.org/10.3390/plants12142648

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