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Article

Induction of Somatic Embryogenesis in Araucaria araucana (Molina) K. Koch: Considerations for Ex Situ Conservation of Ancient Tree in Chile

by
Daniela Riffo-Agurto
1,*,
Neusa Steiner
2,
Priscila Cartes
3,
Pamela Quiroga
3,
Jaime Espejo
4,
Ester San Martin
5,
Jean-Pierre Lasserre
5,
Marcos Edel Martínez-Montero
6,*,
Martha Hernández de la Torre
1,
Darcy Ríos-Leal
1,
Roberto Ipinza
7,
Simón Sandoval
8 and
Manuel Sánchez-Olate
1
1
Laboratorio de Cultivo de Tejidos Vegetales, Facultad de Ciencias Forestales y Centro de Biotecnología, Universidad de Concepción, Concepción 40730386, Chile
2
Laboratorio de Fisiología Vegetal, Universidad Federal de Santa Catarina, Florianópolis 88040-900, SC, Brazil
3
Laboratorio Bio-Vitro SpA, Concepción 4600000, Chile
4
Laboratorio de Botánica y Productos Naturales, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Santiago 7820436, Chile
5
Forestal Mininco SpA, Los Ángeles 4440000, Chile
6
Facultad de Ciencias Agrícolas, Universidad de Ciego de Ávila, Ciego de Ávila 65200, Cuba
7
Instituto Forestal, Valdivia 5090000, Chile
8
Facultad de Ciencias Forestales, Universidad de Concepción, Concepción 40730386, Chile
*
Authors to whom correspondence should be addressed.
Forests 2025, 16(5), 732; https://doi.org/10.3390/f16050732
Submission received: 26 November 2024 / Revised: 7 April 2025 / Accepted: 14 April 2025 / Published: 25 April 2025
(This article belongs to the Special Issue Somatic Embryogenesis and Organogenesis on Tree Species: 2nd Edition)
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Versions Notes

Abstract

Araucaria araucana is an emblematic native conifer from Chile and Argentina that has been classified as threatened due to anthropogenic activities. Somatic embryogenesis (SE) is a biotechnological tool used for both the preservation of genetic material and the propagation of valuable genotypes. The present study investigates the effects of explant source and culture medium on SE induction in A. araucana genotypes from three wild plant populations. Immature strobili were collected from different geographical provenances: a coastal area (Villa Araucarias, “VA”), Cordillera de Nahuelbuta (Trongol Alto, “TR”), and the Andes Mountains (Malalcahuello, “MA”). SE induction was observed after 45 days in a basal medium (BM) supplemented with 1-naphthaleneacetic acid (NAA—11 µM), 6-benzylaminopurine (6-BA—2.8 µM), and Kinetin (Kin—2.8 µM). The highest induction rate (75%) was achieved for seeds from VA. Embryogenic cell line (ECL) proliferation requires auxins but is genotype-dependent, as not all genotypes survive. Cytochemical analysis revealed the presence of pro-embryogenic masses (PEMs) in the ECLs, indicating an efficient SE induction protocol. The progression of PEMs to early embryos was observed in the presence of maltose (3% w/v), polyethylene glycol 3350 (PEG—7% w/v), and abscisic acid (ABA—68 µM). Our results establish a baseline for the establishment of in vitro cultures for a diverse range of A. araucana genotypes, enabling the initiation of ex situ preservation programs and further investigation into embryo maturation.

1. Introduction

Araucaria araucana (Mol) Koch is native to sub-Antarctic rainy temperate forests of South America (Argentina and Chile) and occurs mainly in the Andes Mountains and Nahuelbuta, Chile [1,2]. It is commonly known as the Monkey Puzzle or Mapuche Pehuén tree and is closely associated with the culture of native indigenous inhabitants from south-central Chile and southwestern Argentina [3]. A. araucana is an ancient species, and its ecological, esthetic, landscape, and cultural value in South America is undeniable [4]. However, like 66% of Araucariaceae species, A. araucana is threatened and produces only recalcitrant seeds, which cannot be easily stored in gene banks. This is the reason why it is given high conservation priority in national and international committees for rare and iconic plants worldwide [4,5]. The situation is even more alarming because the original ecosystems of A. araucana are at high risk due to anthropogenic activities, including indiscriminate seed collection, intensive livestock farming, invasion by exotic species, and forest fires. Additionally, the remaining A. araucana trees are facing a serious fungal disease [6] that causes the gradual deterioration of the tree crown, leading to death. A. araucana seeds are recalcitrant and do not form soil seed banks, resulting in little natural regeneration in disturbed ecosystems. Some efforts have been made in terms of tree disease control, plant breeding, and the establishment of in situ conservation areas [7]. However, due to biodiversity loss in these forests, A. araucana was declared a Natural Monument of Chile [8].
Plant conservation programs worldwide are primarily based on in situ and ex situ strategies [9]. It is well established that in situ conservation is appropriate for species with recalcitrant seeds. However, ex situ conservation strategies have become urgent and are widely used for the preservation of the leftover genetic diversity of rare plants, as in the case of A. araucana. Considering that seeds cannot be stored for long periods in conventional seed banks, in vitro culture propagation techniques such as somatic embryogenesis (SE) have become crucial for multiplying and preserving this rare plant germplasm [10,11]. SE has been used in plant conservation and breeding programs, with great potential to regenerate wild plants or select trees for large-scale propagation [12]. In conifers, SE is a multi-step process starting with the induction of pro-embryogenic masses (PEMs), followed by early and late somatic embryo development and maturation and finally plant regeneration [13,14]. However, the induction step remains one of the main challenges for establishing SE protocols in woody species, and juvenile explants such as immature zygotic embryos (ZEs) are typically used [13]. In wild, rare, and poorly studied plants like A. araucana, identifying the appropriate time to collect samples and selecting the right explant are often difficult tasks requiring constant monitoring by experts in the field.
In recent decades, attempts have been made to determine the factors that control SE, recognizing the selection of the appropriate initial explant and the choice of plant growth regulators (PGRs) incorporated into the induction medium as important factors for the successful induction of PEMs [15,16,17]. Overall, there are two stimuli that induce the reprogramming of differentiated plant cells into competent cells: (i) strong stress and (ii) changes in the internal and/or external cellular levels of growth regulators [18,19]. It is recognized that high doses of auxins, such as 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram) [20], with or without cytokinins such as 6-benzylaminopurine (6-BA) and Kinetin (Kin) at low concentrations, are crucial as an initial trigger for acquiring cellular competence. These PGRs first promote the dedifferentiation of somatic cells, followed by a new embryogenic differentiation program [21]. In addition to auxins, both the explant source and its developmental stage appear to significantly affect the induction of SE [15,22,23]. It has also been reported that SE induction is genotype-dependent, which may require different culture media to successfully capture genetic diversity [24]. The quality of the embryogenic cell lines (ECLs) obtained during the induction process is usually defined by the presence of PEMs [14,25]. PEMs are formed by aggregates of embryogenic and suspensor cells that are able to progressively develop somatic embryos [14,24]. While there are no previous reports on SE in A. araucana, studies have been conducted on other rare conifers, such as A. angustifolia [26,27], Torreya taxifolia [28], and Cedrus libani [29], as well as on commercially important species like Picea abies [30], Pseudotsuga menziesii [31], and Pinus taeda [32,33].
Considering the threatened conservation status of A. araucana, as well as the high priority conservation of this iconic Natural Monument in Chile, this study investigated the establishment of an SE protocol in order to capture the genetic diversity from three remnant wild plant populations. Our hypothesis is that the induction of SE in A. araucana will be possible using ZEs and a culture medium supplemented with PGRs. We investigated the effects of culture media using (i) two auxins (NAA and 2,4-D) and (ii) the duration of exposure to auxins/cytokinins on the induction of embryogenic cultures. The proliferation of embryogenic cultures and the morphology of PEMs and somatic embryos were evaluated and discussed in relation to the development of a successful protocol.

2. Material and Methods

2.1. Plant Material

Female strobili with seeds were collected from wild open-pollinated tree populations at three different geographical locations (provenances) of the Chilean territory. The first location was Trongol Alto (TR) (Nahuelbuta Mountain Range, Biobío Region, 1000 m altitude, 37°37′ south latitude and 73°07′ west longitude). The second location was Villa Araucarias (VA) (Carahue, Araucanía Region, 651 m altitude, 38°29′ south latitude and 73°14′ west longitude). The third location was Malalcahuello (MA) (Andes Mountain Range, Araucanía Region, 1300 m altitude, 38°26′ south latitude and 71°31′ west longitude). Seeds were collected every 15 days at the three locations between October 2021 and January 2022.

2.2. Induction of Embryogenic Culture

Seeds of three different plant genotypes, one from each provenance, were individualized from the cones and soaked in a fungicide solution Captan and Benomyl- Arysta LifeScience, Salto de Pirapora, Brazil, (0.1% w/v) for 10 min. The seeds were then transferred to NaClO (10% v/v) and kept under agitation for 5 min, followed by three rinses with sterile distilled water. In a laminar flow cabinet, using a LEICA EZ4 W (Wetzlar, Germany) stereomicroscope, megagametophytes were excised from the seeds, and six isolated ZEs were placed in individual Petri dishes (90 × 15 mm), each containing an induction medium. A BM (basal medium), as described by Gupta and Pullman [32] and modified by Pullman [34], was used. It was supplemented with hydrolyzed casein (0.05% w/v), myo-inositol (2% w/v), maltose (1.5% w/v), 6-BA (2.8 µM), and Kin (2.8 µM). Two different auxins were evaluated: the induction media were designated as MIP when NAA (11 µM) was added, MIN when 2,4-D (23 µM) was used, and IC for media free of PGRs. All media had their pH adjusted to 5.8 before being autoclaved at 101.3 kPa for 15 min. Gelrite®, Saint Louis, MO, USA (0.2% w/v) was used as the gelling agent. Petri dishes containing the immature ZEs were incubated in the dark at 25 ± 2 °C for 45 days. Considering the three provenances, a total of 1296 immature ZEs were isolated and used in the induction experiments. The percentage of ZEs able to form friable embryogenic cultures was evaluated after 30, 45, and 60 days. Each embryogenic culture induced from a single ZE was designated as an embryogenic cell line (ECL).

2.3. Embryogenic Culture Proliferation

The embryogenic cultures that reached a size of approximately 10 mm in diameter during the induction step were transferred to the proliferation medium and maintained in darkness at 25 ± 2 °C, with subculturing performed every 21 days. The proliferation culture medium (MP) was the same BM (basal medium) used in the induction step, but only the PGRs were modified. The MP1 medium was supplemented with 2,4-D (5 µM), 6-BA (2 µM), and Kin (2 µM), while the MP2 medium was supplemented with 2,4-D (23 µM), 6-BA (2 µM), and Kin (2 µM). In both cases, sucrose (3% w/v) and Gelrite® (0.25% w/v) were added to the culture medium. Each embryogenic culture induced from a single ZE was separated and designated as an ECL. At the proliferation step, no PGR-free medium was used because not all embryogenic cultures induced and maintained without PGRs were able to proliferate and develop into ECLs. The ECLs from the PGR-free medium were subcultured in MP1 and MP2 media. To evaluate the growth rate of the ECLs, the increase in relative fresh mass was measured every twenty-one days over five successive subcultures. The growth rate was assessed using seven ECLs, representing genotypes from all three provenances. The quality of the ECLs was evaluated using cytochemical staining, as described in Section 2.5.

2.4. Somatic Embryo Development in Pre-Maturation and Maturation

Based on the high proliferation rates and the presence of PEMs observed in the previous steps, the ECLs (L1) derived from Villa Araucarias seeds were selected and tested in pre-maturation and maturation treatments. During the pre-maturation phase, the ECLs (L1) were subcultured in a PGR-free BM supplemented with maltose (3% w/v) and activated charcoal (0.01% w/v) for 42 days (Step 1). Subsequently, for maturation, the ECLs were transferred to a BM supplemented with maltose (3% w/v), myo-inositol (2% w/v), polyethylene glycol (PEG 3350) (7% w/v), and abscisic acid (ABA) at concentrations of 34, 68, and 95 µM (Step 2). A total of twelve ECL (L1) aggregates, each weighing 0.2 g, were used for each ABA treatment. The ECLs were evaluated every 21 days and maintained in the dark at 25 ± 2 °C for 63 days.

2.5. Morpho-Cytochemical Analysis

The morphology of PEMs and embryos from different embryogenic cultures and seed provenances was evaluated using double staining. A small cell sample was stained with acetocarmine, followed by Evans blue, as described by Gupta and Durzan [35] and Steiner et al. [13]. The PEMs were imaged using an Olympus BX 51 microscope equipped with an image capture camera (Image Focus V 2.0 system software), Qimaging Corporation, Austin, TX, USA. This analysis was performed on all embryogenic cultures from the induction experiment and every two months during the proliferation step for the ECLs. At least three cell samples from each embryogenic culture, later designated as ECLs, were evaluated at each time point. The morphology and developmental stage of ZEs were assessed using a LEICA EZ4 W stereomicroscope.

2.6. Statistical Analysis

All treatments were carried out in triplicate. Data were evaluated using an ANOVA (p ≤ 0.05), and graphs were generated using the SigmaPlot program V4 (2021). For the frequency of cellular activation (induction), the data were transformed using a Poisson distribution incorporating random effects in a generalized linear model (GLM) using R Studio statistical software (2021). The comparison of PGR treatments during fresh biomass subcultures (proliferation) was analyzed using Tukey’s test (p ≤ 0.05).

3. Results

3.1. Zygotic Embryo Development Stage Is Affected by Provenance and Embryogenic Culture Induction

No ZEs were identified in the megagametophytes from seeds collected in October 2021. In November 2021, however, zygotic pro-embryo stages were observed in seeds from all provenances (MA, VA, and TR). This embryo stage is characterized by the presence of long suspensor cells attached to a group of embryonic cells in the apical region (Table 1a–c). By December 2021, further progression in ZE development was observed, and all seeds from all provenances exhibited the early zygotic embryonic stage (Table 1d–f). Early embryo stages are characterized by an increase in the embryonic apex and the beginning of suspensor cell regression. Finally, in January 2022, the late ZE stage was observed in seeds from all provenances but with slight differences in the embryo developmental stage. Seeds from MA showed larger cotyledons compared to those from VA and TR (Table 1g–i). In the late stage, suspensor cells were no longer present, and the ZE displayed an axis, meristems, and cotyledons. Across all seed provenances and collection times, a slight degree of variation was observed in the developmental stages of embryos within the same strobilus, necessitating a rigorous explant selection process.
The ZE stage significantly affects SE induction in A. araucana for all three seed provenances (Table 2). Early ZEs exhibited the highest and most significant induction percentages compared to pro-embryos and late embryo stages across all culture media from different provenances. However, the combination of early ZE stages and MIP medium led to the highest embryogenic culture induction rates for all three A. araucana provenances (Table 2). The highest value was observed in VA seeds, with 75% induction (Table 2). Despite this, it was still possible to induce embryogenic cultures in MIN and IC media for all three populations when the appropriate ZE stage was used as the explant. The megagametophytes isolated from seeds collected in October 2021 did not show any embryogenic culture induction.

3.2. Embryogenic Culture Proliferation and Characterization of Pro-Embryogenic Masses

The induction of embryogenic cultures was observed after 45 days and was characterized by the extrusion of a friable and translucent group of cells from the apical region of the ZE (Figure 1a,b). Proliferation was observed through an increase in the size of the embryogenic cultures (Figure 1c). Through cytochemical analysis, it was observed that the ECLs were composed of PEMs containing two cell types: embryogenic cells (Ecs) and suspensor cells (Scs). Embryogenic cells stained red were round and exhibited dense cytoplasm. Suspensor cells were elongated, vacuolated, and permeable to Evans blue (Figure 1e). Different degrees of PEM organization were observed: PEM I consisted of small groups of Ecs linked to one or two Scs. PEM II showed aggregates similar to those in PEM I but with more than one vacuolated cell. In PEM III, an increase in the groups of Ecs and Scs was observed, which proliferated without polarity (Figure 1d–f).
Overall, 121 ECLs were induced; however, only 37 ECLs proliferated and were selected based on the presence of varying degrees of PEM organization. These ECLs were proliferated in the MIP culture medium, which exhibited the highest growth rate. All ECLs induced in IC medium, which was free of PGRs, had to be subcultured into the MIP medium to enable proliferation. Of these ECLs, 9 were derived from Malalcahuello seeds, 10 from Trongol Alto, and 18 from Villa Araucarias (Figure 2a). After 105 days in the MIP proliferation medium, ECLs from Villa Araucarias (lines 1, 4, and 5) showed the greatest increase in fresh mass, increasing from 0.2 g to 109.2 g (Figure 2b). The ECLs from Trongol Alto (lines 3, 6, and 7) exhibited an intermediate proliferation rate, while those from Malalcahuello (line 2) displayed the smallest increase in fresh mass (Figure 2b).

3.3. Pre-Maturation and Maturation of Embryogenic Culture

In the first step of the pre-maturation treatment, after 42 days in PGR-free medium, ECLs with early embryos were observed on the surface of the embryogenic culture (Table 3). In the second step, after 21 days, the beginning of embryo polarization was observed in the BM supplemented with PEG 3350 (7% w/v), maltose (3% w/v), and ABA (34 and 68 µM). In these two ABA treatments, the apical region of the somatic pro-embryo was observed developing in the opposite direction from the long suspensor system (Table 3). However, in the ECLs treated with ABA at 95 µM, no PEM polarization was observed, and the PEMs appeared to continue to proliferate without further development (Table 3).
Finally, after 63 days in the maturation medium, early somatic embryos were observed on the surface of the embryogenic culture (Figure 3a,b) in the medium supplemented with ABA at 68 µM. In our study, the early somatic embryo development of A. araucana exhibited polarity, with a smooth apical region attached to a group of suspensor cells (Figure 3c).

4. Discussion

Preserving the plant genetic material of threatened species, which produce recalcitrant seeds, in ex situ gene bank collections requires an understanding of how to obtain and proliferate in vitro cultures. In this study, we explored SE as a method for preserving A. araucana, a critically endangered conifer. We examined the effects of explant source and PGRs on SE induction in A. araucana. Our results indicate that the developmental stage of ZEs from three seed provenances significantly affects the induction rate. This issue can be addressed by selecting the appropriate ZE stage and culture medium composition. We found that a BM culture medium supplemented with auxins and early embryos successfully induced SE in all seed provenances (Table 2). The in vitro response to SE induction is influenced by factors such as genotype, tissue type, media composition, and culture conditions, all of which contribute to varying outcomes and determine the success or failure of the technique.
In other conifer species, pre-cotyledonary embryos have been used as explants, as reported for Pinus taeda [34,35,36], P. pinaster [37], and P. strobus [38]. Similarly, our results confirm the findings by Silveira et al. [27], who demonstrated that embryogenic cultures could be obtained from immature ZEs of Araucaria angustifolia, while mature embryos formed a whitish, non-embryogenic tissue. Additionally, Astarita and Guerra [39] showed that the developmental stage of ZEs used as initial explants influenced the induction of cell masses in A. angustifolia. Younger explants (pre-cotyledonary) responded to induction, whereas cotyledonary (mature) embryos lost this capacity. Since Haberlandt [40] proposed the concept of totipotency, it has been understood that plant cells can transition between types, enabling entire plants to be regenerated from somatic cells. Generally, younger plant tissues contain totipotent cells capable of such transitions, as observed in meristems and somatic plant regeneration [41].
Although ZEs are common explant sources in conifers, early ZEs in gymnosperms are tiny and surrounded by megagametophytes. Moreover, within the same plant, ovules develop asynchronously [38], complicating the collection of embryos at a uniform developmental stage. Despite these challenges, our results indicate that the early ZEs of A. araucana can be easily identified and successfully used as explants from three different wild seed populations.
The efficiency of SE in A. araucana is not solely dependent on the developmental stage of ZEs but also on the composition of the culture medium (Table 2), which appears to be a key factor for ensuring the success of “genetic cloning” programs [42]. PGRs play a critical role in determining the fate of plant cells in vitro. While the proliferation of embryogenic cultures requires auxins and cytokinins [43], the development and maturation of individual somatic embryos require ABA [44].
To the best of our knowledge, no prior studies have investigated SE in A. araucana. Here, we established a reproducible protocol for SE using BM culture medium supplemented with NAA (11 µM), achieving a 75% induction rate of embryogenic cultures. Our results align with previous reports on SE in P. taeda [30] and A. angustifolia [26], where BM and auxins such as NAA (11 µM) and 2,4-D (23 µM) were also effective. These PGRs and their concentrations directly influence SE induction. Silveira et al. [27] reported embryogenic induction rates ranging from 33.3% (PGR-free medium) to 43.8% (medium with PGRs), whereas in our study, we achieved only 11% induction in PGR-free medium (Table 2). However, these authors noted that ECLs in PGR-free medium initially exhibited typical conifer PEMs but quickly began to brown and lose proliferation capacity. Similarly, dos Santos et al. [45] reported that ECLs maintained with PGRs (2,4-D, 6-BA, and Kin) appeared translucent white, while those in PGR-free medium underwent progressive browning. These findings suggest that the culture medium environment imposes selective pressures driving cell proliferation.
The use of 2,4-D during the maintenance stage enhances auxin activity but may also induce stress responses and block later stages of embryo maturation [46,47]. Our results corroborate this observation, as some ECLs exhibited increased fresh mass up to a certain point before ceasing growth or beginning to die (Figure 2). These findings highlight one of the main challenges in woody plants: the interaction between genotype and medium composition, which determines the success of in vitro culture protocols. Nevertheless, in this study, we successfully established an SE induction protocol for three wild populations of a threatened woody plant species.
The quality of ECLs is not only evaluated based on proliferation capacity but also on the presence and morphology of PEMs [13,46,47]. In A. araucana, ECLs were characterized by the presence of PEMs (Figure 1d–f), distinguished by cellular organization and cell number (PEM stages I, II, and III), which are considered suitable for developing into early somatic embryos [46,47]. Early embryo development arises de novo from PEM III and progresses through the same sequence of stages described for zygotic embryogenesis in Araucariaceae [48]. In this study, early somatic embryo stages were observed arising from PEMs when the culture medium was supplemented with PEG 3350 (7% w/v), maltose (3% w/v), and ABA (68 µM) (Figure 3). However, these embryos did not progress to late stages. Previous studies in conifers have shown that osmotic agents in maturation media promote embryonic development, often combined with ABA [30,49,50]. For example, the use of maltose combined with ABA improved somatic embryo development in Abies alba [51], P. taeda [52], and P. nigra [53]. Conversely, dos Santos et al. [45] demonstrated that during the maturation phase, only treatments with maltose (6% and 9% w/v) and PEG 3350 (6% and 9% w/v), combined with 6-BA and Kin (1 µM), allowed for progression from globular to torpedo embryos in A. angustifolia. According to dos Santos et al. [45], the use of ABA in combination with osmotic agents was ineffective for somatic embryo development because ABA in the early morphological stages of zygotic embryogenesis inhibits cleavage polyembryony. Furthermore, embryonic maturation in P. taeda [34] subcultured in medium with ABA (38 µM) resulted in the excessive proliferation of embryogenic tissue and fewer early embryos. A similar outcome was observed in our study when using ABA (95 µM) (Table 3), as a cytochemical analysis of proliferated ECLs (Step 1) revealed only PEMs II–III without early embryos.
An interesting study on SE in Picea abies demonstrated that PEMs must reach a specific morphological stage to respond effectively to ABA supplementation [46]. The pro-embryo-to-early embryo transition is a critical stage in SE, as it establishes embryo polarity, a process in which auxin also plays a role. Thus, the pro-embryo-to-early somatic embryo transition is fundamental to the SE of conifers [13]. Although the inability of many embryogenic cultures to form well-developed embryos is largely associated with morphological alterations, the biochemical state of the cultures also plays a role and may result from inadequate culture conditions [46,54,55]. Therefore, clarifying and optimizing this stage is essential for advancing to the later stages of somatic embryo maturation and their conversion into plants.

5. Conclusions

This study represents the first report of SE in Araucaria araucana, a threatened, ancient, and iconic plant species worldwide. Early immature ZEs can be successfully used for SE induction in A. araucana. ECLs were successfully established and proliferated in culture media supplemented with PGRs, such as NAA and 2,4-D, enabling the rescue of genotypes from three wild plant populations: Villa Las Araucarias, Trongol Alto, and Malalcahuello. However, the varying induction rates indicate that A. araucana exhibits genotype dependency for in vitro culture establishment. The progression of PEMs to early and late embryo stages requires further investigation. The cytochemical analysis performed in this study suggests that it may be premature to expose PEMs to ABA. Our work provides a foundation for developing an SE protocol for iconic and threatened plant species (Figure A1). These results can be applied to ex situ preservation programs by combining cryobiotechnologies and SE, not only for A. araucana but also for other threatened species within the Araucariaceae family.

Author Contributions

Conceptualization, D.R.-A., N.S., M.S.-O., M.H.d.l.T., D.R.-L. and R.I.; conducting of experiments, D.R.-A.; methodology, D.R.-A., N.S., M.S.-O., P.C., P.Q. and J.E.; formal analysis, D.R.-A., N.S. and M.S.-O.; writing—draft preparation, D.R.-A., N.S. and M.S.-O.; writing—review and editing, D.R.-A., N.S., M.S.-O., S.S. and M.E.M.-M.; software, D.R.-A., J.E. and S.S.; supervision, N.S., M.H.d.l.T., D.R.-L. and R.I.; project administration, D.R.-A., E.S.M. and P.C.; funding acquisition, E.S.M. and J.-P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received financing from an agreement between Forestal Mininco SpA and the University of Concepción.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Acknowledgments

The authors would like to thank the University of Concepcion (Biotechnology Center) for the formation of human capital in support of the lead author (D.R.) in his master’s thesis in Forestry Sciences. All the authors wish to express their thanks to Forestal Mininco SpA for their role in providing the plant material and financial support for this study. Special thanks to Rebeca Sanhueza for her motivation to carry out this project.

Conflicts of Interest

Authors Priscila Cartes and Pamela Quiroga were employed by the company Laboratorio Bio-Vitro SpA. Jean-Pierre Lasserre and Ester San Martin has received research grants from Forestal Mininco SpA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

ABAAbscisic acid
BMBasal medium [7]
EcEmbryogenic cell
ScSuspensor cell
ECLsEmbryogenic cell lines
FMFresh mass
ICInduction medium control (PGR-free BM)
KinKinetin
MAMalalcahuello
MIPInduction medium (BM with 11 μM NAA and others)
MINInduction medium (BM with 23 μM 2,4-D and others)
MP1Proliferation medium 1 (BM with 5 μM 2,4-D and others)
MP2Proliferation medium 2 (BM with 23 μM 2,4-D and others)
NAA1-naftalenacetic acid
NaClOSodium hypochlorite
PEGPolyethyleneglycol 3350
PEMsPro-embryogenic masses
PGRPlant growth regulator
scSuspensor-like cell
SESomatic embryogenesis
TRTrongol
VAVilla las Araucarias
ZEZygotic embryo
2,4-D2,4-diclorofenoxiacetic acid
6-BA6-benzylaminopurine

Appendix A

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Figure A1. Schematic representation of progress in somatic embryogenesis of Araucaria araucana and main factors evaluated. Established stages (induction and proliferation). Partially developed (pre-maturation/maturation) and unevaluated stages (complete maturation and conversion).
Figure A1. Schematic representation of progress in somatic embryogenesis of Araucaria araucana and main factors evaluated. Established stages (induction and proliferation). Partially developed (pre-maturation/maturation) and unevaluated stages (complete maturation and conversion).
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Forests 16 00732 g001
Figure 1. Induction of somatic embryogenesis (a–c) and cytochemical analysis of pro-embryogenic masses (d–f) of A. araucana. (a) Early zygotic embryo (ZE), (b) extrusion of embryogenic culture (ECS) from ZE, (c) proliferation, (d) pro-embryogenic mass II (PEM II) composed of groups of embryogenic cells (ec) surrounded by suspensor-like cells (sc), (e) PEM I with small group of ecs attached to one or two sc, (f) PEM II with clusters of ec connected to sc. Embryogenic cell stained by acetocarmine (red) and sc stained by Evans blue.
Figure 1. Induction of somatic embryogenesis (a–c) and cytochemical analysis of pro-embryogenic masses (d–f) of A. araucana. (a) Early zygotic embryo (ZE), (b) extrusion of embryogenic culture (ECS) from ZE, (c) proliferation, (d) pro-embryogenic mass II (PEM II) composed of groups of embryogenic cells (ec) surrounded by suspensor-like cells (sc), (e) PEM I with small group of ecs attached to one or two sc, (f) PEM II with clusters of ec connected to sc. Embryogenic cell stained by acetocarmine (red) and sc stained by Evans blue.
Forests 16 00732 g001
Forests 16 00732 g002
Figure 2. Number (a) and fresh mass (FM) increase (b) in embryogenic cell lines (ECLs) of A. araucana during proliferation. ECL subculture was conducted in BM (basal medium) supplemented with 23 μM 2,4-D + 2.8 μM 6-BA and Kin (MP1). L1-7: different ECLs from seed provenance. MA: Malalcahuello; TR: Trongol; VA: Villa Araucarias; SE: somatic embryogenesis. Different letter in (a) mean statistical differences according Tukey’s test (p < 0.05).
Figure 2. Number (a) and fresh mass (FM) increase (b) in embryogenic cell lines (ECLs) of A. araucana during proliferation. ECL subculture was conducted in BM (basal medium) supplemented with 23 μM 2,4-D + 2.8 μM 6-BA and Kin (MP1). L1-7: different ECLs from seed provenance. MA: Malalcahuello; TR: Trongol; VA: Villa Araucarias; SE: somatic embryogenesis. Different letter in (a) mean statistical differences according Tukey’s test (p < 0.05).
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Forests 16 00732 g003
Figure 3. Embryogenic cell line (ECL) L1 of A. Araucana showing early somatic embryos on surface after 63 days in maturation treatments with PEG 3350 (7% w/v), maltose (3% w/v), and ABA 68 μM. (a) ECL L1 from Villa Araucarias (VA) seeds with early embryos on surface (yellow asterisks); (b,c) details of early somatic embryo development.
Figure 3. Embryogenic cell line (ECL) L1 of A. Araucana showing early somatic embryos on surface after 63 days in maturation treatments with PEG 3350 (7% w/v), maltose (3% w/v), and ABA 68 μM. (a) ECL L1 from Villa Araucarias (VA) seeds with early embryos on surface (yellow asterisks); (b,c) details of early somatic embryo development.
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Table 1. Zygotic embryo development stages of Araucaria araucana seeds collected between november and january in plants from three different geographical locations.
Table 1. Zygotic embryo development stages of Araucaria araucana seeds collected between november and january in plants from three different geographical locations.
S. America and Chile Geographical Location Plant Population
Provenance		Zygotic Embryo Development Stage (Months)
Pro-Embryo
(November)		Early
(December)		Late
(January)
Forests 16 00732 i001Malalcahuello
38°26’ S –
Forests 16 00732 i002 71 °31’ W		Forests 16 00732 i003Forests 16 00732 i004Forests 16 00732 i005
Trongol
Forests 16 00732 i006 37°37’ S –
73°07’ W		Forests 16 00732 i007Forests 16 00732 i008Forests 16 00732 i009
Villa Las
Forests 16 00732 i010 Araucarias
38°29’ S –
73°14’ W		Forests 16 00732 i011Forests 16 00732 i012Forests 16 00732 i013
Scale: 1.0 mm
Table 2. The effects of the zygotic embryo stage and BM culture medium on the induction of somatic embryogenesis in Araucaria araucana from seeds collected in three plant populations (provenances). The BM (basal medium) was supplemented with NAA (11 µM) (MIP) or 2,4-D (23 µM) (MIN) or was free of plant growth regulators (PGRs) (IC).
Table 2. The effects of the zygotic embryo stage and BM culture medium on the induction of somatic embryogenesis in Araucaria araucana from seeds collected in three plant populations (provenances). The BM (basal medium) was supplemented with NAA (11 µM) (MIP) or 2,4-D (23 µM) (MIN) or was free of plant growth regulators (PGRs) (IC).
Plant
Population Provenance		Pro-
Embryo
(%)		Early
(%)		Late
(%)		Pro-
Embryo
(%)		Early
(%)		Late
(%)		Pro-
Embryo
(%)		Early
(%)		Late
(%)
ICMIPMIN
Malalca
huello		6 Bb *3 Cb0 Cc0 Cc39 Aa0 Cc0 Cc3 Cb0 Cc
Trongol0 Cc17 Bb0 Cc0 Cc22 Ba0 Cc0 Cc19 Ba0 Cc
Villa Las
Araucarias		0 Cc11 Bb0 Cc0 Cc75 Aa0 Cc0 Cc17 Bb0 Cc
* Comparison capital letter (A: columns) and lowercase (a: row). Poisson distribution in GLM incorporating random effects.
Table 3. Embryogenic cell lines (ECL L1) and cytochemical analysis of pro-embryogenic masses (PEMs) of A. araucana. Pre-maturation (Step 1—PGR-free medium), maturation (Step 2 with maltose (3% w/v), PEG 3350 (7% w/v), and ABA (34, 68, and 95 μM)). Embryogenic cell (ec) stained by acetocarmine (red) and sc stained by Evans blue.
Table 3. Embryogenic cell lines (ECL L1) and cytochemical analysis of pro-embryogenic masses (PEMs) of A. araucana. Pre-maturation (Step 1—PGR-free medium), maturation (Step 2 with maltose (3% w/v), PEG 3350 (7% w/v), and ABA (34, 68, and 95 μM)). Embryogenic cell (ec) stained by acetocarmine (red) and sc stained by Evans blue.
Morpho-Cytological Response of PEMs in Pre-Maturation and Maturation
Step 1
(PGR-free-42 days)		Step 2
(ABA-21 days)		PEMs Cytology
(ABA-21 days)
Forests 16 00732 i01434 μM ABA
Forests 16 00732 i015Forests 16 00732 i016
Forests 16 00732 i01768 μM ABA
Forests 16 00732 i018Forests 16 00732 i019
Forests 16 00732 i02095 μM ABA
Forests 16 00732 i021Forests 16 00732 i022
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Riffo-Agurto, D.; Steiner, N.; Cartes, P.; Quiroga, P.; Espejo, J.; San Martin, E.; Lasserre, J.-P.; Martínez-Montero, M.E.; Hernández de la Torre, M.; Ríos-Leal, D.; et al. Induction of Somatic Embryogenesis in Araucaria araucana (Molina) K. Koch: Considerations for Ex Situ Conservation of Ancient Tree in Chile. Forests 2025, 16, 732. https://doi.org/10.3390/f16050732

AMA Style

Riffo-Agurto D, Steiner N, Cartes P, Quiroga P, Espejo J, San Martin E, Lasserre J-P, Martínez-Montero ME, Hernández de la Torre M, Ríos-Leal D, et al. Induction of Somatic Embryogenesis in Araucaria araucana (Molina) K. Koch: Considerations for Ex Situ Conservation of Ancient Tree in Chile. Forests. 2025; 16(5):732. https://doi.org/10.3390/f16050732

Chicago/Turabian Style

Riffo-Agurto, Daniela, Neusa Steiner, Priscila Cartes, Pamela Quiroga, Jaime Espejo, Ester San Martin, Jean-Pierre Lasserre, Marcos Edel Martínez-Montero, Martha Hernández de la Torre, Darcy Ríos-Leal, and et al. 2025. "Induction of Somatic Embryogenesis in Araucaria araucana (Molina) K. Koch: Considerations for Ex Situ Conservation of Ancient Tree in Chile" Forests 16, no. 5: 732. https://doi.org/10.3390/f16050732

APA Style

Riffo-Agurto, D., Steiner, N., Cartes, P., Quiroga, P., Espejo, J., San Martin, E., Lasserre, J.-P., Martínez-Montero, M. E., Hernández de la Torre, M., Ríos-Leal, D., Ipinza, R., Sandoval, S., & Sánchez-Olate, M. (2025). Induction of Somatic Embryogenesis in Araucaria araucana (Molina) K. Koch: Considerations for Ex Situ Conservation of Ancient Tree in Chile. Forests, 16(5), 732. https://doi.org/10.3390/f16050732

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