Search Results (106)

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

Secondary somatic embryogenesis (SSE) is a powerful tool in plant biotechnology, enabling the continuous production of embryos from primary somatic embryos (PSEs) and offering broad applications across agriculture, forestry, horticulture, and pharmaceutical industries. Depending on culture conditions, SSE may proceed directly from the surface of PSEs or indirectly via callus formation, with the outcome strongly influenced by exogenous plant growth regulators (PGRs). A key advantage of SSE is its cyclic nature, which offers a valuable strategy to maintain embryogenic potential over extended culture periods, generating true-to-type embryos without reliance on the original explant, while significantly increasing the multiplication rate, often making SSE more productive than PSE in many species. This review explores in detail the cellular origin and developmental pathways of secondary embryos, the maintenance of embryogenic competence through cyclic embryogenesis, as well as genetic and epigenetic aspects and the biotechnological applications of this process. Moreover, it addresses challenges regarding strong genotype dependence, variability in embryo quality and morphology, limitations in maturation and conversion potential, and the gradual decline of embryogenic competence with successive cycles, all of which need to be overcome to ensure the stability and reproducibility of SSE and maximize its impact. Full article

Agave species possess substantial cultural, ecological, and economic significance, particularly in Mexico, where they are traditionally utilized for food, fiber, and beverages. Their industrial relevance has expanded to include bioenergy, nutraceuticals, and sustainable agriculture. However, conventional propagation methods are constrained by long life cycles, low seed germination rates, and susceptibility to phytopathogens. In vitro culture has emerged as a pivotal biotechnological strategy for clonal propagation, germplasm conservation, and physiological enhancement. This review presents a critical synthesis of plant growth regulators (PGRs) employed in agave micropropagation, emphasizing their roles in organogenesis, somatic embryogenesis, shoot proliferation, and rooting. Classical PGRs such as 6-benzylaminopurine (BAP), benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) are widely utilized, with BA + 2,4-D and BA + IAA combinations demonstrating high efficiency in embryogenic callus induction and shoot multiplication. Additionally, non-traditional regulators such as abscisic acid (ABA) and putrescine (Put) have been shown to affect embryo maturation. This review synthesizes recent studies on agave in vitro culture protocols, identifies trends in PGR use, and highlights key research gaps. These insights reveal opportunities for innovation and underscore the need for species-specific optimization and molecular validation to improve reproducibility and scalability. Full article

Propagation of rubber tree (Hevea brasiliensis) via secondary somatic embryogenesis (SSEis) is a reliable method. However, its efficiency is relatively low. The aim of this study was to understand more about the factors related to SSEis in rubber trees, trying to improve the efficiency of somatic embryo (SE) yield. Our study showed that the orientations of explants, i.e., the fragments of primary SE (PSE), on the medium affected secondary SE (SSE) yield significantly. Among five experimental tests, the highest yield was 2.6 ± 0.9 secondary somatic embryos (SSEs) per explant, which was achieved by orienting the abaxial side of the explant in contact with the medium and then the adaxial side after a period of culture time. Based on histological evidence, SSEis was induced from the epidermal cells and adjacent cells on the adaxial side of the explants. A remarkable difference in embryogenic capacity difference existed among individual PSE. The concentrations of soluble proteins, starch, soluble sugars, and the superoxide dismutase activity (SOD) levels in the explants were measured during a 25-day long SSEis induction treatment and compared between explants of high and low embryogenic capacity. This study proves that the explant orientation toward the culture medium plays a crucial role in SSEis, while the concentration changes of these biochemical compounds correlate to morphological changes in the explants during induction, as do the changes in SOD activity. Furthermore, the trend of the dynamic changes in the explants reflected a process of de-differentiation and re-differentiation, which started from mature SE tissues during SSE induction. Full article

Somatic embryogenesis (SE) is a powerful biotechnological tool for large-scale clonal plant propagation. However, most woody species exhibit a recalcitrant response. Olea europaea L., a highly valuable tree crop, is among these recalcitrant species. Unravelling the molecular mechanisms underlying SE efficiency in O. europaea is, therefore, essential. Access to embryogenic lines with contrasting capacities for somatic embryo differentiation is a key requirement for such studies. Mature fruits of olive cultivars ‘Galega Vulgar’ and ‘Arbequina’ were collected from open-pollinated orchard-grown plants, and various explants taken from zygotic embryos were used to establish SE. A differentiated response was observed both within each cultivar and between cultivars, with cv. ‘Arbequina’ showing the highest embryogenic induction, particularly when radicles were used. Secondary SE was successfully established for both high- and low-efficiency lines, providing valuable material for future molecular studies. Somatic embryo conversion into plants, a key indicator of embryo quality, was successfully achieved in both cultivars. Flow cytometry analysis revealed a high degree of chromosomal stability. This study presents a reliable procedure to obtain and maintain distinct embryogenic responses in O. europaea, identifying lines with both high and low embryogenic efficiency that can serve as model systems for future molecular investigations. Full article

Catalpa bungei C.A.Mey is an economically significant deciduous tree valued for timber production and landscaping applications. An efficient regeneration system is crucial for clonal propagation and serves as a foundation for future molecular breeding in C. bungei. This study established two in vitro regeneration pathways—indirect somatic embryogenesis and shoot organogenesis utilizing mature zygotic embryos as explants. Primary callus was induced from cotyledon, hypocotyl, and plumule explants. A high frequency (45.73%) of yellow-green compact callus was achieved on De-Klerk and Walton (DKW) medium supplemented with 2.0 mg/L 6-BA, 1.0 mg/L zeatin (ZT), and 0.1 mg/L NAA. Subsequent transfer to 1.5× Murashige and Skoog (MS) medium containing 1.5 mg/L 6-BA, 0.2 mg/L ZT, and 0.1 mg/L NAA yielded the highest embryogenic callus induction rate (16.67%). Embryogenic callus demonstrated bipotent potential, generating both adventitious shoots and somatic embryos under specific hormonal conditions. Histological analyses confirmed the typical developmental stages of somatic embryos, from globular to cotyledonary forms, validating the embryogenic origin of regenerated structures. Furthermore, hormone or osmotic additives such as abscisic acid (ABA), Phytagel, and polyethylene glycol 4000 (PEG4000) significantly enhanced somatic embryo induction, with Phytagel at 5.0 g/L achieving the highest rate (76.31%). For shoot organogenesis, the optimal hormonal combination of the 0.6 mg/L 6-BA, 0.4 mg/L KT, and 0.15 mg/L NAA achieved the highest bud induction rate (88.89%) and produced an average of 4.07 adventitious buds per explant. This study presents an efficient regeneration system for C. bungei, providing a practical platform for large-scale propagation and basis for biotechnological applications in woody plants. Full article

Because of the effects of climate change, a shortage of seeds from superior mother trees for forestry breeding programmes can occur. While biotechnological tools can help address this challenge, the benefits and drawbacks of each technique need to be evaluated. In this work, a comparative Pinus radiata plantlet production study was performed using two different approaches: seeds versus somatic embryos. For each procedure, the number of plantlets obtained was recorded, and the manual labour time was calculated. The skills and time required were higher for somatic embryogenesis, but so was the number of plantlets obtained: 3660 acclimatized somatic plantlets versus 1124 acclimatized zygotic plantlets. The number of different genotypes obtained was higher when germinating mature seeds, but somatic embryogenesis coupled with cryopreservation offers the advantage of having a backup for field-tested material. Additionally, analyses of carbohydrates, total protein and dry weight of somatic and zygotic embryos were carried out. These analyses revealed considerable differences between somatic and zygotic embryos but underlined the importance of high sucrose levels for germination in both systems. In conclusion, each technique offers advantages and disadvantages, and the choice will depend on the objective, the species and the value of the material to be propagated. Full article

The miR156 family, crucial for phase transition and stress responses in plants, remains functionally uncharacterized in the ecologically and commercially important gymnosperm Larix kaempferi. This study systematically investigated L. kaempferi miR156 through phylogenetic analysis, structural prediction, expression profiling during somatic embryogenesis, and heterologous functional validation in Arabidopsis. Four MIR156 family members (LkMIR156s) were identified in Larix kaempferi, each with a characteristic stem-loop structure and highly conserved mature sequences. Computational predictions indicated that these LkMIR156s target four LkSPL family genes (LkSPL1, LkSPL2, LkSPL3, and LkSPL9). qRT-PCR analysis showed that mature LkmiR156s expression remained relatively low during early embryonic development but was significantly upregulated at the cotyledonary stage (21–42 days). Precursor transcript levels peaked earlier (around 28 days) than those of the mature LkmiR156, which remained highly expressed throughout cotyledonary embryo development. This sustained high expression coincided with cotyledon morphogenesis and embryonic dormancy. Functional validation via heterologous overexpression of LkMIR156b1 in Arabidopsis resulted in increased rosette leaf numbers (42.86% ± 6.19%) and individual leaf area (54.90% ± 6.86%), phenotypically consistent with the established role of miR156 in growth regulation. This study reveals the temporal expression dynamics of LkmiR156s during L. kaempferi somatic embryogenesis and its coordinated expression patterns with cotyledon development and embryonic dormancy. The functional conservation of the miR156-SPL module was confirmed in a model plant, providing key molecular insights into the developmental regulatory network of conifers. These findings offer potential strategies for optimizing somatic embryogenesis techniques in conifer species. Full article

Malania oleifera Chun & S.K. Lee, an endemic monotypic species that belongs to the family Olacaceae, is under continuous pressure of decline owing to several ecological and physiological factors. The present study aimed to establish an efficient in vitro protocol for callus-mediated indirect somatic embryogenesis in M. oleifera by alleviating tissue browning. Internodes and leaves obtained from seedlings were used as explants. Antioxidant pre-treatment (ascorbic acid, AA) followed by different carbon sources (sucrose, maltose, glucose, and fructose) and plant growth regulators in various concentrations and combinations were employed in Woody Plant Medium (WPM) to alleviate explant browning and induce callus formation from the explants. AA pre-treatment and subsequent culture on maltose at a concentration of 116.8 mM were optimal for controlling phenolic exudation on >90% of both explants. The highest responses of 53.77% and 57.43% for embryogenic calli were induced from internode and leaf explants, respectively. The highest responses, 85.22% and 93.80%, were observed for somatic embryos that matured into the globular, heart-shaped and torpedo stages at different percentages on NAA 2.5 mg/L in combination with BA 1.0 mg/L for both explants. The matured somatic embryos were finally germinated at a maximum concentration of GA₃, 2.0 mg/L. All plantlets were successfully hardened and acclimatized under culture room conditions and then transferred to the greenhouse. The current study suggests an efficient protocol for indirect somatic embryogenesis by alleviating phenolic exudation from the explants of M. oleifera. This first successful report of in vitro culture establishment in M. oleifera may offer an effective alternative measure to conserve this species and provide a system for analyzing bioactive chemicals and for use in the oil industry. Full article

In the severe environment of Hunshandake Sandy Land, the uncommon and indigenous Chinese tree species Picea mongolica is an important biological component. Conventional seed propagation in P. mongolica is constrained by low germination rates, prolonged breeding cycles, and hybrid offspring genetic instability, limiting efficient varietal improvement. In contrast, somatic embryogenesis (SE) offers superior propagation efficiency, exceptional germination synchrony, and strict genetic fidelity, enabling rapid mass production of elite regenerants. However, SE in P. mongolica is hampered by severe genotype dependence, poor mature embryo induction rates, and loss of embryogenic potential during long-term cultures, restricting the production of high-quality seedlings. In this study, we aimed to analyze the transcriptome and metabolome of three crucial phases of SE: mature somatic embryos (MSEs), globular somatic embryos (GSEs), and embryogenic calli (EC). Numerous differentially expressed genes (DEGs) were found, especially in pathways linked to ribosomal functions, flavonoid biosynthesis, and the metabolism of starch and sucrose. Additionally, 141 differentially accumulated metabolites (DAMs) belonging to flavonoids, organic acids, carbohydrates, lipids, amino acids, and other metabolites were identified. An integrated study of metabolomic and transcriptome data indicated considerable enrichment of DEGs and DAMs in starch and sucrose metabolism, as well as phenylpropanoid biosynthesis pathways, all of which are required for somatic embryo start and development. This study revealed a number of metabolites and genes linked with SE, offering important insights into the molecular mechanisms driving SE in P. mongolica and laying the groundwork for the development of an efficient SE system. Full article

The asynchrony of cytoplasmic and nuclear maturation in cumulus–oocyte complexes (COCs) due to prematurely declining concentrations of cyclic adenosine monophosphate (cAMP) has been shown to result in reduced oocyte developmental competence. The objective of this study was to evaluate the effect of pre-IVM treatment with cAMP modulators for different durations on the developmental potential of equine oocytes used for cloned embryo production. Collected COCs were transferred to cryovials filled with transport medium at 20–22 °C. Within the cryovials, the COCs were either untreated (Control) for 18 h or treated with 50 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine for the first 4 h (Pre-IVM 4 h) or the entire 18 h (Pre-IVM 18 h). Oocytes were then transferred to maturation medium and incubated for a further 22–24 h at 38.5 °C in 5% CO₂ in air. Somatic cell nuclear transfer embryos were then produced using the meiotically mature oocytes and donor cells from six different fibroblast cell lines. The rates of maturation and embryo development did not differ significantly between the groups, though blastocyst formation tended to be inferior in the Pre-IVM 4 h group compared with the Control group (p = 0.06). Of 67 blastocysts produced, 23 were transferred to recipient mares on Day 4 or 5 post-ovulation. Regarding the pregnancy outcomes, no significant differences were found between the groups, and four viable foals were born, each derived from a different donor cell line. The findings expand on those from previous evaluations of this biphasic IVM system, and indicate that the cAMP-modulating treatments exert limited effects under the pre-IVM conditions used here. Full article

Pinus elliottii, a key economic conifer in southern China, requires efficient propagation methods to meet demand for elite germplasm in resin and timber production. While somatic embryogenesis-based plant regeneration has been successfully achieved in Pinus elliottii, large-scale production remains challenging. Our results demonstrate that the genotype of Pinus elliottii significantly influences the induction rate of embryogenic callus. During somatic embryo maturation, the liquid–solid induction method increased the number of mature embryos by 25.85 times. Maturation efficiency was further enhanced by a 3-week pretreatment followed by the application of 9 mg/L ABA, 0.5 mg/L PSK, and 6 mg/L COS. Additionally, the incorporation of activated carbon significantly promoted both the maturation and germination of somatic embryos. In the somatic embryo maturation stage, 1 g/L activated carbon induced 288.67 mature embryos per gram of embryogenic callus, resulting in a total of 1452 embryos. During germination, the application of 4 g/L activated carbon achieved a germination rate of 63%, and the survival rate of somatic embryo-derived seedlings reached 85%. This study not only identifies the optimal conditions for somatic embryogenesis in Pinus elliottii but also establishes an efficient protocol for somatic embryo maturation induction, providing a crucial scientific foundation for the rapid propagation and seedling production of Pinus elliottii. Full article

Pinus massoniana Lamb. (masson pine) is a critical species for afforestation in southern China but faces severe threats from pine wilt disease (PWD) caused by Bursaphelenchus xylophilus. To accelerate disease-resistant breeding, this study investigated the effects of cryopreservation on the embryonic capacity of the embryogenic callus as well as the effects of abscisic acid (ABA), polyethylene glycol 8000 (PEG 8000) and phytagel concentration on the somatic embryo’s maturation and germination. Furthermore, the impact of transplanting substrates on the survival and growth of regenerated plantlets were evaluated. The results showed that cryopreservation at −196 °C effectively maintained the embryogenic potential of the callus, with post-thaw tissues exhibiting superior somatic embryo maturation capacity compared to the long-term subcultured callus (38.4 vs. 13.2 embryos/mL). Key maturation parameters were systematically optimized: ABA concentration at 6 mg/L in the suspension culture maximized embryo yield of 24.1 somatic embryos/mL, while PEG 8000 at 130 g/L in solid medium achieved peak embryo production of 38.4 somatic embryos/mL, and the maximum of 26.6 somatic embryos/mL when the concentration of phytagel was 3.5 g/L. The highest germination rate of 29.8% was observed with 130 g/L PEG in the maturation medium. The highest survival rate (56.5%) and maximum plant height (22.3 cm) after 12 months of transplantation were achieved in substrates consisting of soil and vermiculite, which outperformed those containing varying proportions of mushroom residue. This study establishes a scalable protocol for the mass propagation of PWD-resistant P. massoniana, integrating cryopreservation and maturation media optimization, which offers dual benefits for disease-resistant breeding and sustainable germplasm conservation. Full article

DNA methylation plays a crucial role in regulating the developmental processes of plants. Particularly, it is closely associated with the development of embryogenic cells (EC) and somatic embryos (SE). In this study, we investigated the effects of 5-azaCytidine (5-azaC) treatment on somatic embryogenesis proliferation and maturation of Taxodium hybrid ‘zhongshanshan’. The results showed that the callus proliferation was inhibited when the concentration of 5-azaC exceeded 30 μM, while treatment with 5 μM 5-azaC improved the maturation rate and expedited the process of SE formation. It was also noted that 5-azaC influenced somatic embryogenesis during the second week of embryo induction, substantially enhancing the maturation rate of somatic embryos and the germination rate of Taxodium hybrid ‘zhongshanshan’. Furthermore, the analysis revealed that treatment with 5-azaC resulted in elevated levels of H₂O₂, SOD, POD, and AsA during the cotyledonary embryo period in Taxodium hybrid ‘zhongshanshan’, indicating its potential to promote somatic embryogenesis by regulating redox homeostasis. This study concluded that 5-azaC could improve the efficiency of somatic embryogenesis in Taxodium hybrid ‘zhongshanshan’, as well as provide a solid foundation for investigating the effects of 5-azaC on somatic embryogenesis in other conifer species. Full article

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. Full article