Somatic embryogenesis is one of the key ways to achieve asexual reproduction of plants under in vitro culture conditions. The process undergoes somatic cell, treated by the exogenous hormone, reprogramming to form a zygote-like structure that eventually develops into a complete plant. As an efficient plant regeneration technology, somatic embryogenesis has the potential to be applied in plant Agrobacterium-mediated genetic transformation and CRISPR-based genome editing technologies, and is of great value in genetic improvement of plants. Recent advances have revealed that somatic embryogenesis is governed by a hierarchical molecular network involving multiple regulatory tiers. In this review, we review the recent research progress on the molecular regulatory mechanisms of somatic embryogenesis in plants, focusing on the key transcription factors, such as WOX (WUSCHEL-RELATED HOMEOBOX) gene family, AP2/ERF (APETALA2/ETHYLENE-RESPONSIVE FACTOR) and MADS-box gene family, as well as the related signaling pathways in which they are involved in. The members of WOX gene family, particularly WUS and WOX2/5/8/9, play a central role in maintaining stem cell pluripotency and establishing polarity during the development of embryos. The LAFL network (LEC1-ABI3-FUS3-LEC2), integrates hormonal signals, which regulate auxin biosynthesis (YUCs and TAA1) and the balance of endogenous ABA/GA, and epigenetic signals, which modified epigenetically, to regulate somatic embryogenesis. AP2/ERF superfamily members, notably BABY BOOM and PLETHORA 5(PLT5), are essential for somatic cells to acquire totipotency. Members of the MADS-box gene family, including AGL15 and AGL18, regulate somatic embryogenesis mainly by modulating GA-related pathways. These transcription factors play crucial roles in somatic embryogenesis. This paper delves into the broad application prospects of somatic embryogenesis in plant breeding and other biotechnology field. With the deepening of research, this technology will occupy a central position in plant biotechnology and agricultural development.
【Objective】This study aims to elucidate the expression patterns and functional roles of the auxin efflux carrier gene PIN1 during somatic embryogenesis in Liriodendron chinense.【Method】The temporal expression profiles of three homologous genes, LcPIN1a, LcPIN1b, and LcPIN1c, during somatic embryogenesis were analyzed using quantitative real-time PCR (qRT-PCR) at key developmental stages, including the globular, heart-shaped, torpedo-shaped, and cotyledon embryo stages. To investigate spatial expression, about 3.5 kb upstream promoter regions of each LcPIN1 gene were cloned and fused to the β-glucuronidase (GUS) reporter gene and the mCherry fluorescent protein reporter gene. The recombinant constructs were introduced into embryogenic callus of Liriodendron hybrid via Agrobacterium tumefaciens-mediated transformation. Transgenic calli were selected and induced to undergo somatic embryogenesis, followed by histochemical GUS staining and fluorescence microscopy to visualize the spatiotemporal expression patterns. In parallel, the full-length coding sequences of LcPIN1a, LcPIN1b and LcPIN1c were cloned into overexpression vectors driven by the constitutive Cauliflower Mosaic Virus 35S (CaMV35S) promoter and similarly transformed into embryogenic callus of Liriodendron hybrid. The effects of overexpression on somatic embryo formation were assessed by calculating the somatic embryogenesis efficiency, defined as the number of somatic embryos produced by a certain amount of embryogenic callus, and comparing the data statistically across independent transgenic lines.【Result】The qRT-PCR data revealed that all three LcPIN1 homologs were dynamically expressed during somatic embryogenesis, displaying both temporal overlap and distinct peaks at specific developmental stages. GUS staining and mCherry fluorescence assays provided high-resolution insights into their spatial expression patterns. During the globular embryo stage, LcPIN1a and LcPIN1c exhibited pronounced polar localization, with signals concentrated at one pole of the embryo, suggesting their early involvement in establishing embryonic polarity and the apical-basal axis. By contrast, LcPIN1b showed weak and diffuse expression at this stage. At the heart-shaped embryo stage, clear expression divergence was observed: LcPIN1a was predominantly expressed in the developing vascular tissues; LcPIN1b exhibited uniform expression throughout the embryo body; and LcPIN1c was specifically localized in the shoot apical meristem region and the nascent cotyledon primordia. As the embryos progressed into the torpedo-shaped and cotyledon stages, the three homologs displayed convergent expression patterns, with signals extending continuously from the cotyledon tips along the vascular strands down to the embryonic root tip. This suggests that LcPIN1a, LcPIN1b and LcPIN1c may coordinately maintain the auxin gradient required for proper somatic embryo elongation and differentiation during late embryogenesis. Overexpression experiments demonstrated that ectopic expression of each LcPIN1 homolog significantly inhibited somatic embryogenesis. Transgenic calli overexpressing LcPIN1a, LcPIN1b or LcPIN1c exhibited reduced somatic embryogenesis efficiency compared to wild-type controls, with a negative correlation between transgene expression levels and embryogenic potential. Notably, transgenic lines with higher levels of LcPIN1 overexpression showed a pronounced decline in somatic embryo formation, indicating that excessive auxin efflux may disrupt the finely tuned auxin gradients required for embryogenic competence and proper morphological development.【Conclusion】This study provides comprehensive evidence that the spatiotemporal precision of PIN1-mediated auxin transport plays a pivotal role in somatic embryogenesis of L. chinense. The redundant yet specialized expression patterns of LcPIN1a, LcPIN1b and LcPIN1c highlight their coordinated function in regulating auxin distribution at distinct developmental stages and tissue sites. Overexpression of LcPIN1 homologs perturbs auxin homeostasis, leading to impaired somatic embryogenesis. These findings advance our understanding of the molecular mechanisms underlying somatic embryogenesis in woody species and underscore the importance of auxin transport regulation for optimizing plant regeneration systems. This work also lays a foundation for future studies on functional diversification of PIN proteins and their potential applications in improving somatic embryogenesis efficiency in forest tree breeding.
【Objective】This study aims to elucidate the effects of exogenousabscisic acid(ABA) combined with low-temperature pretreatment on the maturation of Pinus koraiensis somatic embryos, unravel the mechanisms regulating embryogenic cell differentiation and metabolite accumulation, and identify the optimal culture conditions.【Method】Using P. koraiensis embryogenic callus as the material, four cold durations (0, 2, 4 and 6 days) at 4 ℃ were combined with six ABA concentrations (0, 5, 10, 20, 50, 100 mg/L) medium. The activity of nutrients and related physiological and biochemical indicators the callus were measured, and the effects of different treatment combinations on somatic embryo maturation were systematically evaluated.【Result】(1) 20 mg/L ABA with two days cold pretreatment increased somatic embryo yield 9.90 times versus control. (2) In this treatment group, the contents of storage substances reached the highest levels, and activities of peroxidase (POD) and catalase (CAT) were also at their peak, reaching 59.45 and 98.96 U/g, respectively. Meanwhile, H2O2 content was the lowest at 0.53 μmol/mg, indicating that the antioxidant system effectively mitigated oxidative damage. (3) Somatic embryos obtained under optimized culture conditions exhibited a regeneration rate of 45% after germination and successful transplantation.【Conclusion】The combination of 20 mg/L exogenous ABA with low-temperature pretreatment at 4 ℃ for two days was identified as the optimal condition for somatic embryo induction and maturation in P. koraiensis. This study provides technical support for the efficient propagation of elite P. koraiensis varieties, contributing to the effective conservation of genetic resources, the selection of superior cultivars, and the development of a rapid propagation system.
【Objective】This study aims to develop a complete somatic embryogenesis system for Tilia cordata by systematically optimizing the culture medium composition to identify optimal conditions for somatic embryo induction and subsequent plant regeneration.【Method】Immature zygotic embryos of T. cordata were used as explants. The effects of different concentrations and combinations of plant growth regulators 2,4-dichlorophenoxyacetic acid(2,4-D and 6-benzylaminopurine(6-BA) on callus induction, proliferation, and somatic embryo induction efficiency were systematically investigated. Additionally, the impacts of different basal media, indole-3-butyric acid(IBA) concentrations, and activated charcoal supplementation on somatic embryo germination and plant regeneration were evaluated.【Result】Optimal callus induction was achieved at a concentration of 3.0 mg/L 2,4-D combined with 0.2 mg/L 6-BA, yielding an induction efficiency of 64.25%. Subsequent proliferation of embryogenic callus demonstrated maximal growth potential under identical hormonal conditions (3.0 mg/L 2,4-D+0.2 mg/L 6-BA), as evidenced by an exceptional proliferation coefficient of 1 063.72%. For somatic embryo induction, the same hormonal formulation (3.0 mg/L 2,4-D+0.2 mg/L 6-BA) proved most effective, generating somatic embryos at a frequency of 46.41%. Complete plant regeneration was successfully accomplished through transfering of developed somatic embryos to MS basal medium supplemented with 0.5 mg/L IBA in the absence of activated charcoal, with all regenerated plants exhibiting normal morphological characteristics.【Conclusion】In this study, a complete somatic embryogenesis system of T. cordata was initially established, providing a basis for subsequent establishment of an efficient propagation and genetic transformation system for Tilia.
【Objective】To systematically investigate the impact of 6-(2-hydroxy-3-methylbenzylamino) purine (PI-55) on the development of embryogenic callus tissues in European spruce (Picea abies), this study examines both its role during extended periods of callus proliferation and its influence on the subsequent stages of somatic embryo differentiation. By analyzing the morphogenetic responses, cellular proliferation rates, and molecular regulatory mechanisms triggered by PI-55 treatment, this research aims to elucidate the compound’s potential to modulate key developmental processes in coniferous embryogenesis. The findings are anticipated to provide critical insights into optimizing somatic embryogenesis protocols for P. abies, offering a robust scientific foundation for enhancing the efficiency and reproducibility of large-scale plant regeneration techniques in this ecologically and economically important conifer species.【Method】Embryogenic callus tissue derived from mature zygotic embryos of the P. abies genotype 33 was selected as the experimental substrate to investigate the impact of 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) on callus proliferation and somatic embryo development. During the proliferation phase, callus cultures were supplemented with varying concentrations of PI-55 [0 (control), 0.5, 1.0 and 2.0 μmol/L] and maintained under controlled conditions for a duration of 14 days. Subsequent analyses involved comprehensive assessments of physiological and biochemical parameters, including endogenous phytohormone profiles (indole-3-acetic acid [IAA], cytokinins (CTK), zeatin riboside (ZR), and abscisic acid (ABA), antioxidant enzyme activities (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), soluble protein content, and somatic embryo maturation efficiency. These metrics were systematically quantified and compared across treatment groups to elucidate the concentration-dependent effects of PI-55 on callus tissue viability, hormonal balance, oxidative stress tolerance and embryogenic potential.【Result】The application of 0.5-1.0 μmol/L PI-55 has been shown to significantly enhance the differentiation rate of European spruce embryogenic callus. This effect is mediated through PI-55 ability to elevate endogenous hormone levels, antioxidant enzyme activities, and soluble protein content in the embryogenic callus. Notably, comparative analysis revealed that cell lines with weaker somatic differentiation capacity exhibited higher concentrations of cytokinins (CTK) and zeatin riboside (ZR), as well as greater peroxidase (POD) and catalase (CAT) activities compared to those with stronger differentiation potential. These findings suggest that while a certain threshold of endogenous substances is required to promote somatic embryogenesis, supraoptimal levels may conversely inhibit the differentiation process.【Conclusion】These insights provide a robust scientific framework for improving somatic embryogenesis protocols in European spruce. The elucidation of PI-55 regulatory role, in particular, offers valuable opportunities for developing enhanced tissue culture systems capable of large-scale propagation of superior genotypes. Such advancements will significantly contribute to modern breeding initiatives focused on producing improved spruce varieties, thereby meeting both ecological conservation objectives and the growing commercial requirements of the forestry and timber sectors. The potential applications of these findings extend to addressing current challenges in sustainable forest management and wood production.
