Search Results (98)

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

Embryos propagated from vegetative organs can maintain the excellent characteristics of the ortet tree and can make full use of the advantages of somatic embryogenesis technology in the large-scale clonal propagation of forest trees. However, in forest trees, a major obstacle to reproducing seedlings through somatic embryogenesis is the challenge of inducing somatic embryos using vegetative organs as explants. In this study, we have successfully developed a procedure to induce somatic embryogenesis (SE) in adult hybrid sweetgum trees for the first time. Leaves, petioles, and stem segments isolated from test-tube seedlings of three genotypes of hybrid sweetgum trees were used as explants to induce SE. The induction of SE was significantly influenced by genotype, explant type, and medium composition. The highest induction and proliferation efficiencies were achieved using a modified Blaydes’ medium supplemented with 1.0 mg/L 2,4-D and 0.5 mg/L 6-BA. Mature somatic embryos were obtained in media without plant growth regulators (PGRs). Among the three genotypes, only FX-12 failed to induce somatic embryos in all the explants. Petiole explants of FX-2 yielded 22 somatic embryos per gram. In FX-54, somatic embryos were induced from both leaf and petiole explants. The PGR concentration in the germination medium significantly affected the efficiency of somatic embryo germination, with the best germination results observed in modified Blaydes’ medium containing 0.5 mg/L 6-BA. This procedure resulted in over 60% of somatic embryos developing normally into plantlets. This study develops an SE system using vegetative organs as explants for the first time, providing technical support for large-scale asexual propagation and molecular breeding in hybrid sweetgum. Full article

For nearly three decades, interspecies somatic cell nuclear transfer (iSCNT) has been explored as a potential tool for cloning, regenerative medicine, and wildlife conservation. However, developmental inefficiencies remain a major challenge, largely due to persistent barriers in nucleocytoplasmic transport, mitonuclear communication, and epigenome crosstalk. This review synthesized peer-reviewed English articles from PubMed, Web of Science, and Scopus, spanning nearly three decades, using relevant keywords to explore the molecular mechanisms underlying iSCNT inefficiencies and potential improvement strategies. We highlight recent findings deepening the understanding of interspecies barriers in iSCNT, emphasizing their interconnected complexities, including the following: (1) nucleocytoplasmic incompatibility may disrupt nuclear pore complex (NPC) assembly and maturation, impairing the nuclear transport of essential transcription factors (TFs), embryonic genome activation (EGA), and nuclear reprogramming; (2) mitonuclear incompatibility could lead to nuclear and mitochondrial DNA (nDNA-mtDNA) mismatches, affecting electron transport chain (ETC) assembly, oxidative phosphorylation, and energy metabolism; (3) these interrelated incompatibilities can further influence epigenetic regulation, potentially leading to incomplete epigenetic reprogramming in iSCNT embryos. Addressing these challenges requires a multifaceted, species-specific approach that balances multiple incompatibilities rather than isolating a single factor. Gaining insight into the molecular interactions between the donor nucleus and recipient cytoplast, coupled with optimizing strategies tailored to specific pairings, could significantly enhance iSCNT efficiency, ultimately transforming experimental breakthroughs into real-world applications in reproductive biotechnology, regenerative medicine, and species conservation. Full article

Donkeys and mules have historically played an important role in agriculture and are now gaining recognition for their contributions to animal conservation, milk production, tourism, and equid-assisted services. However, their distinctive reproductive challenges pose obstacles to breeding management. As a result, the application of assisted reproductive technologies (ARTs) could help address these challenges, enhancing their roles in both traditional and emerging industries. This review examines the current and emerging in vitro techniques for breeding donkeys and mules. Key methodologies such as sperm cryopreservation, innovative sperm preservation technologies, embryo transfer, ovum pick-up (OPU), oocyte maturation, and vitrification are discussed, emphasizing their importance in optimizing ARTs. Advances in in vitro embryo production technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer (SCNT) are reviewed, with particular attention to its success in producing the first donkey and mule blastocysts or foals. Despite significant progress in the last decade, standardization of protocols for gamete conservation and embryo transfer are still required in long-ear equids. Advancing these technologies in combination with established in vitro embryo production could significantly improve reproductive outcomes and enhance the genetic management of donkey and mule populations. Full article

The nuclear factor Y (NF-Y) transcription factor is widely involved in various plant biological processes, such as embryogenesis, abscisic acid signaling, and abiotic stress responses. This study presents a comprehensive genome-wide identification and expression profile of transcription factors NF-YB and NF-YC in Pinus koraiensis. Eight NF-YB and seven NF-YC transcription factors were identified through bioinformatics analysis, including sequence alignment, phylogenetic tree construction, and conserved motif analysis. We evaluate the expression patterns of NF-YB/C genes in various tissues and somatic embryo maturation processes through the transcriptomics of ABA-treated tissues from multiple nutritional tissues, reproductive tissues, and somatic embryo maturation processes. The Leafy cotyledon1 (LEC1) gene belongs to the LEC1-type gene in the NF-YB family, numbered PkNF-YB7. In this study, we characterized the function of PkLEC1 during somatic embryonic development using genetic transformation techniques. The results indicate that PkNF-YB/C transcription factors are involved in the growth and development of nutritional tissues and reproductive organs, with specific high expression in PkNF-YB7 embryogenic callus, somatic embryos, zygotic embryos, and macropores. Most PkNF YB/C genes do not respond to ABA treatment during the maturation culture process. Compared with the absence of ABA, PkNF-YB8 was up-regulated in ABA treatment for one week (4.1 times) and two weeks (11.6 times). However, PkNF-YC5 was down-regulated in both one week (0.6 times) and two weeks (0.36 times) of culture, but the down-regulation trend was weakened in tissues treated with ABA (0.72–0.83 times). In addition, the promoter of PkNF YB/Cs was rich in elements that respond to various plant hormones, indicating their critical role in hormone pathways. The overexpression of PkLEC1 stimulated the generation of early somatic embryos from callus tissue with no potential for embryogenesis, enhancing the somatic embryogenesis ability of P. koraiensis callus tissue. Full article

Arabidopsis thaliana LEAFY COTYLEDON1 (LEC1) gene is shown to have numerous diverse functions in plant development, including the regulation of embryo morphogenesis and maturation, hypocotyl elongation, flowering transition, etc. However, the functions of LEC1 orthologs in different plant species have not been extensively studied. In this study, we obtained a line of Medicago truncatula, a model leguminous plant, carrying the loss-of-function mutation in the MtLEC1 (MtNF-YB10) gene, orthologous to LEC1, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas9) genome editing system. Edited plants with loss of MtNF-YB10 function did not demonstrate any severe abnormalities during their normal growth and gave viable seeds, but their capability for somatic embryogenesis in vitro was dramatically reduced. The T1 progeny of unedited plants with a Cas9-gRNA cassette insertion was also analyzed based on the suggestion that editing could occur during seed formation. However, no edited plants were found in the T1 generation. These results suggest divergent functions of LEC1 orthologs and make it possible to investigate potential specific MtNF-YB10 functions. Full article

Recombination-activating genes (RAGs) play a crucial role in the V(D)J recombination process and the development of immune cells. The development of the immune system and its mechanisms in pigs exhibit greater similarity to those of humans compared to other animals, thus rendering pigs a valuable tool for biomedical research. In this study, we utilized CRISPR/Cas9 gene editing and somatic cell nuclear transfer technology to generate RAG2 knockout (KO) pigs. Furthermore, we evaluated the impact of RAG2 KO on the immune organs and immune cell development through morphological observations, blood analysis and flow cytometry technology. RAG2 KO cell lines were used as donors for cloning. The reconstructed embryos were transplanted into 4 surrogate sows, and after 116 days of gestation, 2 sows gave birth to 12 live piglets, all of which were confirmed to be RAG2 KO. The thymus and spleen sizes of RAG2 KO pigs were significantly smaller than those of wild-type (WT) pigs. Hematoxylin-eosin staining results revealed that the thymus and spleen tissue structures of RAG2 KO pigs were disorganized and lacked the characteristic structures, indicating that RAG2 KO leads to dysplasia of the thymus and spleen. Hematological analysis demonstrated that the total number of white blood cells and lymphocytes in the circulation of RAG2 KO pigs was significantly lower, while the number of eosinophils was higher. Flow cytometry results indicated that the proportions of mature T and B lymphocytes were significantly reduced compared to WT pigs. These findings successfully verified the immunodeficiency phenotype of RAG2 KO pigs. This study may provide experimental animals for the development of tumor models and humanized animals. Full article

Larix principis-rupprechtii Mayr, a coniferous species indigenous to Northern China, possesses significant ecological and economic value. Somatic embryogenesis offers a pathway with significant potential for large-scale propagation, long-term germplasm conservation, and genetic transformation in L. principis-rupprechtii Mayr. However, it remains unclear whether significant variations occur in embryogenic tissues during long-term successive subculturing, which could impact the productivity of somatic embryos. This is a pivotal concern that lacks comprehensive understanding. In this study, three embryogenic cell lines were used to explore the dynamics and relationships among proliferation rate, pre-treatment proliferation rate, and embryogenic capabilities across a series of 32 subculturing cycles. Proliferation rate, pre-treatment proliferation rate, and somatic embryo maturation rate showed no significant correlation with subculturing cycles. However, there was a positive correlation between subculturing cycles and pre-treatment proliferation rate and a negative correlation with somatic embryo maturation rate in the BFU1 cell line. In addition, we utilized ten SSR molecular markers to investigate the genetic stability in embryogenic tissues during long-term subculturing. No genomic variations were detected in any of the three embryogenic cell lines, which suggests that the observed phenotypic dynamics during subculturing may not be primarily driven by genomic alterations. This study provides novel insights into the dynamics of the long-term culture of embryogenic tissues, laying a foundation for the optimization and application of somatic embryogenesis techniques in L. principis-rupprechtii Mayr and potentially other coniferous species. Full article

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with β-naphthoxyacetic acid (5 μM) and 6-benzylaminopurine (BAP—9.0 μM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar. Full article