Search Results (414)

To overcome the seasonal constraints of explant availability and facilitate genetic improvement in Catalpa ovata, this study established a dual-pathway in vitro regeneration system (encompassing adventitious shoot organogenesis and somatic embryogenesis) using mature zygotic embryos. We systematically evaluated the synergistic effects of maternal genotypes, plant growth regulators (PGRs), basal media, and the histone deacetylase inhibitor Trichostatin A (TSA). Genotype screening revealed significant divergence in regenerative potential, with the half-sib family 32F17 exhibiting superior responsiveness (84.7% callus induction). A high cytokinin-to-auxin ratio (ZA3 medium) optimally drove direct shoot organogenesis. For adventitious shoot proliferation, the addition of TDZ significantly improved the multiplication coefficient (up to 2.99 on ZB4 medium), although a physiological trade-off with shoot elongation was observed. In parallel, the application of 10 µM TSA significantly enhanced somatic embryogenesis from embryogenic calli, effectively alleviating the inhibitory constraints of exogenous PGRs. For rhizogenesis, the DKW basal medium proved superior to half-strength MS, with the ZE3 treatment (0.1 mg·L⁻¹ NAA + 0.1 mg·L⁻¹ IBA) yielding the highest rooting frequency (69.6%) and robust root architecture. Notably, while somatic embryo conversion remained recalcitrant, plantlets derived exclusively from the adventitious shoot organogenesis pathway were successfully acclimatized ex vitro. These transplanted plantlets exhibited consistently high survival rates (83.1–84.4%) across all tested genotypes, effectively overcoming the initial genotype-dependent recalcitrance. Collectively, this optimized protocol provides a reliable technical platform for the large-scale clonal propagation and biotechnological breeding of C. ovata. Full article

The walnut cultivar ‘Yunxin No. 14’ is an early fruiting, high-yielding, and widely adaptable fruit tree with compact growth and superior nuts. Establishing a successful tissue culture system for this cultivar is crucial for its rapid clonal propagation and as a foundation for future genetic transformation. Using young fruits as explants, 3% NaClO sterilization for 20 min effectively controlled contamination and browning. Somatic embryos induced from zygotic embryos cultured on DKW medium with 30 g·L⁻¹ sucrose showed high proliferation and minimal browning. After a 4-day dehydration treatment using saturated NH₄NO₃, mature somatic embryos germinated rapidly on differentiation medium (DKW containing 1 mg·L⁻¹ 6-BA and 0.1 mg·L⁻¹ IBA), reaching 90.0% germination. Optimal shoot multiplication was achieved on DKW medium supplemented with 2 mg·L⁻¹ 6-BA and 0.3 mg·L⁻¹ IBA, yielding a proliferation rate of 91.1% and a proliferation index of 3.1. For rooting, shoots (~3 cm) treated with Clonex^® rooting gel were transferred to a low-cost, sugar-free vermiculite medium with gaseous CO₂ as the sole carbon source. Root initiation occurred within two weeks at a rate of 54.2%, significantly shortening the rooting phase. Rooted plantlets were acclimatized in a peat:perlite:vermiculite (2:2:1, v/v/v) mixture under high humidity for two weeks before outdoor transfer, achieving an 88.6% survival rate. This study provides a reliable protocol for the micropropagation of ‘Yunxin No. 14’ and a valuable reference for other difficult-to-root woody species. Full article

Embryonic stem cells represent a valuable germplasm resource with significant implications for breed conservation, development, and utilization. However, the scarcity of genetic resources in endangered species poses a fundamental constraint on obtaining gametes for embryonic stem cell derivation. Therefore, generating embryonic stem cells from somatic cell nuclear transfer blastocysts offers an optimal alternative for conservation cloning. In this study, we established ApèiJiaza somatic cell nuclear transfer ESCs (APNT-ESCs) from cloned embryos, using ApèiJiaza cattle ear fibroblasts as nuclear donors. APNT-ESCs could be passaged for over 30 generations in vitro, exhibiting high expression of key pluripotency markers, genomic stability, and the ability to form embryoid bodies and differentiate into cell types of all three germ layers. This research established an effective biotechnological framework for the genetic conservation of other endangered species lacking accessible gametes. Full article

Anther-derived somatic embryogenesis is a valuable approach in rubber tree (Hevea brasiliensis) breeding; however, its effectiveness is highly influenced by the developmental stage of the microspores. The present investigation focused on male flower buds of the cultivar Reyan 73397 at successive developmental stages to examine the relationship between visible bud characteristics and internal microspore development, assess how microspore developmental stage affects callus induction and somatic embryo formation, and identify the stage with the greatest embryogenic potential. Cytological observations distinguished six well-defined phases of microspore development, spanning from microspore mother cells to fully mature pollen grains, each reliably linked to particular bud diameters, coloration, and anther morphology. Anthers corresponding to each developmental phase were cultured in vitro, and their ability to initiate callus and produce somatic embryos was systematically evaluated. Anthers containing uninucleate microspores exhibited the highest rates of both callus formation and somatic embryogenesis, with the early-uninucleate stage showing the strongest response. This stage consistently matched flower buds measuring 1.42–1.57 mm in transverse diameter and displaying a green to yellowish-green appearance. In contrast, anthers collected at the microspore mother cell and tetrad stages did not produce embryogenic responses. Histological evidence has indicated that both callus and somatic embryos originate from diploid somatic tissues of the anther wall, particularly connective parenchyma cells, rather than from microspores themselves. Based on these findings, a rapid, non-destructive selection method integrating bud diameter, bud color, and sieve-based size separation was developed to identify responsive explants efficiently. Overall, this study defines the optimal developmental window for anther culture in rubber trees, verifies the somatic origin of embryogenic tissues, and provides a practical morphological and cytological basis for improving anther culture efficiency in rubber tree breeding programs. Full article

Papaya (Carica papaya L.) is a highly cross-pollinated crop that exhibits considerable genetic variability when propagated through seeds, resulting in non-true-to-type progeny. Therefore, the development of an efficient in vitro regeneration system is essential for large-scale clonal propagation of elite cultivars. In the present study, a highly efficient and reproducible somatic embryogenesis protocol was developed for C. papaya var. TNAU Papaya CO 8 using immature zygotic embryos as explants. This study provides the first comprehensive comparative evaluation of three basal media, viz., Murashige and Skoog Medium, N6 Medium, and Woody Plant Medium, for somatic embryogenesis and plant regeneration in this variety, along with the optimization of polyamine-enriched media for enhanced plantlet recovery. The embryogenic potential of explants was assessed across different stages, including callus induction, somatic embryo development, plant regeneration, shoot elongation, rooting, and acclimatization. Maximum callus induction (81.96%) was observed on half-strength MS medium supplemented with 2,4-Dichlorophenoxyacetic acid under dark conditions, followed by ½ N6 (63.00%) and ½ WPM (58.02%). Somatic embryo initiation was highest on ½ MS medium containing 2.0 mgL⁻¹ 2,4-D (77.82%). Somatic embryos developed through distinct globular, heart, torpedo, and cotyledonary stages. Embryo maturation was significantly enhanced on MS medium supplemented with abscisic acid, polyethylene glycol, benzylaminopurine, and proline. The highest plantlet regeneration (85.02%) was achieved on MS medium enriched with putrescine, whereas comparatively lower regeneration was recorded on N6 (75.99%) and WPM (57.97%). Shoot elongation was significantly improved by supplementation with gibberellic acid (1.0 mgL⁻¹). Root induction was optimal on half-strength MS medium containing Indole-3-butyric acid, 1-Naphthaleneacetic acid, phloroglucinol, and activated charcoal, resulting in well-developed roots. Regenerated plantlets were successfully acclimatized in a cocopeat–vermicompost substrate with a survival rate of 74.01%. The optimized protocol provides a reliable and efficient system for large-scale clonal propagation and offers promising applications in genetic transformation and commercial production of papaya var. TNAU papaya CO 8. Full article

The use of seven biomaterials was examined in the study: polyethylene (PE), polypropylene (PP), polyurethane (PU), teflon (PTFE), polycaprolactone (PCL), polylactide (PLLA), and a copolymer of poly L-lactide and dibutyryl chitin (PLLA/DBC). The use of these materials has not been discussed in the context of animal reproduction so far. Due to the specific nature of the reproductive system and the high sensitivity of reproductive cells, at the outset the biocompatibility and cytotoxicity of the materials were tested in somatic cell and embryo cultures. Additionally, the material properties of the catheters were determined in terms of the roughness of the internal and external surfaces, the stability of the shape of the catheters, their elasticity, durability, and ductility. Finally, clinical testing of the developed catheters was performed in laparoscopic transplantation of embryos into the uterine tubes of the sows. Significant toxicity of PTFE, PCL and PLLA/DBC towards the embryos was indicated in the cytotoxicity testing. In the materials testing, numerous flaws of the PP, PLLA, and PLLA/DBC catheters were indicated. In the final stage, catheters were developed using PE and PU. In clinical testing, these latter catheters exhibited high effectiveness in transferring embryos. Among the seven biomaterials tested, only polyethylene and polyurethane exhibited high biocompatibility and the material properties mentioned above. There is thus good indication for the introduction of these catheters for embryo transfer in animal reproduction biotechnology. Full article

Genetic engineering tools have the potential to rapidly and precisely improve the genome of slow-to-breed cacao. We previously developed an efficient protocol for transforming cacao using cotyledonary explants derived from secondary somatic embryos via Agrobacterium tumefaciens. In this study, we demonstrate that our transformation protocol is successful in elite cultivars, INIAPG-038 and Matina 1-6, producing fertile seeds with stable visual marker inheritance regardless of whether the transgenic plants were used as the pollen or ovule donor. Three vectors were used in the transformations, each containing genes for enhanced yellow fluorescent protein (eyfp) and neomycin phosphotransferase II (nptII). Three transgenic INIAPG-038 events and one transgenic Matina 1-6 event were used to evaluate seed fertility and the stability of transgene inheritance in cacao seeds and plants. The T₁ progeny of these four transgenic events were analyzed for YFP expression and transgene presence. YFP expression segregated at a 1:1 ratio in all events when the transgenic plants were crossed with non-transgenic plants, while a 3:1 segregation was observed when transgenic events were crossed with each other. The transgenic plants exhibited a normal phenotype compared to non-transgenic control plants, producing seeds with a 97% germination rate. Full article

Based on prior transcriptome data, we established a core gene interaction network for Korean pine somatic embryo maturation and screened 18 core genes. These genes showed distinct differential expression in early somatic embryogenesis. In particular, PkGLP1-2-6 (Pkor04G01180) and PkGLP-1-2-21 (Pkor04G01200) were highly correlated in the network and can be regarded as key genes mediating Korean pine somatic embryo maturation. A total of 92 members of the PkGLP gene family were identified in the Korean pine genome, which can be classified into 8 subfamilies based on evolutionary relationships. Both PkGLP1-2-6 and PkGLP1-2-21 were localized in the cell membrane and nucleus. By means of a stable genetic transformation system, transgenic Korean pine calli overexpressing PkGLP1-2-6 and PkGLP1-2-21 were successfully established. The results demonstrated that the overexpression of PkGLP1-2-6 and PkGLP1-2-21 could effectively promote somatic embryogenesis and enhance the yield of somatic embryos. In the presence of exogenous abscisic acid (ABA), the somatic embryo yield of the transgenic lines was significantly higher than that of the wild-type controls. Compared with the wild-type controls, the SOD activity in the cell lines overexpressing PkGLP1-2-6 and PkGLP1-2-21 was significantly increased, whereas the activities of POD and CAT were decreased, and the contents of H₂O₂ and superoxide anion (O₂⁻) were significantly reduced. These results indicate that PkGLP1-2-6 and PkGLP1-2-21 are actively involved in the reactive oxygen species (ROS) scavenging process during somatic embryogenesis of Korean pine. The overexpression of PkGLP1-2-6 and PkGLP1-2-21 contributes to enhancing the antioxidant capacity of cells, thereby increasing the yield of somatic embryos. Full article

Somatic embryogenesis (SE) represents the most efficient and scalable technology for the mass clonal propagation and genetic improvement of superior conifer genotypes, which is crucial for meeting global wood demand and supporting forest adaptation to climate change. Despite its immense potential, SE in the genus Pinus still faces major limitations, including low initiation frequencies, restricted explant availability, and pronounced genotype dependence. This review synthesizes current knowledge on the factors influencing SE in Pinus species, with a specific focus on two ecologically vital Tertiary relicts endemic to the Balkan Peninsula: Pinus peuce (Macedonian pine) and Pinus heldreichii (Bosnian pine). For these species, traditional vegetative propagation methods are difficult or ineffective, making SE the priority approach for clonal propagation. Detailed studies on these species revealed that SE induction is highly dependent on the explant type and developmental stage. Successful embryogenic tissue formation was achieved only from whole megagametophytes containing immature zygotic embryos, within a narrow developmental window spanning 4–10 weeks post-fertilization. Furthermore, medium composition, particularly reduced ammonium concentration, proved critical for P. heldreichii success. These findings underscore the need for continued, species-specific optimization to overcome current bottlenecks and realize the full potential of SE for the conservation and sustainable clonal forestry of these high-value pines. Full article

Pig somatic cell nuclear transfer (SCNT) has valuable applications in agriculture, biomedicine, and life sciences, yet low cloning efficiency remains a major constraint limiting its application. To systematically investigate factors related to the production efficiency of pig cloning, this study conducted a retrospective analysis of 367,701 SCNT embryos transferred into 2019 surrogate sows over five years, focusing on breeds of donor cells, the season of embryo transfers, and the number of embryos transferred per surrogate. Our data demonstrate that the genetic background of donor cells is a critical determinant. SCNT embryos generated by wild-type (WT) Pietrain and Duroc pigs yielded significantly higher cloning efficiencies compared to those from Large White and Yorkshire pigs. This breed-specific influence was also observed with genetically modified (GM) donor cells. Nevertheless, within the GM groups, GM-Duroc and GM-Yorkshire showed superior efficiency compared to GM-Large White and GM-Bama. Furthermore, Summer was identified as the least favorable season for embryo transfer, with significantly lower pregnancy rates, delivery rates, and cloning efficiency compared to the other seasons. Importantly, we established that transferring 100–150 embryos per recipient optimized cloning efficiency, significantly outperforming groups receiving higher embryo numbers without compromising pregnancy rates, delivery rates, or average litter sizes. Our findings provide valuable guidance for optimizing large-scale SCNT protocols in swine. Full article

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

Clubroot disease, caused by Plasmodiophora brassicae, is a major global threat, causing severe yield losses of up to 100% in heavily infested fields. Interspecific hybridization is essential for the transfer of clubroot resistance genes among the Brassica species. This review aimed to describe the sources of clubroot resistance, categorize their types in Brassica crops, and identify the most effective techniques and underutilized sources for both intergeneric and interspecific hybridization. A systematic literature review served as the foundation for expert analysis, encompassing a comprehensive list of known sources of resistance and a detailed description of their characteristics, including monogenic, polygenic, dominant, and recessive traits. In addition, this review specifies techniques suitable for gene transfer, such as markers, embryo rescue, somatic hybridization, and CRISPR/Cas. Based on the literature, underutilized directions for genetic crosses have been proposed. These conclusions suggest that combining biotechnological methods, including markers, CRISPR/Cas, and embryo rescue, with intergeneric crosses offers the potential to transfer resistance genes from previously untapped sources. Full article

In Korea, a persistent shortage of Japanese larch (Larix kaempferi) seeds and the high costs of managing seed orchards have created a significant demand for alternative reforestation methods. This pilot study, conducted over nine years, evaluated the field performance of somatic embryo-derived larch seedlings (emblings) across 14.4 hectares in nine different locations. The study addressed challenges with SE technology, such as limited genetic diversity and the inconsistent quality of seedlings due to year-round production. Despite these initial issues and other environmental interferences, the statistical analysis revealed age to be the sole significant fixed factor driving tree growth and root collar diameter (RCD) increase (p < 0.001 for both). Crucially, the growth rate (slope) for height and RCD was not statistically different between the embling and seed-derived groups (seedlings). Furthermore, the GLMM for survival confirmed that age was not a significant predictor (p > 0.35 for both types). Instead, site-specific factors were the primary drivers of overall survival and growth variation. The random effects analysis showed that site heterogeneity was substantial for height (${\mathit{\sigma }}_{\mathrm{Site}}=0.8256$, indicating that somatic embryo-derived larch plantlets were more sensitive to site-specific environmental conditions than seed-derived seedlings (${\mathit{\sigma }}^2$ was 1.078 for embling survival and 0.4074 for seedling survival). We also found no significant difference in overall tree form or evidence that emblings developed dominant side branches. This research demonstrates that SE technology can produce high-quality larch emblings that are statistically equivalent to their seedling counterparts in long-term growth trajectory and RCD development. It confirms that this method offers a viable and cost-effective solution to Korea’s seed shortage without sacrificing long-term growth or survival. Full article

In vitro germplasm conservation provides an alternative method for preserving plant species that are vulnerable to natural hazards or for which in situ conservation is costly and challenging to manage. This review examines the significance and challenges associated with various in vitro conservation methods. It also provides an overview of the current advances in cryopreservation technology for oil palm. In vitro conservation approaches include two strategies: medium-term conservation, in which plants are maintained through the slow growth of explants, facilitated by the gradual release of nutrients, and low-temperature storage. The second approach involves long-term preservation via cryopreservation in liquid nitrogen. Cryopreservation enables the storage of pollen, calli, somatic embryos, and zygotic embryos. Significant progress has been made in cryopreservation, which was initially limited to cold-tolerant species. New techniques focus on conserving sensitive species, such as oil palm, through rapid dehydration and vitrification procedures using various plant materials, particularly polyembryoids and zygotic embryos. Additionally, hardening of plant material is to be induced through pre-culture techniques to enhance their survival under osmotic stress and ultralow temperature. The mechanisms underlying the adaptability of various plant materials, i.e., somatic embryoids and zygotic embryos under cryopreservation, need to be understood. Full article

Somatic embryogenesis (SE) is a morphogenetic pathway widely employed in the commercial micropropagation of plants. This route enables the generation of somatic embryos from somatic tissues, which give rise to complete (bipolar) plants that develop like zygotic embryos. SE can proceed via direct or indirect pathways, and both approaches have been adapted not only for large-scale clonal propagation but also for the regeneration of genetically modified plants. In this context, SE can be harnessed as a versatile platform for recombinant protein production, including vaccine antigens and therapeutic proteins, by combining plant tissue culture with genetic transformation strategies. Successful examples include non-model plants, as Daucus carota and Eleutherococcus senticosus expressing the cholera and heat-labile enterotoxin B subunits, respectively; Oryza sativa, Nicotiana tabacum, and Medicago sativa producing complex proteins such as human serum albumin (HSA), α₁-antitrypsin (AAT), and monoclonal antibodies. However, challenges remain in optimizing transformation efficiency, scaling up bioreactor-based suspension cultures, and ensuring proper post-translational modifications under Good Manufacturing Practice (GMP) standards. Recent advances in synthetic biology, modular vector design, and glycoengineering have begun to address these limitations, improving control over transcriptional regulation and protein quality. This review highlights the application of SE as a biotechnological route for recombinant protein production, discusses current challenges, and presents innovative strategies and perspectives for the development of sustainable plant-derived biopharmaceutical systems. Full article