Search Results (429)

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

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

Members of the Rosaceae family possess substantial economic and ornamental value, making their effective propagation and genetic improvement critical. Plant regeneration represents a foundational technology for efficient breeding, genetic transformation, functional genomics, molecular breeding, germplasm conservation, and large-scale commercial propagation. The regenerative capacity of explants in many Rosaceae taxa remains limited, despite significant progress. This review systematically synthesized conventional and emerging plant regeneration strategies and critically examined the principal biological and technical constraints affecting regenerative efficiency. A comprehensive comparison was first made among the various genera of the Rosaceae family regarding regeneration processes, environmental conditions, PGRs, exogenous additives, basal media, common obstacles and regeneration suggestions. The application of molecular biotechnology approaches in elucidating the mechanisms underlying regeneration and in enhancing regeneration capacity is also evaluated. Finally, this review assesses the future potential of these advanced technologies for improving regeneration systems in Rosaceae plants, providing a comprehensive reference framework for both academic research and industrial applications. 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

Efficient in vitro regeneration remains a major constraint in the genetic transformation, genome editing, and molecular breeding of wheat (Triticum aestivum L.), largely due to strong genotype-dependent recalcitrance and limited activation of developmental programs required for somatic embryogenesis. Plant regeneration relies on extensive transcriptional reprogramming and epigenetic remodeling orchestrated by morphogenic regulators that modulate meristem identity, as well as cellular pluri- and totipotency. In this review, we synthesize current molecular knowledge on key transcription factors (BBM, WUS/WUS2, GRF-GIF, WOX, LAX1, SERK, WIND1/ERF115) and signaling peptides (CLE/CLV-WUS module, phytosulfokine/PSK) that regulate embryogenic competence in monocot cereals, with emphasis on their orthologs and functional relevance in wheat. We highlight how controlled expression of these morphogenic genes, promoter engineering, and transient or excisable induction systems can significantly enhance regeneration capacity, reduce chimerism in CRISPR-Cas-edited plants, and facilitate genotype-independent transformation. We also discuss epigenetic and metabolic constraints underlying wheat recalcitrance and their potential modulation to improve culture responsiveness. By integrating evidence from wheat, rice, maize, and barley, we outline conserved gene-regulatory networks that reinitiate totipotency and propose strategies to accelerate doubled haploid production and speed-breeding pipelines. Collectively, morphogenic factors emerge as central molecular tools for overcoming regeneration bottlenecks and enabling next-generation wheat improvement. The objective of this review is to synthesize and critically evaluate current molecular knowledge on morphogenic regulators controlling in vitro regeneration in wheat (Triticum aestivum L.), with particular emphasis on their roles in genetic transformation and genome editing. Full article

Somatic embryogenesis (SE) plays a pivotal role in the propagation and genetic improvement of coniferous trees; however, its efficiency is frequently limited by the reduced embryogenic potential of callus cultures. Here, we investigated the metabolic determinants underlying this phenomenon in Picea mongolica by conducting a comparative metabolomic analysis of embryogenic calli (EC) and non-embryogenic calli (NEC). We observed significant metabolic differences between EC and NEC using an integrated approach combining morphological observations and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. EC exhibited increased central carbon metabolism, characterized by enhanced citrate cycle (TCA) flux, with significantly increased levels of the key TCA intermediates, citric acid and L-malic acid—18.8- and 3.6-fold higher, respectively, than those in NEC. Conversely, NEC displayed a divergent metabolic state, characterized by the accumulation of various amino acids and the activation of secondary metabolic pathways, especially alkaloid biosynthesis. These results indicate that embryogenic competence in P. mongolica is supported by a distinct metabolic program that prioritizes energy generation and efficient carbon-nitrogen allocation for biosynthetic processes. Conversely, the non-embryogenic state arises from a shift in metabolic resources toward secondary metabolism. These findings provide key metabolic insights and a theoretical basis for enhancing conifer SE systems. Full article

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is a globally destructive threat to coniferous forests, causing severe ecological and economic losses. Conventional resistance breeding is critically hampered by long life cycles of trees and field evaluation challenges. To address these limitations, we developed a three-tier biotechnology pipeline with a dual-output goal (generating both resistant germplasm and mechanistic insights) designed to bridge the in vitro–field gap. This strategy is founded upon the resolution of a longstanding pathogenesis debate, which established aseptic PWNs as a standardized research tool. The pipeline integrates high-throughput in vitro cellular screening (Tier 1), whole-plant validation via organogenesis (Tier 2), and scaled production coupled with mechanistic investigation through somatic embryogenesis (Tier 3). Tier 1 enables rapid phenotypic screening, Tier 2 validates resistance in whole plants, and Tier 3 facilitates mass production and in-depth study. It operates as a closed-loop, knowledge-driven system, simultaneously accelerating PWN-resistant germplasm development and empowering molecular mechanism discovery. Validated across Pinus massoniana and P. densiflora, this work provides a concrete, community-usable model system that directly addresses a core methodological bottleneck in forest pathology. This strategy effectively bridges the in vitro–field gap, offering a replicable model for perennial crop breeding and contributing to resilient forest management. 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

Garlic’s vegetative reproduction limits genetic improvement, necessitating advanced biotechnological tools like protoplast culture. However, efficient protoplast regeneration in monocots such as garlic remains a significant challenge. This study establishes an optimized protocol for embryogenic callus induction and subsequent protoplast-to-plant regeneration in garlic (Allium sativum L.), aiming to overcome current limitations using suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, and phytosulfokine-alpha (PSK). We successfully induced embryogenic callus from four garlic accessions and refined protoplast isolation and culture conditions. Key optimizations included using a specific enzyme mixture (2% cellulase R-10 and 0.2% pectolyase Y23) for high yields (from 0.8 to 2.1 × 10⁶ protoplasts per g FM) of viable (approx. 90%) protoplasts and employing the enriched K8M culture medium. Short exposure of protoplasts to SAHA (0.05 or 0.1 µM) significantly improved microcallus formation and plant regeneration. Notably, only callus derived from SAHA-treated cultures displayed regeneration potential, highlighting its pivotal role in embryo differentiation and development. This optimized protocol achieved a 70% success rate for plant acclimatization to ex vitro conditions, with 97% of regenerated plants retaining the ploidy of the donor accession. We demonstrate that SAHA and PSK application enhances garlic protoplast regeneration efficiency. This reliable system provides the groundwork for advanced biotechnological applications, including gene editing technologies in garlic. Full article

Traditional methods for grapevine (Vitis spp.) breeding are marked by lengthy breeding cycles with usually low efficiency, rendering them inadequate for the demands of the rapidly evolving grapevine industry. While grapevine genetic transformation holds significant potential for improvement, its application is hampered by bottlenecks in efficiency, speed, and genotype dependence. In this context, this review systematically examines the factors influencing and challenges associated with key steps in grapevine genetic transformation—specifically, gene delivery and plant regeneration. It posits that the development and application of marker genes, the exploration and utilization of developmental regulators, and the establishment of novel genetic transformation systems are effective strategies to overcome current limitations. In this paper, we present a foundation and methodological guidance for creating efficient and stable genetic transformation systems for grapevine, with significant theoretical and practical implications. Full article

The Japanese cedar (Cryptomeria japonica), also known as sugi, is one of the most important trees in Japanese forests. It covers 44% of artificial forests, spanning approximately 4.5 million ha. It is cultivated in East Asia, the Azores archipelago, and some islands in the Indian Ocean. It is also grown worldwide as an ornamental tree in parks and gardens. The cultivation and use of sugi in Japan dates back centuries, and clonal forestry through cuttings has been practiced since the early 15th century. Its broad adaptability, genetic diversity, rapid growth, easy propagation, and precocious flowering—enabling early generational crosses—combined with their advanced genomic resources and efficient biotechnological tools, make sugi an outstanding conifer model. This review aims to provide an overview of the biotechnology and genetics of sugi for researchers and stakeholders. Full article

Roses (Rosa spp.) are among the most economically and ornamentally important floricultural crops worldwide, yet their improvement is constrained by inefficient breeding methods. Tissue culture regeneration based plant transformation and genome editing technologies provide innovative and increasingly effective approaches to surmount these longstanding challenges; however, rose tissue culture regeneration remains notoriously recalcitrant. Successful plant regeneration in roses depends on multiple factors, including genotype, explant source, physiological status, and the precise combination of plant growth regulators and culture conditions. Over the past three decades, numerous efforts have focused on optimizing rose organogenesis and somatic embryogenesis systems. Despite progress, low regeneration frequencies, strong genotype dependency continue to limit molecular breeding and genome editing application in rose. This review synthesizes current advances in in vitro regeneration methods for roses, emphasizing key determinants of morphogenic response, including explant selection, hormonal balance, media composition, light and temperature regimes, and the organic and inorganic additives. The underlying causes of recalcitrance are discussed in relation to tissue physiology, biochemical and molecular regulation of morphogenesis. Finally, strategies for overcoming regeneration barriers—such as the use of morphogenic regulators and in planta transformation—are highlighted as emerging avenues toward cultivar independent transformation and genome editing for rose. 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