Search Results (158)

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

Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, the optimal conditions for callus induction were identified, and formation of the embryogenic callus was confirmed by histological analysis. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using suitable embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5 + 1.5 mg/L 6-BA + 0.5 mg/L NAA + 30 g/L sucrose + 7 g/L agar (pH = 5.8). In this medium, the induction rate of callus achieved 97.50%, and the rate of embryogenic callus formation was 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82 × 10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridisation and transient gene expression in this species. 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

Gynogenesis offers a promising route for doubled haploid (DH) production in Cucurbita, yet efficient protocols remain scarce. This study established a reproducible ovary culture system for Cucurbita pepo and evaluated zeatin riboside (ZR) as an alternative cytokinin. Ovaries collected at anthesis and one day before were cultured to screen nine media with different cytokinin–auxin combinations. Subsequently, four optimized ZR-based formulations were evaluated. Both floral stages showed morphogenic activity, but embryo formation occurred almost exclusively in pre-anthesis ovaries. Among ZR treatments, E6.1 (1 mg·L⁻¹ ZR + 3 mg·L⁻¹ NAA, 30 g·L⁻¹ sucrose) achieved the highest embryogenic output (approximately 97 embryos per 100 explants), while high-sucrose media (120 g·L⁻¹) induced abundant swollen ovules but poor conversion, suggesting that excessive osmotic pressure promotes morphogenesis but hampers embryogenic transition. In total, 415 embryos were obtained, and 52 regenerants were analyzed by flow cytometry, confirming haploid, diploid, and mixoploid plants and evidencing spontaneous chromosome doubling during in vitro development. A categorical A–D scoring system enabled early prediction of embryogenic potential. This represents the first successful application of ZR in cucurbit gynogenesis and highlights its value as a biologically compatible cytokinin for DH production. The findings open new avenues for testing ZR-based formulations in other Cucurbita species under different auxin and sucrose regimes. 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

Efficiency in vitro regeneration is a crucial prerequisite for the application of New Nenomics Techniques (NGTs) in grapevine (Vitis vinifera L.) for improving resistance to biotic and abiotic stresses. This is especially true given that their management must be addressed sustainably, considering the impact of climate change. Unfortunately, in vitro plant regeneration and the establishment of embryogenic calluses are two genotype-dependent processes. Up to now, extensive research has been conducted on major international cultivars, whereas studies on the application of in vitro protocols for autochthonous cultivars remain limited. In this study, protocols for the acquisition of embryogenic calluses were applied on the most relevant Sicilian grapevine cultivars: the red-skinned ‘Frappato’, ‘Nerello mascalese’, and ‘Nero d’Avola’, and the white-skinned ‘Grillo’, ‘Carricante’, and ‘Catarratto’. Stamens and pistils were cultured in two different induction media (PIV and MSII) and at three stages (mother cells in the late premeiotic phase, tetrads, and mature pollen) to induce embryogenic calluses. Five thousand explants per cultivar were cultured, forming calluses in four selected cultivars. Plantlets were successfully generated from calluses of ‘Carricante’, ‘Frappato’, and ‘Nero d’Avola’. Moreover, protoplasts were isolated from ‘Frappato’ and ‘Nero d’Avola’. Our results establish a critical foundation for developing successful regeneration protocols for the future application of NGTs in Sicilian grapevine cultivars. Full article

Somatic embryogenesis (SE) in Narcissus offers significant potential for both horticultural propagation and pharmaceutical applications. In this study, embryogenic callus lines derived via primary and secondary SE were evaluated under different in vitro conditions to assess the effects of medium type (liquid vs. solid) and composition (proliferation vs. regeneration) on biomass growth and somatic embryo formation. Lines derived from primary SE (LC1–LC4) were less efficient compared to those obtained through secondary SE (LC5–LC7). Cultures cultivated in liquid proliferation medium for eight weeks showed a greater biomass accumulation than those grown on solid medium. Multivariate analyses revealed distinct growth patterns and responses to medium type among the callus lines. The LC5 and LC7 lines formed a separate cluster characterized by superior biomass proliferation and embryogenic competence. An eight-week culture in a liquid proliferation medium followed by a transfer to a solid medium of the same composition resulted in the highest somatic embryo yield in the LC5 line (54.4 embryos per 0.5 g of callus). Under the same conditions, the LC7 line showed the highest biomass growth (a 23.4-fold increase), but its embryogenic response was more effectively stimulated when the callus was initially proliferated on a solid medium and then transferred to a regeneration medium. Full article

Grammatophyllum speciosum Blume is an endangered wild orchid with medicinal properties. In this research, we propagated G. speciosum from vegetative organs grown under aseptic conditions. Subsequently, salinity stress was applied at the plantlet stage to investigate its effect on the accumulation of bioactive compounds. Half-strength Murashige and Skoog (½ MS) medium supplemented with a combination of 1 mg of L⁻¹ 1-naphthaleneacetic acid (NAA) and 0.5 mg of L⁻¹ 6-benzylaminopurine (BAP) proved to be a more suitable medium for shoot formation (32.33 ± 2.52 shoots per explant). The protocorm-like bodies, derived from embryogenic callus, were transferred into a temporary immersion bioreactor (TIB) system; 10-min of immersion every 3 h enhanced the maximum number of shoots, shoot height, and the fresh growth index (127.00 ± 2.16, 5.00 ± 0.51 cm and 4.26 ± 0.52, respectively). The proliferated plantlets from the TIB system successfully rooted in Vacin and Went medium. Furthermore, the plantlets were maintained in ½ MS medium supplemented with sodium chloride (NaCl) (0, 50, 100 or 200 µM) under a white light-emitting diode for 72 h to determine the total phenolic content (TPC) in the in vitro cultures. The TPC was highest in the medium with 100 µM of NaCl (111.06 ± 2.24 mg gallic acid equivalent g⁻¹ dry weight), the diphenyl picrylhydrazyl antioxidant activity was 24.50 ± 0.76% and ferric-reducing antioxidant power values were in the range 2441.79 ± 1.21 to 2491.96 ± 3.23 µM ascorbic acid equivalent g⁻¹ dry weight. The G. speciosum extracts showed antibacterial activity against acne pathogens, with minimum inhibitory concentration and minimum bactericidal concentration values in the ranges 6.4–12.8 mg mL⁻¹ and 12.8–25.6 mg mL⁻¹, respectively. Full article

Napier grass (Cenchrus purpureus Schumach) is an important forage crop and livestock feed. However, its yield and quality in Kenya are often limited by Napier grass headsmut and stunt disease. Napier grass genetic improvements through mutation breeding and selection could avail cultivars with increased forage. This study investigated the response of embryogenic calli to different levels of colchicine in inducing polyploidy in the two germplasms of Napier grass; South africa and Bana grass. The experiments were carried out as a factorial experiment in a completely randomized design (CRD). The colchicine concentrations used were 0, 0.05, 0.1, and 0.2%, and the exposure durations were 24, 48, and 72 h. During the shoot regeneration stage, culturing explants on an MS medium (Murashige and Skoog) supplemented with 0.2 mg L⁻¹ Benzyl Adenine (BAP), 0.1 mg L⁻¹ dichlorophenoxyacetic acid (2, 4-D), and 0.1 mg L⁻¹ indole-3-butyric acid (IBA) was more suitable for shoot regeneration. Chromosome doubling was confirmed by genomic DNA and the stomata size and number. Culturing explants on an MS medium supplemented with 1 mg L⁻¹ IBA, 1 mg L⁻¹ 2, 4-D, and 0.5 mg L⁻¹ BAP was more suitable in inducing embryogenic calli in both genotypes. Polyploidy results revealed that a 0.1% concentration of colchicine with two days of treatment established the maximum number of octoploid plantlets induced in vitro, while a 0.2% concentration was very toxic. The stomata size and number of derived octoploid plantlets were bigger with a lower density, a shorter plant height, and a smaller stem diameter, and despite being the first to produce tillers, they were significantly higher than their progenitors. Induced mutants also had a significantly higher number of chromosomes and showed different band patterns and distances during gel electrophoresis. However, we recommend the use of flow cytometry to confirm the ploidy level. The superior mutant plantlets can be selected and recommended for characterization across representative agro-ecologies for large-scale production and used in Cenchrus purpureus breeding programs in Kenya and its environments. Full article

This study elucidates the key molecular features underlying the embryogenic initiation divergence between japonica rice Chongxiangjing (CXJ) and indica rice 9311 during isolated microspore culture. Comparative transcriptome analysis across critical timepoints (0, 5, and 10 days post-culture initiation) revealed that while both varieties initially exhibit comparable microspore viability, CXJ maintains transcriptional stability and activates developmental programs (e.g., hormone signaling, DNA replication, cell morphogenesis), enabling sustained callus formation. In contrast, 9311 undergoes drastic transcriptome reorganization by 5 days, characterized by maladaptive activation of stress-response pathways (glutathione metabolism, MAPK signaling, ER stress) and futile metabolic reactivation (photosynthesis, starch degradation), culminating in near-total cell death and failed callus induction. Transcription factor dynamics further explain this divergence: CXJ specifically upregulates regulators coordinating development and stress resilience (NAC, ERF, HSF, GRAS, bZIP), while 9311 exhibits detrimental upregulation of FAR1 and B3, leading to catastrophic energy misallocation. These findings identify master transcriptional networks and stress-response pathways as pivotal indicators of embryogenic initiation efficiency, providing strategic targets for enhancing indica rice microspore culture technology. Full article

Vitellaria paradoxa (shea tree) is an economically and medicinally important species indigenous to sub-Saharan Africa. Although the species holds substantial value, domestication efforts have been constrained, primarily due to the absence of efficient propagation alternatives, especially for the East African subspecies (V. paradoxa subsp. nilotica) which remains understudied in tissue culture research. This study investigated the influence of leaf explant developmental stage and media composition on callogenesis and embryogenic potential in V. paradoxa subsp. nilotica. Thus, leaf explants from six distinct growth stages were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 2,4-D, TDZ, NAA, and BAP. Callogenesis was significantly influenced by explant age, media strength, and specific PGR combinations. Results revealed that explants from Stage III (11–15 days) and Stage IV (16–20 days) exhibited the highest callus induction rates (up to 100%), particularly on half-strength MS media containing 2.0 mg/L 2,4-D and 0.5–1.0 mg/L TDZ. Histological analysis suggests that varying responses at the different stages relate to chloroplast distribution, trichome density/orientation, and vascular tissue maturity. Pro-embryogenic structures were successfully induced, representing a developmental milestone with strong prospects for advanced stages of differentiation. The findings also emphasize the importance of explant physiology and media formulation in developing regeneration protocols for V. paradoxa from leaf explants. Full article