Search Results (91)

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

Rice (Oryza sativa L.), a staple food for over half of the global population, plays a pivotal role in food security. Among its two primary groups, japonica and indica, temperate japonica varieties are particularly valued for their high-quality grain and culinary uses. Although some of these varieties are adapted to cooler climates, they often suffer from reduced productivity or increased disease susceptibility when cultivated in warmer productive environments. These limitations underscore the need for breeding programs to incorporate biotechnological tools that can enhance the adaptability and resilience of the plants. However, New Genomic Techniques (NGTs), including CRISPR-Cas9, require robust in vitro systems, which are still underdeveloped for temperate japonica genotypes. In this study, we developed a reproducible and adaptable protocol for protoplast isolation and regeneration from the temperate japonica cultivars ‘Ónix’ and ‘Platino’ using somatic embryos as the starting tissue. Protoplasts were isolated via enzymatic digestion (1.5% Cellulase Onozuka R-10 and 0.75% Macerozyme R-10) in 0.6 M AA medium over 18–20 h at 28 °C. Regeneration was achieved through encapsulation in alginate beads and coculture with feeder extracts in 2N6 medium, leading to embryogenic callus formation within 35 days. Seedlings were regenerated in N6R and N6F media and acclimatized under greenhouse conditions within three months. The isolated protoplast quality displayed viability rates of 70–99% within 48 h and supported transient PEG-mediated transfection with GFP. Additionally, the transient expression of a gene editing CRISPR-Cas9 construct targeting the DROUGHT AND SALT TOLERANCE (OsDST) gene confirmed genome editing capability. This protocol offers a scalable and genotype-adaptable system for protoplast-based regeneration and gene editing in temperate japonica rice, supporting the application of NGTs in the breeding of cold-adapted cultivars. Full article

The application of non-thermal (cold) plasmas is considered an environmentally friendly method that could affect plant metabolism and cellular development or can be used for the commercial production of natural products that cannot be chemically synthesized. In the present study, the non-embryogenic callus of iris (Iris reichenbachii Heuff.) was treated with a Radio Frequency (RF) plasma needle device using He as a working gas. We investigated short-term (up to seven days) and long-term (up to one year) changes on morphological, physiological and biochemical levels. An increased production of O₂⁻ and H₂O₂ was observed in the callus tissue after plasma treatment. The enzymes SOD and CAT represented the frontline in the antioxidant defense against reactive oxygen species (ROS) produced during the first hour of treatment, while POX was the leading antioxidant enzyme seven days after plasma treatment. Significant long-term morphological changes were observed in the calli due to the increased mitotic activity of the plant cells. In addition, three flavonoids (naringenin, apigenin and acacetin) and two isoflavonoids (irisolidone and irilone) were detected only in the plasma-treated tissue even one year after plasma treatment. The present study emphasizes the application of the plasma technique to promote meristematic activity and stimulate the production of specialized metabolites in iris calli. 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

Caragana intermedia is a perennial shrub species in the genus Caragana (Fabaceae), demonstrating remarkable stress resistance and adaptability. However, research on its somatic embryogenesis (SE) and genetic transformation techniques remains limited. In this study, we established an SE system by utilizing immature cotyledons isolated from young C. intermedia seeds. Our findings demonstrated that the immature cotyledons at 6–7 weeks after flowering (WAF) were the best explants for SE. The optimal embryo induction medium consisted of an MS basal medium supplemented with 5 mg/L α-naphthaleneacetic acid (NAA), 3 mg/L 6-benzylaminopurine (6-BA), 30 g/L sucrose, 7 g/L agar, and 500 mg/L hydrolyzed casein. Cotyledon-stage embryos germinated on a half-strength MS medium, exhibiting a 34.36% germination rate. Based on the SE system, we developed a preliminary genetic transformation system using the RUBY reporter gene, which successfully generated transgenic calli and cotyledon-stage embryos. The establishment of the SE system is expected to shorten breeding cycles, facilitate propagation of superior cultivars, and support large-scale industrial applications in C. intermedia. Furthermore, the stable transformation system provides a platform for molecular breeding and gene function verification. Full article

Background: The development of an efficient tissue culture system is essential for advancing genetic transformation and genome editing in commercially important pineapple cultivars. However, a robust tissue culture workflow for the elite pineapple cultivar 73–50, enabling reliable transformation and plant regeneration is not established. Methods: A comparative analysis of hormone combinations, including 6-benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA), picloram, and abscisic acid (ABA) was conducted. Transformation competence of 73–50 callus was tested using the iGUS reporter gene. Results: We established that 1 mg/L picloram and 0.5 µg/L ABA was the most effective combination for inducing friable embryogenic callus (FEC). FEC, composed of small, loosely associated cell clusters, is highly suitable for transformation but prone to browning during long-term culture. We optimized the conditions to minimize browning and support prolonged maintenance using a medium supplemented with 5 mg/L NAA. Transformation efficiency was demonstrated using the iGUS reporter gene, showing that FEC can be effectively transformed via both biolistic and Agrobacterium-mediated methods. For shoot regeneration, the optimal medium was found to contain 2 mg/L BAP. To standardize the assessment of callus development, we introduce a classification system describing distinct developmental stages. Conclusions: A detailed step-by-step protocol optimized for 73–50 cultivar facilitates efficient genetic improvement in pineapple, supporting both conventional transformation and DNA-free genome editing approaches. Full article

Somatic embryogenesis (SE) involves the formation of embryo-like structures from somatic cells without fertilization and is widely used for clonal propagation and genetic transformation. However, in olive (Olea europaea sp. europaea), SE remains challenging due to the recalcitrant behavior of adult tissues when used as initial explants. Bioactive molecules released into the culture medium (conditioned medium, CM) by embryogenic cultures have been identified as modulators of the SE response. However, their potential role in enhancing SE efficiency in olive and overcoming tissue recalcitrance remains largely unexplored. To investigate the role of these biomolecules in olive SE, a protocol was established using SE cultures of cv. ‘Galega Vulgar’. Proteins and metabolites were separated by filtration, concentrated through lyophilization, and precipitated using three methods: Acetone, TCA/Acetone, and Methanol/Chloroform. The efficiency of these methods was evaluated through total protein quantification and via SDS-PAGE electrophoresis. LC-MS/MS was employed to analyze secretome composition using the TCA/Acetone precipitation method. Additionally, metabolite profiles were analyzed using ¹H NMR spectroscopy. The results led to the identification of 1096 (526 protein groups) Olea europaea proteins, including well-known SE biomarkers such as kinases and peroxidases. NMR spectroscopy identified several metabolites secreted into the medium or resulting from the metabolic activity of secreted enzymes, confirming the applicability of the procedure. Although extracting secreted biomolecules from the culture medium presents significant challenges, the protocol established in this study successfully enabled the isolation and identification of both proteins and metabolites, revealing a valuable workflow for future in-depth analyses of secreted biomolecules in olive SE. Full article

Lonicera japonica Thunb has significant edible and medicinal value, possessing heat clearing, detoxification, antibacterial, and blood pressure reduction properties. Currently, its quality is constrained by factors such as climate, environment, flowering period, and germplasm degradation. The strategy of using bioreactors and abiotic inducers to produce bioactive metabolites has not yet been implemented. This study reports, for the first time, the induction of an embryogenic callus from L. japonica anthers, the identification of tissue morphological structures, and the effects of light induction on the callus morphology, metabolite accumulation, and antioxidant activity. The results showed that the MS medium, supplemented with 1.0 mg·L⁻¹ 6-BA, 1.5 mg·L⁻¹ NAA, 1.5 mg·L⁻¹ 2,4-D, and 0.2 mg·L⁻¹ KT, induced 89% embryogenic callus formation. Uniform callus lines were obtained using 2.0 mg·L⁻¹ 6-BA, 0.5 mg·L⁻¹ NAA, and 0.2 mg·L⁻¹ KT in each subcultivation. Embryogenic cells were observed to have closely arranged spherical protruding granules on their surface, along with visible nuclei and numerous starch grains. After 15 days of blue light induction, active metabolites and antioxidant activities peaked. This experimental system not only provides support for germplasm innovation but also indicates that abiotic inducers can be utilized as a means to achieve higher yields of metabolic products. Full article

The Mediator complex (MED) functions as a co-activator in plants, transmitting transcriptional signals to regulate gene expression, including responses to environmental stresses. While the MED gene family has been identified in several species, it has not yet been reported in cassava. In this study, we identified 32 members of the MeMED gene family in cassava (Manihot esculenta Crantz) distributed across 13 chromosomes. These genes were categorized into distinct Mediator subunits based on their similarity to Arabidopsis modules. Promoter analysis revealed the presence of various cis-regulatory elements, which likely play key roles in regulating plant growth, development, and stress responses. RNA-seq data showed tissue-specific expression patterns for the MeMED genes, with significant expression observed in leaves, roots, petioles, stems, friable embryogenic callus, and shoot apical meristems. Further RT-qPCR analysis under various abiotic stress conditions—including drought, exogenous hydrogen peroxide, cold, heat, and salt—demonstrated that 10 selected MeMED genes exhibited significant differential expression, indicating their potential functional involvement in stress adaptation. These findings offer insights into the biological roles of the MeMED gene family in cassava, with implications for improving stress tolerance in future breeding programs. 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

Liquid culture systems, including bioreactors, are valuable tools for the scaling up of production. Their involvement leads to the automation of the highly efficient, reproducible somatic embryogenesis of Narcissus L. ‘Carlton’. Alternative procedures for efficient embryogenic tissue and early somatic embryo multiplication have been developed. The long-term embryogenic callus of narcissus ‘Carlton’, obtained by repetitive somatic embryogenesis, was multiplicated and differentiated in different liquid culture systems. For multiplication, the Rita^® temporary immersion bioreactor and the rotary shaker at 60 rpm and 100 rpm were used, and, for differentiation, the rotary shaker at 60 rpm and solid cultures were investigated. Cultures immersed with a frequency of 15 min every 24 h during multiplication were characterized by the greatest increase in biomass (1.3), and the greatest number of embryos (152.6 embryos per 1 g of inoculum) was seen during differentiation. Higher immersion frequencies (15 min every 8 and 12 h) decreased the tissue quality and yield. The use of a bioreactor during multiplication promoted the number of embryos obtained during differentiation. In turn, cultivation in a rotary shaker during differentiation, regardless of the multiplication system, stimulated the multiplication of embryogenic tissue. The liquid medium used for the multiplication and differentiation of somatic embryos improved the synchronization of their development, which reached up to 95–99% depending on the system. Full article

Centaurium erythraea Rafn. is a medicinal plant used as a model for studying plant developmental processes due to its developmental plasticity and ease of manipulation in vitro. Identifying the genes involved in its organogenesis and somatic embryogenesis (SE) is the first step toward unraveling the molecular mechanisms underlying its morphogenic plasticity. Although SE is the most common method of centaury regeneration, the genes involved in this have not yet been identified. The aim of this study was to identify the differentially expressed genes (DEGs) during key stages of SE and organogenesis using transcriptome data, with a focus on novel SE-related genes. The transcriptomic analysis revealed a total of 4040 DEGs during SE and 12,708 during organogenesis. Gene Ontology (GO) annotation showed that the highest number of SE-related genes was involved in defense responses. The expression of fifteen selected SE-related candidate genes was assessed by RT-qPCR across nine centaury developmental stages, including embryogenic tissues. Notably, a newly reported transcript, named CeNA1, was specifically activated during embryogenic callus (ec) induction, making it a potential novel marker for early SE. These findings provide, for the first time, insight into SE-related transcriptional patterns, representing a step closer to uncovering the molecular basis of centaury’s developmental plasticity. Full article

Biomanufacturing enables novel sources of compounds with constant demand, such as food coloring and preservatives, as well as new compounds with peak demand, such as diagnostics and vaccines. The COVID-19 pandemic has highlighted the need for alternative sources of research materials, thrusting research on diversification of biomanufacturing platforms. Here, we show initial results exploring the walnut somatic embryogenic system expressing the recombinant receptor binding domain (RBD) and ectodomain of the spike protein (Spike) from the SARS-CoV-2 virus. Stably transformed walnut embryo lines were selected and propagated in vitro. Both recombinant proteins were detected at 3–14 µg/g dry weight of tissue culture material. Although higher yields of recombinant protein have been obtained using more conventional biomanufacturing platforms, we also report on the production of the red pigment betanin in somatic embryos, reaching yields of 650 mg/g, even higher than red beet Beta vulgaris. This first iteration shows the potential of biomanufacturing using somatic walnut embryos that can now be further optimized for different applications sourcing specialized proteins and metabolites. Full article

Somatic embryogenesis (SE) provides alternative methodologies for the propagation of grapevine (Vitis spp.) cultivars, conservation of their germplasm resources, and crop improvement. In this review, the current state of knowledge regarding grapevine SE as applied to these technologies is presented, with a focus on the benefits, challenges, and limitations of this method. The paper provides a comprehensive overview of the different steps involved in the grapevine SE process, including callus induction, maintenance of embryogenic cultures, and the production of plantlets. Additionally, the review explores the development of high-health plant material through SE; the molecular and biochemical mechanisms underlying SE, including the regulation of gene expression, hormone signaling pathways, and metabolic pathways; as well as its use in crop improvement programs. The review concludes by highlighting the future directions for grapevine SE research, including the development of new and improved protocols, the integration of SE with other plant tissue culture techniques, and the application of SE for the production of elite grapevine cultivars, for the conservation of endangered grapevine species as well as for cultivars with unique traits that are valuable for breeding programs. Full article

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species. Full article