Search Results (125)

Protoplasts offer a promising alternative to embryogenic cell suspensions (ECS) for gene editing in banana, potentially overcoming several limitations associated with ECS-based transformation systems. This study aimed to optimize protoplast isolation and regeneration in Cavendish banana (cv. Williams) and to assess their suitability for transient gene expression. Enzymatic digestion of ECS using cellulase and macerozyme consistently yielded approximately 3 × 10⁶ protoplasts per milliliter of settled cell volume. Protoplast yield was further enhanced, by approximately threefold, through the addition of an antioxidant mixture (ascorbic acid, citric acid and L-cysteine) combined with 0.01% bovine serum albumin. Polyethylene glycol-mediated transfection with a green fluorescent protein reporter gene yielded transient expression in approximately 0.75% of protoplasts five days post-transfection. While phenotypically normal plants were regenerated from untransfected protoplasts after 12 weeks in agarose bead culture with conditioned liquid medium, no regeneration was observed from transfected cells. These findings establish a reproducible protocol for protoplast isolation and plant regeneration in Cavendish banana and provide insight into the barriers limiting successful regeneration following transfection. Full article

Somatic embryogenesis (SE) is a key plant regeneration technique involving the reprogramming of somatic cells into embryogenic structures. This developmental transition is regulated by complex genetic and epigenetic mechanisms, including post-translational modifications such as SUMOylation—the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, influencing their function, stability, and interactions. While SUMOylation is known to regulate plant development and stress responses, its role in SE has remained unknown. In this study, we investigated the involvement of the SUMOylation pathway in SE induction in Medicago truncatula. Using BLASTp analysis with known SUMO pathway proteins from Arabidopsis thaliana and Glycine max, we identified 10 homologous genes in M. truncatula. Phylogenetic relationships, gene structure, and conserved motif analyses confirmed their evolutionary conservation and characteristic domains. Expression profiling revealed significant upregulation of SUMO pathway genes—including Mt SUMO2, Mt SAE1-2, Mt SCE1a-b, Mt MMS21, and Mt PIAL2—in embryogenic cell lines during early SE induction. Additionally, in silico prediction of SUMOylation sites and SUMO-interacting motifs (SIMs) in 12 key SE regulatory proteins indicated a broad potential for SUMO-mediated regulation. These findings suggest that SUMOylation may contribute to the acquisition of embryogenic competence during somatic cell reprogramming in plants. 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

Castanea mollissima Bl. is rich in nutrition and strong in stress resistance, and has nutritional, economic, and ecological values. A protoplast is impactful in somatic fusion and germplasm creation. Here, we propose an effective scheme for the construction of an embryonic suspension cell, protoplast isolation, and fusion. Studies have shown that when 1.0 g yellow loose embryonic callus was inoculated into MS + 1.5 mg∙L⁻¹ 6-BA + 0.2 mg∙L⁻¹ NAA + 0.5 mg∙L⁻¹ 2, 4-D liquid medium, a stable suspension cell line can be obtained. After further culturing for 2–4 days, protoplast isolation was performed. First, single-factor screening was conducted on the four enzymes, and then a two-factor random block was further set up to screen the enzyme combinations based on the results. We found that 1.0%cellulase R-10 + 0.5%pectolase Y-23 led to the highest protoplast yield (9.27 × 10⁶/g FW) and the highest activity (92.49%). Furthermore, the protoplast yield could be increased to 9.47 × 10⁶/g FW by adding 0.4 M mannitol and shaking for 8 h. The protoplasts were purified by centrifuging at 40× g for 4 min and then mixed with 30% PEG 6000 at a volume ratio of 1.5:1 for 25 min. The fusion rate could reach 70.00%. This study laid a foundation for the creation of new germplasm by Castanea mollissima Bl. 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

DNA methylation plays a crucial role in regulating the developmental processes of plants. Particularly, it is closely associated with the development of embryogenic cells (EC) and somatic embryos (SE). In this study, we investigated the effects of 5-azaCytidine (5-azaC) treatment on somatic embryogenesis proliferation and maturation of Taxodium hybrid ‘zhongshanshan’. The results showed that the callus proliferation was inhibited when the concentration of 5-azaC exceeded 30 μM, while treatment with 5 μM 5-azaC improved the maturation rate and expedited the process of SE formation. It was also noted that 5-azaC influenced somatic embryogenesis during the second week of embryo induction, substantially enhancing the maturation rate of somatic embryos and the germination rate of Taxodium hybrid ‘zhongshanshan’. Furthermore, the analysis revealed that treatment with 5-azaC resulted in elevated levels of H₂O₂, SOD, POD, and AsA during the cotyledonary embryo period in Taxodium hybrid ‘zhongshanshan’, indicating its potential to promote somatic embryogenesis by regulating redox homeostasis. This study concluded that 5-azaC could improve the efficiency of somatic embryogenesis in Taxodium hybrid ‘zhongshanshan’, as well as provide a solid foundation for investigating the effects of 5-azaC on somatic embryogenesis in other conifer species. Full article

Araucaria araucana is an emblematic native conifer from Chile and Argentina that has been classified as threatened due to anthropogenic activities. Somatic embryogenesis (SE) is a biotechnological tool used for both the preservation of genetic material and the propagation of valuable genotypes. The present study investigates the effects of explant source and culture medium on SE induction in A. araucana genotypes from three wild plant populations. Immature strobili were collected from different geographical provenances: a coastal area (Villa Araucarias, “VA”), Cordillera de Nahuelbuta (Trongol Alto, “TR”), and the Andes Mountains (Malalcahuello, “MA”). SE induction was observed after 45 days in a basal medium (BM) supplemented with 1-naphthaleneacetic acid (NAA—11 µM), 6-benzylaminopurine (6-BA—2.8 µM), and Kinetin (Kin—2.8 µM). The highest induction rate (75%) was achieved for seeds from VA. Embryogenic cell line (ECL) proliferation requires auxins but is genotype-dependent, as not all genotypes survive. Cytochemical analysis revealed the presence of pro-embryogenic masses (PEMs) in the ECLs, indicating an efficient SE induction protocol. The progression of PEMs to early embryos was observed in the presence of maltose (3% w/v), polyethylene glycol 3350 (PEG—7% w/v), and abscisic acid (ABA—68 µM). Our results establish a baseline for the establishment of in vitro cultures for a diverse range of A. araucana genotypes, enabling the initiation of ex situ preservation programs and further investigation into embryo maturation. Full article

Miscanthus × giganteus (Greef and Deuter ex Hodkinson and Renvoize) is a perennial, rhizomatous grass that has gained significant attention as an industrial crop, particularly as a bioenergy feedstock. It is a natural interspecific hybrid with 57 chromosomes (2n = 3x = 57). Due to its sterility, M. × giganteus has limited genetic variability, making traditional breeding methods ineffective for its improvement. Consequently, alternative approaches are being explored to enhance its cultivation and utility. The study aimed to investigate the potential for M. × giganteus plant regeneration through ovary and flower bud cultures. Indirect in vitro regeneration of M. × giganteus plants was successfully achieved using flower bud cultures. Embryogenic-like callus was derived from explants originating from inflorescences that had undergone a four-day pretreating at 10 °C. The most effective medium for callus induction was a modified MS medium supplemented with 5 mg·dm⁻³ dicamba, 0.2 mg dm⁻³ 6-benzylaminopurine, 30 g dm⁻³ sucrose, and solidified with 8 g dm⁻³ agar or agarose. The optimal conditions for callus induction were achieved by culturing in the dark. The regenerated plants exhibited the characteristic chromosome number of the species, confirming that the regenerants did not develop from embryo sac cells. In contrast, ovary culture failed to produce callus or regenerated plants, highlighting its ineffectiveness for M. × giganteus regeneration. These findings underscore the potential of flower bud culture as a successful in vitro regeneration method while demonstrating the limitations of ovary culture for this species. Full article

In Coffea canephora, a direct somatic embryogenesis (SE) protocol has been established by pretreating plants with NAA and kinetin, followed by the induction of leaf explants in a liquid medium with BA. Through a transcriptomic analysis of 10 key moments of the induction of SE in C. canephora, we were able to establish that the transcriptional responses of this process are divided into four stages. These stages correspond to the pretreatment, characterized by the positive regulation of genes associated with cell wall remodeling, flavonoid biosynthesis, and antioxidant activity that prepare the explants for the intense cellular activity that represents the induction of SE. During the first few hours, the early response to induction occurs, characterized by the highest number of differentially expressed genes, most related to the response to multiple stimuli. At 24 h, a late response begins with the upregulation of genes related to energy production and the biosynthesis of amino acids. Finally, WOX, BBM, and ABI3 genes are upregulated during the embryogenic response. The downregulation of genes related to the circadian cycle, photomorphogenesis, photosynthesis, and chloroplast components were observed throughout the process. The detailed analysis of the primary transcriptional responses that occur during the SE of C. canephora helps us to clarify how auxins and cytokinins orchestrate the integration of different networks of plant metabolism and lead to the development of somatic embryos. 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

Bananas and plantains are important staple food crops affected by biotic and abiotic stresses. The gene editing technique via Clustered Regularly Interspaced Short Palindromic Repeats associated with the Cas protein (CRISPR/Cas) has been used as an important tool for development of cultivars with high tolerance to stresses. This study sought to develop a protocol for the construction of vectors for gene knockout. Here we use the phytoene desaturase (PDS) gene as a case study in Prata-Anã banana by the nonhomologous end junction (NHEJ) method. PDS is a key gene in the carotenoid production pathway in plants and its knockout leads to easily visualized phenotypes such as dwarfism and albinism in plants. Agrobacterium-mediated transformation delivered CRISPR/Cas9 constructs containing gRNAs were inserted into embryogenic cell suspension cultures. This is the first study to provide an effective method/protocol for constructing gene knockout vectors, demonstrating gene editing potential in a Brazilian banana variety. The constitutive (CaMV 35S) and root-specific vectors were successfully assembled and confirmed in transformed Agrobacterium by DNA extraction and PCR. The specificity of transformation protocols makes it possible to use the CRISPR-Cas9 technique to develop Prata-Anã banana plants with enhanced tolerance/resistance to major biotic and abiotic factors. Full article

The negative effects of climate change impact both farmers and consumers. This is exemplified in coffee, one of the most widely consumed beverages in the world. Yield loss in high-quality Coffea arabica L., due to the spread of coffee leaf rust (Hemileia vastatrix), results in lower income for subsistence farmers and volatile prices in markets and cafes. Genetic improvement of crops is a proven approach to support sustainable production while mitigating the effects of biotic and abiotic stresses and simultaneously maintaining or improving quality. However, the improvement of many species, including coffee, is hindered by low genetic diversity. This can be overcome by inducing novel genetic variation via treatment of seeds or cells with mutagens. To evaluate this approach in coffee, mutant populations created by incubating seed or embryogenic calli with the chemical mutagens ethyl methanesulphonate or sodium azide were subject to reduced-representation DNA sequencing using the ddRADseq approach. More than 10,000 novel variants were recovered. Functional analysis revealed hundreds of sequence changes predicted to be deleterious for gene function. We discuss the challenges of unambiguously assigning these variants as being caused by the mutagenic treatment and describe purpose-built computational tools to facilitate the recovery of novel genetic variation from mutant plant populations. Full article

The embryogenic transition of plant somatic cells to produce somatic embryos requires extensive reprogramming of the cell transcriptome. The prominent role of transcription factors (TFs) and miRNAs in controlling somatic embryogenesis (SE) induction in plants was documented. The profiling of MIRNA expression in the embryogenic culture of Arabidopsis implied the contribution of the miR156 and miR169 to the embryogenic induction. In the present study, the function of miR156 and miR169 and the candidate targets, SPL and NF-YA genes, were investigated in Arabidopsis SE. The results showed that misexpression of MIRNA156 and candidate SPL target genes (SPL2, 3, 4, 5, 9, 10, 11, 13, 15) negatively affected the embryogenic potential of transgenic explants, suggesting that specific fine-tuning of the miR156 and target genes expression levels seems essential for efficient SE induction. The results revealed that SPL11 under the control of miR156 might contribute to SE induction by regulating the master regulators of SE, the LEC (LEAFY COTYLEDON) genes (LEC1, LEC2, FUS3). Moreover, the role of miR169 and its candidate NF-YA targets in SE induction was demonstrated. The results showed that several miR169 targets, including NF-YA1, 3, 5, 8, and 10, positively regulated SE. We found, that miR169 via NF-YA5 seems to modulate the expression of a master SE regulator LEC1/NF-YA and other auxin-related genes: YUCCA (YUC4, 10) and PIN1 in SE induction. The study provided new insights into miR156-SPL and miR169-NF-YA functions in the auxin-related and LEC-controlled regulatory network of SE. Full article

Somatic embryogenesis (SE) is a biotechnological tool used to generate new individuals and is the preferred method for rapid plant regeneration. However, the molecular basis underlying somatic cell regeneration through SE is not yet fully understood, particularly regarding interactions between the proteome and post-translational modifications. Here, we performed association analysis of high-throughput proteomics and phosphoproteomics in three representative samples (non-embryogenic calli, NEC; primary embryogenic calli, PEC; globular embryos, GE) during the initiation of plant regeneration in cotton, a pioneer crop for genetic biotechnology applications. Our results showed that protein accumulation is positively regulated by phosphorylation during SE, as revealed by correlation analyses. Of the 1418 proteins that were differentially accumulated in the proteome and the 1106 phosphoproteins that were differentially regulated in the phosphoproteome, 115 proteins with 229 phosphorylation sites overlapped (co-differential). Furthermore, seven dynamic trajectory patterns of differentially accumulated proteins (DAPs) and the correlated differentially regulated phosphoproteins (DRPPs) pairs with enrichment features were observed. During the initiation of plant regeneration, functional enrichment analysis revealed that the overlapping proteins (DAPs-DRPPs) were considerably enriched in cellular nitrogen metabolism, spliceosome formation, and reproductive structure development. Moreover, 198 DRPPs (387 phosphorylation sites) were specifically regulated at the phosphorylation level and showed four patterns of stage-enriched phosphorylation susceptibility. Furthermore, enrichment annotation analysis revealed that these phosphoproteins were significantly enriched in endosomal transport and nucleus organization processes. During embryogenic differentiation, we identified five DAPs-DRPPs with significantly enriched characteristic patterns. These proteins may play essential roles in transcriptional regulation and signaling events that initiate plant regeneration through protein accumulation and/or phosphorylation modification. This study enriched the understanding of key proteins and their correlated phosphorylation patterns during plant regeneration, and also provided a reference for improving plant regeneration efficiency. Full article