Search Results (17)

Triadica sebifera, an economically and medicinally valuable tree species native to China, was investigated for its in vitro regeneration potential using leaf explants from nodal cuttings of young stems and sprouts. This study evaluated the effects of basal media, plant growth regulators (PGRs), explant sources, and incision methods on adventitious shoot induction, supplemented by histological analysis. The highest shoot regeneration frequency (98.89%) and maximum shoot number (72) were achieved via direct organogenesis using sucker-derived nodal cuttings cultured on MS medium with 2 mg/L 6- benzyladenine (6-BA), 0.3 mg/L kinetin (KT), and 0.2 mg/L α-naphthaleneacetic acid (NAA). Under identical conditions, branch-derived explants showed lower regeneration (84.44%, 64 shoots). Transverse midvein incision proved most effective, with sucker-derived leaves exhibiting superior regeneration. Shoots elongated completely (100%) on Murashige and Skoog (MS) medium containing 0.3 mg/L 6-BA, 0.03 mg/L NAA, and activated charcoal. Rooting was optimal on MS medium with 0.3 mg/L indole-3-butyric acid (IBA), yielding a 98% acclimatization survival rate. Histological analysis revealed de novo meristem formation from parenchyma cells, confirming direct organogenesis without callus intermediation, further validating the enhanced regenerative capacity of sprout-derived explants. This efficient in vitro regeneration system provides a foundation for large-scale propagation and germplasm conservation of T. sebifera, while offering insights for woody plant regeneration studies. Full article

Cannabis sativa L. cv. ‘Cheungsam’ is an industrial hemp plant of Republic of Korea origin, primarily cultivated for fiber and seed production. In vitro seed germination and tissue culture are valuable tools for developing various biotechnological techniques. In the present study, we aimed to develop a tissue culture process for hemp plants using Cheungsam as a model plant and examine the secondary metabolites produced from its callus. We also developed a method to prepare pathogen-free seedlings from field-derived seeds using hydrogen peroxide (H₂O₂) solution as a liquid germination medium. Treating seedlings with removed seed coat in 3% H₂O₂ significantly reduced the contamination rate. Callus formation and de novo organogenesis of shoots and roots from callus were successfully achieved using cotyledon and leaf tissues prepared from the pathogen-free seedlings. The most effective in vitro regeneration results were obtained using the Murashige and Skoog (MS) medium supplemented with certain targeted growth regulators. An optimal combination of 0.5 mg/L thidiazuron (TDZ) and 1.0 mg/L 1-naphthalene acetic acid proved highly effective for callus induction. The addition of 0.5 mg/L TDZ in the MS medium significantly stimulated shoot proliferation, while robust root development was best supported by MS medium supplemented with 2.5 mg/L indole-3-butyric acid for both cotyledon and leaf explants. Finally, gas chromatography–mass spectrometry (GC–MS) analysis of ethanol extract from Cheungsam leaf callus revealed the presence of different secondary metabolites, including 9-octadecenamide, methyl salicylate, dodecane, tetradecane, and phenol, 2,4-bis-(1,1-dimethylethyl). This study provides a comprehensive de novo regeneration protocol for Cheungsam plants and insight into the secondary metabolite profiles of its callus. Full article

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output—adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members. Full article

Improving plant regeneration ability and shortening regeneration time can promote the development of genetic transformation breeding technology for horticultural crops. We optimized several culture conditions, including explant type, mother plant genotype, and medium, to improve shoot formation in winter squash (Cucurbita maxima Duch.). Histological analysis of the occurrence of shoots was also carried out. The results indicate that cotyledon was the most suitable explant for inducing the shoot regeneration of winter squash. We found that ‘Jin-li’ had a shorter shoot induction time and a higher average number of shoots. The highest induction rate of 95.23% among the five lines. The average shoot induction rate of five lines was the highest (84.85%) on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.2 mg/L indole-3-acetic acid (IAA). We also found that there was an interaction between genotypes and induction media, and their interaction had a greater impact on the shoot induction rate than individual effects. Histological observation revealed that the induced shoots of winter squash cotyledons originated from subepidermal cells. We also found that the optimal medium for de novo root regeneration was 1/2 MS. We acclimatized and cultivated regenerated plants and harvested their fruits, which maintained the characteristics of mother plants. These findings lay an important foundation for further research on direct shoot regeneration and accelerate its application in winter squash genetic transformation. Full article

Cultivated tomato (Solanum lycopersicum L.) is one of the most important horticultural crops in the world. The optimization of culture media for callus formation and tissue regeneration of different tomato genotypes presents numerous biotechnological applications. In this work, we have analyzed the effect of different concentrations of zeatin and indole-3-acetic acid on the regeneration of cotyledon explants in tomato cultivars M82 and Micro-Tom. We evaluated regeneration parameters such as the percentage of callus formation and the area of callus formed, as well as the initiation percentage and the number of adventitious shoots. The best hormone combination produced shoot-like structures after 2–3 weeks. We observed the formation of leaf primordia from these structures after about 3–4 weeks. Upon transferring the regenerating micro-stems to a defined growth medium, it was possible to obtain whole plantlets between 4 and 6 weeks. This hormone combination was applied to other genotypes of S. lycopersicum, including commercial varieties and ancestral tomato varieties. Our method is suitable for obtaining many plantlets of different tomato genotypes from cotyledon explants in a very short time, with direct applications for plant transformation, use of gene editing techniques, and vegetative propagation of elite cultivars. Full article

Kohlrabi (Brassica oleracea var. gongylodes) is a vegetable cultivated for its edible stem tuber. Although valued for its nutritional properties and tolerance to abiotic stress, kohlrabi is one of the least studied brassicas. In this review, we summarize the results of our decade-long research on in vitro morphogenesis of kohlrabi, starting from 2013. Protocols for efficient in vitro regeneration with minimal requirements for external application of plant growth regulators (PGRs) have been developed, both by somatic embryogenesis (SE) and by de novo shoot organogenesis (DNSO). Efficient regeneration by direct SE was achieved using immature zygotic embryos as explants incubated on PGR-free media, and the production process was maintained in culture thanks to highly efficient secondary SE. Conversely, efficient regeneration by indirect DNSO was achieved using entire seedlings as explants and adding only cytokinin (CK) without the need for exogenous auxin. Comprehensive phytohormone analyses revealed that different exogenously applied CKs differentially affected the composition of endogenous phytohormones and induced changes in the expression of cell cycle-related genes and other genes involved in the organogenic response. The addition of high sucrose concentrations to the nutrient media failed to induce the formation of stem tubers in in vitro culture, but revealed a complex interaction with exogenously applied CKs, interfering with both the endogenous phytohormonome and the expression of organogenesis-related genes. Our work has provided substantial biotechnological advances in the field of in vitro regeneration of kohlrabi, as well as in understanding the underlying phytohormonal regulation. The review aims to introduce kohlrabi to the scientific community as a model system for both basic and applied research, while we continue seeking answers to the outstanding questions and trying to pave the way for the development of more resistant varieties. Full article

Coconut (Cocos nucifera L.) is widely recognized as one of nature’s most beneficial plants. Makapuno, a special type of coconut with a soft, jelly-like endosperm, is a high-value commercial coconut and an expensive delicacy with a high cost of planting material. The embryo rescue technique is a very useful tool to support mass propagation of makapuno coconut. Nevertheless, transplanting the seedlings is a challenge due to poor root development, which results in the inability of the plant to acclimatize. In this study, primary root excision was used in makapuno to observe the effects of primary root excision on lateral root development. The overall results showed that seedlings with roots excised had a significantly higher number of lateral roots, and shoot length also increased significantly. Using de novo transcriptome assembly and differential gene expression analysis, we identified 512 differentially expressed genes in the excised and intact root samples. ERF071, encoding an ethylene-responsive transcription factor, was identified as a highly expressed gene in excised roots compared to intact roots, and was considered a candidate gene associated with lateral root formation induced by root excision in makapuno coconut. This study provides insight into the mechanism and candidate genes involved in the development of lateral roots in coconut, which may be useful for the future breeding and mass propagation of makapuno coconut through tissue culture. Full article

Some of the hormone crosstalk and transcription factors (TFs) involved in wound-induced organ regeneration have been extensively studied in the model plant Arabidopsis thaliana. In previous work, we established Solanum lycopersicum “Micro-Tom” explants without the addition of exogenous hormones as a model to investigate wound-induced de novo organ formation. The current working model indicates that cell reprogramming and founder cell activation requires spatial and temporal regulation of auxin-to-cytokinin (CK) gradients in the apical and basal regions of the hypocotyl combined with extensive metabolic reprogramming of some cells in the apical region. In this work, we extended our transcriptomic analysis to identify some of the gene regulatory networks involved in wound-induced organ regeneration in tomato. Our results highlight a functional conservation of key TF modules whose function is conserved during de novo organ formation in plants, which will serve as a valuable resource for future studies. Full article

Syngonium podophyllum Schott. cv. ‘White Butterfly’ is recognized as a valuable ornamental plant, and today it is also an important plant species of medicinal interest due to its high contents of phenolic compounds. The purpose of this article is to review the main scientific publications from our laboratory with regard to new scientific achievements dealing with Syngonium species or topics of interest, such as callus formation and further de novo shoot regeneration. The principles and stages necessary to start an industrial-level micropropagation protocol are discussed based on our experience. Different media compositions induced different morphogenetic responses inside the callus—particularly those related to the development of xylematic elements in the organogenetic areas, such as those for rooting, protocorms, and de novo shoot formation. The re-evaluation of old histological images revealed for the first time that xylematic elements are constantly closely positioned to all organogenetic centers, and that their development is closely dependent on the composition of the culture medium. Separate protocorms can be identified only when xylematic tracheary elements are well developed and closely connected to them. The formation of protocorms is strongly dependent on the mineral composition of the culture medium and the balance of plant growth regulators. Full article

The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1–T4), additionally including the effects of cytokinins (CKs)—trans-zeatin (transZ) and thidiazuron (TDZ)—and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA₂₀ increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses. Full article

In plants, stem cells are embedded in structures called meristems. Meristems can be formed either during embryogenesis or during the plant’s life such as, for instance, axillary meristems. While the regulation of the stem cell population in an established meristem is well described, how it is initiated in newly formed meristems is less well understood. Recently, two transcription factors of the NGATHA-like family, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2 have been shown to facilitate de novo stem cell initiation in Arabidopsis thaliana axillary meristems. Here, we tested whether the DPA4 and SOD7 genes had a similar role during stem cell formation in embryo shoot apical meristems. Using DPA4 and SOD7 reporter lines, we characterized the expression pattern of these genes during embryo development, revealing only a partial overlap with the stem cell population. In addition, we showed that the expression of a stem cell reporter was not modified in dpa4-2 sod7-2 double mutant embryos compared to the wild type. Together, these observations suggest that DPA4 and SOD7 are not required for stem cell specification during embryo shoot apical meristem initiation. This work stresses the difference in the regulatory network leading to meristem formation during the embryonic and post-embryonic phases. Full article

Plants have a remarkable regenerative capacity, which allows them to survive tissue damage after biotic and abiotic stresses. In this study, we use Solanum lycopersicum ‘Micro-Tom’ explants as a model to investigate wound-induced de novo organ formation, as these explants can regenerate the missing structures without the exogenous application of plant hormones. Here, we performed simultaneous targeted profiling of 22 phytohormone-related metabolites during de novo organ formation and found that endogenous hormone levels dynamically changed after root and shoot excision, according to region-specific patterns. Our results indicate that a defined temporal window of high auxin-to-cytokinin accumulation in the basal region of the explants was required for adventitious root formation and that was dependent on a concerted regulation of polar auxin transport through the hypocotyl, of local induction of auxin biosynthesis, and of local inhibition of auxin degradation. In the apical region, though, a minimum of auxin-to-cytokinin ratio is established shortly after wounding both by decreasing active auxin levels and by draining auxin via its basipetal transport and internalization. Cross-validation with transcriptomic data highlighted the main hormonal gradients involved in wound-induced de novo organ formation in tomato hypocotyl explants. Full article

The development of above-ground lateral organs is initiated at the peripheral zone of the shoot apical meristem (SAM). The coordination of cell fate determination and the maintenance of stem cells are achieved through a complex regulatory network comprised of transcription factors. Two AP2/ERF transcription factor family genes, ESR1/DRN and ESR2/DRNL/SOB/BOL, regulate cotyledon and flower formation and de novo organogenesis in tissue culture. However, their roles in post-embryonic lateral organ development remain elusive. In this study, we analyzed the genetic interactions among SAM-related genes, WUS and STM, two ESR genes, and one of the HD-ZIP III members, REV, whose protein product interacts with ESR1 in planta. We found that esr1 mutations substantially enhanced the wus and stm phenotypes, which bear a striking resemblance to those of the wus rev and stm rev double mutants, respectively. Aberrant adaxial–abaxial polarity is observed in wus esr1 at relatively low penetrance. On the contrary, the esr2 mutation partially suppressed stm phenotypes in the later vegetative phase. Such complex genetic interactions appear to be attributed to the distinct expression pattern of two ESR genes because the ESR1 promoter-driving ESR2 is capable of rescuing phenotypes caused by the esr1 mutation. Our results pose the unique genetic relevance of ESR1 and the SAM-related gene interactions in the development of rosette leaves. Full article

Plants have remarkable regenerative capacity, which allows them to survive tissue damage after exposure to biotic and abiotic stresses. Some of the key transcription factors and hormone crosstalk mechanisms involved in wound-induced organ regeneration have been extensively studied in the model plant Arabidopsis thaliana. However, little is known about the role of metabolism in wound-induced organ formation. Here, we performed detailed transcriptome analysis and used a targeted metabolomics approach to study de novo organ formation in tomato hypocotyl explants and found tissue-specific metabolic differences and divergent developmental pathways. Our results indicate that successful regeneration in the apical region of the hypocotyl depends on a specific metabolic switch involving the upregulation of photorespiratory pathway components and the differential regulation of photosynthesis-related gene expression and gluconeogenesis pathway activation. These findings provide a useful resource for further investigation of the molecular mechanisms involved in wound-induced organ formation in crop species such as tomato. Full article

The metallophyte Imperata cylindrica inhabits copper (Cu) polluted soils in large areas from Central Chile. Here, we subjected clonal vegetative plantlets to 300 mg Cu kg⁻¹ of substrate for 21 days to identify the main molecular pathways involved in the response to Cu stress. Transcriptomic analyses were performed for shoots and roots, with and without Cu supply. RNA-Seq and de novo transcriptome assembly were performed to identify the gene response associated with molecular mechanisms of Cu tolerance in I. cylindrica. De novo transcriptome revealed a total of 200,521 transcripts (1777 bp) comprising ~91% complete ultra-conserved genes in the eukaryote and Plantae database. The differentially expressed genes (DEGs) in roots were 7386, with 3558 of them being up-regulated and the other 3828 down-regulated. The transcriptome response in shoots was significantly less, showing only 13 up-regulated and 23 down-regulated genes. Interestingly, DEGs mainly related with actin and cytoskeleton formation, and to a minor degree, some DEGs associated with metal transporters and superoxide dismutase activity in root tissues were found. These transcriptomic results suggest that cytoskeleton could be acting as a mechanism of Cu-binding in the root, resulting in a high Cu tolerance response in this metallophyte, which deserve to be analyzed ultra-structurally. Our study contributes to reinforcing the potential of I. cylindrica as a candidate plant species to be used as a phytoremediation agent in Cu-contaminated environments. Full article