Effect of gamma irradiation on proliferation and growth of friable embryogenic callus and in vitro nodal cuttings of ugandan cassava genotypes.

Cassava (Manihot esculenta Crantz) production and productivity in Africa is affected by two viral diseases; cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Induced mutagenesis of totipotent/embryogenic tissues or in vitro plant material can lead to the generation of CMD and/or CBSD tolerant mutants. To massively produce non-chimeric plants timely and with less labor, totipotent cells or tissues are a pre-requisite. This study aimed to determine the effect of gamma radiation on the proliferation and growth of friable embryogenic callus (FEC) and in vitro nodal cuttings respectively. To obtain FEC, 2-6 mm sized leaf lobes of nine cassava genotypes were plated on Murashige and Skoog (MS) basal media supplemented with varying levels (37, 50, 70, 100) µM of picloram for production of organized embryogenic structures (OES). The OES of five cassava genotypes (Alado, CV-60444, NASE 3, NASE 13 and TME 204) were crushed and plated in Gresshoff and Doy (GD) basal media in combination with the amino acid tyrosine in varying concentrations for FEC production. FEC from five cassava genotypes and in vitro nodal cuttings of nine genotypes were irradiated using five different gamma doses (0, 5, 10, 15, 20 and 25 Gy) at a dose rate of 81Gy/hr. The lethal dose (LD)50 was determined using the number of roots produced and flow cytometry was done to determine the ploidy status of plants. The highest production of OES was noted in Alado across varying picloram concentrations, while TME 204 obtained the highest amount of FEC. The irradiated FEC gradually died and by 28 days post irradiation, FEC from all five cassava genotypes were lost. Conversely, the irradiated in vitro nodal cuttings survived and some produced roots, while others produced callus. The LD50 based on number of roots varied from genotype to genotype, but plants remained diploid post-irradiation. Accordingly, the effect of gamma irradiation on Ugandan cassava genotypes (UCGs) was genotype-dependent. This information is foundational for the use of in vitro tissues as target material for cassava mutation breeding.

Green synthesized iron oxide nanoparticles as a potential regulator of callus growth, plant physiology, antioxidative and microbial contamination in Oryza sativa L.

In tissue culture, efficient nutrient availability and effective control of callus contamination are crucial for successful plantlet regeneration. This study was aimed to enhance callogenesis, callus regeneration, control callus contamination, and substitute iron (Fe) source with FeO-NPs in Murashige and Skoog (MS) media. Nanogreen iron oxide (FeO-NPs) were synthesized and well characterized with sizes ranging from 2 to 7.5 nm. FeO-NPs as a supplement in MS media at 15 ppm, significantly controlled callus contamination by (80%). Results indicated that FeCl3-based FeO-NPs induced fast callus induction (72%) and regeneration (43%), in contrast FeSO4-based FeO-NPs resulted in increased callus weight (516%), diameter (300%), number of shoots (200%), and roots (114%). Modified media with FeO-NPs as the Fe source induced fast callogenesis and regeneration compared to normal MS media. FeO-NPs, when applied foliar spray, increased Plant fresh biomass by 133% and spike weight by 350%. Plant height increased by 54% and 33%, the number of spikes by 50% and 265%, and Chlorophyll content by 51% and 34% in IRRI-6 and Kissan Basmati, respectively. Additionally, APX (Ascorbate peroxidase), SOD (Superoxide dismutase), POD (peroxidase), and CAT (catalase) increased in IRRI-6 by 27%, 29%, 283%, 62%, while in Kissan Basmati, APX increased by 70%, SOD decreased by 28%, and POD and CAT increased by 89% and 98%, respectively. Finally, FeO-NPs effectively substituted Fe source in MS media, shorten the plant life cycle, and increase chlorophyll content as well as APX, SOD, POD, and CAT activities. This protocol is applicable for tissue culture in other cereal crops as well.

Bone and periosteum protein analysis via tandem mass tag quantitative proteomics in pediatric patients with osteomyelitis.

Bone healing is crucial in managing osteomyelitis after fracture fixation. Understanding the mechanism of extensive callus formation in pediatric osteomyelitis is highly important. This study aims to analyze bone and periosteum samples from pediatric patients to elucidate the essential processes involved in callus formation during osteomyelitis. The study included eight patients from our hospital: four with positive microbial culture who underwent osteomyelitis debridement and four who had osteotomy surgery as contral. We used tandem mass tag quantitative proteomics to investigate proteomic changes in bone and periosteum tissues obtained from these patients. Differential expression proteins were analyzed for their pathways through Gene Ontology (GO) annotation, GO enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction networks. A total of 4737 proteins were successfully identified. About 2224 differentially expressed proteins were detected in the bone tissues group and periosteum tissues group. Among the differentially expressed proteins, 10 protein genes in the bone group were associated with inflammation and osteogenesis, while in the periosteum group were nine. Cytochrome b-245, beta polypeptide (CYBB), nicotinamide phosphoribosyltransferase (NAMPT), tissue inhibitor of metalloproteinases 1 (TIMP-1), Raf-1 proto-oncogene, serine/threonine kinase (RAF-1), RELA proto-oncogene, NF-KB subunit (RELA), and sphingomyelin synthase 2 (SGMS2) may play an important role in callus formation in patients with osteomyelitis. This study provides novel clues for understanding callus formation in pediatric patients with osteomyelitis.

Utility of ultrasound imaging in monitoring fracture healing in rat femur: Comparison with other imaging modalities.

Objective: Fractures are common injuries and various imaging modalities are employed to diagnose and monitor bone union. However, the follow-up of fracture healing using ultrasound imaging (US) remains a topic of debate. In this study, we analyzed of fracture healing process and compared US and radiological analyses with histological analyses to clarify the characteristics and limitations of each modality. Methods: An osteotomy model was created using the femur of Wistar rats, and US, radiological (radiography and micro-computed tomography (micro-CT)), and histological analyses were performed. Radiological assessments were conducted for the evaluation of calcified tissue. The gap between the bony callus and cartilaginous callus was measured. Results: US effectively captured changes on the fracture surface, potentially reflecting the early healing processes. Both US and radiographic findings showed strong correlation in terms of the decrease in the bony callus gap. US was unable to distinguish cartilaginous callus from the surrounding soft tissue. During the remodeling stage, micro-CT offered a detailed assessment of the internal fracture surface, whereas US was limited to evaluating the outer bone surface and lacked accuracy in visualizing the entire fracture site. Radiography provided a general overview of the fractures. The decrease in the bony callus gap measured using US correlated with the reduction in cartilaginous callus observed histologically. Conclusion: This study demonstrated that US could be a valuable tool for evaluating fracture healing. Combining fracture management with US and radiological examinations may provide a more accurate assessment of healing progress.

Elicitor-mediated simultaneous accumulation of phloridzin and ursolic acid in Annurca apple peel-derived calli.

BACKGROUND: Apple peel is rich in natural molecules, many exhibiting a significant bioactivity. In this study, our objective was to establish a novel callus line derived from the apple peel of the Italian local variety Annurca, known to accumulate high levels of dihydrochalcones and terpenes. In this regard, we tested the impact of one elicitor, yeast extract, on the expression of genes encoding key enzymes involved in phloridzin and ursolic acid biosynthesis, leading to the accumulation of these antioxidant compounds. We also assessed the bioactivity of callus extracts enriched in these phytochemicals. RESULTS: After the elicitation, data showed increased expression of genes directly related to the synthesis of phloridzin and ursolic acid that were found to accumulate within the cultures. This presumably could explain the remarkable activity of extracts from the elicited-calli in inhibiting the growth of Staphylococcus aureus and Bacillus cereus. Also, the extracts enriched in antioxidant compounds inhibited reactive oxygen species (ROS) production in human cells exposed to ultraviolet-A (UV-A) radiation. CONCLUSION: Our results underscore the vast potential of the Annurca apple peel cell line in producing natural compounds that can be employed as food components to promote human health. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Targeted regulation of 5-aminolevulinic acid enhances flavonoids, anthocyanins and proanthocyanidins accumulation in Vitis davidii callus.

BACKGROUND: Spine grape (Vitis davidii) is a promising source of high-quality anthocyanins, with vast potential for application in food, pharmaceutical, and cosmetic industries. However, their availability is limited by resource constraints. Plant cell culture has emerged as a valuable approach for anthocyanin production and serves as an ideal model to investigate the regulation of anthocyanin biosynthesis. Elicitors are employed to achieve targeted enhancement of anthocyanin biosynthesis. The present study investigated the impact of 5-aminolevulinic acid (ALA) as an elicitor on the accumulation of anthocyanins and flavonoids during spine grape callus growth. Specifically, we examined the effects of ALA on anthocyanin and its component accumulation in callus, and biosynthetic anthocyanin gene expression. RESULTS: ALA at 25 µg/L increased the biomass of spine grape callus. ALA induction enhanced the levels of flavonoids, anthocyanins and proanthocyanidins in callus, with maximum values reaching 911.11 mg/100 g DW, 604.60 mg/100 g DW, and 5357.00 mg/100 g DW, respectively, after callus culture for 45 days. Notably, those levels were 1.47-, 1.93- and 1.83-fold higher than controls. ALA induction modulated the flavonoid profile, and among 97 differential flavonoid metabolites differing from controls, 77 were upregulated and 20 were downregulated. Six kinds of anthocyanins, namely cyanidin (8), delphinidin (6), peonidin (5), malvidin (4), petunidin (3) and pelargonidin (3), were detected in callus, with peonidin most abundant. Compared with controls, anthocyanin components were increased in ALA-treated callus. The key genes PAL1, PAL2, PAL4, CHI, CHS3, F3'H, F3H, FLS, DFR, UFGT, MYBA1, LDOX, OMT3, GT1 and ACT involved in anthocyanin biosynthesis were upregulated following ALA treatment, resulting in anthocyanin accumulation. CONCLUSION: This study revealed a novel mode of ALA-mediated promotion of plant anthocyanin biosynthesis and accumulation at the cellular level, and a strategy for enhancing anthocyanin content in spine grape callus. The findings advance commercial-scale production of anthocyanins via spine grape callus culture. we also explored the accumulation patterns of flavonoids and anthocyanins under ALA treatment. Augmentation of anthocyanins coincided with elevated expression levels of most genes involved in anthocyanin biosynthesis within spine grape callus following ALA treatment.

H3K4me3 changes occur in cell wall genes during the development of Fagopyrum tataricum morphogenic and non-morphogenic calli.

Epigenetic changes accompany the dynamic changes in the cell wall composition during the development of callus cells. H3K4me3 is responsible for active gene expression and reaction to environmental cues. Chromatin immunoprecipitation (ChIP) is a powerful technique for studying the interplay between epigenetic modifications and the DNA regions of interest. In combination with sequencing, it can provide the genome-wide enrichment of the specific epigenetic mark, providing vital information on its involvement in the plethora of cellular processes. Here, we describe the genome-wide distribution of H3K4me3 in morphogenic and non-morphogenic callus of Fagopyrum tataricum. Levels of H3K4me3 were higher around the transcription start site, in agreement with the role of this mark in transcriptional activation. The global levels of methylation were higher in the non-morphogenic callus, which indicated increased gene activation compared to the morphogenic callus. We also employed ChIP to analyse the changes in the enrichment of this epigenetic mark on the cell wall-related genes in both calli types during the course of the passage. Enrichment of H3K4me3 on cell wall genes was specific for callus type, suggesting that the role of this mark in cell-wall remodelling is complex and involved in many processes related to dedifferentiation and redifferentiation. This intricacy of the cell wall composition was supported by the immunohistochemical analysis of the cell wall epitopes' distribution of pectins and extensins. Together, these data give a novel insight into the involvement of H3K4me3 in the regeneration processes in F. tataricum in vitro callus tissue culture.

Exosome-like Nanoparticles, High in Trans-δ-Viniferin Derivatives, Produced from Grape Cell Cultures: Preparation, Characterization, and Anticancer Properties.

Background: Recent interest in plant-derived exosome-like nanoparticles (ENs) has surged due to their therapeutic potential, which includes antioxidant, anti-inflammatory, and anticancer activities. These properties are attributed to their cargo of bioactive metabolites and other endogenous molecules. However, the properties of ENs isolated from plant cell cultures remain less explored. Methods: In this investigation, grape callus-derived ENs (GCENs) were isolated using differential ultracentrifugation techniques. Structural analysis through electron microscopy, nanoparticle tracking analysis, and western blotting confirmed that GCENs qualify as exosome-like nanovesicles. Results: These GCENs contained significant amounts of microRNAs and proteins characteristic of plant-derived ENs, as well as trans-δ-viniferin, a notable stilbenoid known for its health-promoting properties. Functional assays revealed that the GCENs reduced the viability of the triple-negative breast cancer cell line MDA-MB-231 in a dose-dependent manner. Moreover, the GCENs exhibited negligible effects on the viability of normal human embryonic kidney (HEK) 293 cells, indicating selective cytotoxicity. Notably, treatment with these GCENs led to cell cycle arrest in the G1 phase and triggered apoptosis in the MDA-MB-231 cell line. Conclusions: Overall, this study underscores the potential of grape callus-derived nanovectors as natural carriers of stilbenoids and proposes their application as a novel and effective approach in the management of cancer.

Nicotinamide mononucleotide enhances fracture healing by promoting skeletal stem cell proliferation.

The process of skeletal regeneration initiated by stem cells following injury, especially in fractures, is significantly impaired by aging and adverse factors. Nicotinamide mononucleotide (NMN), a critical endogenous precursor of nicotinamide adenine dinucleotide (NAD), has garnered extensive attention for its multifaceted regulatory functions in living organisms and its wide-ranging therapeutic potential. However, whether NMN contributes to trauma-induced skeletal regeneration remains unclear. Methods: The transverse femoral shaft fracture model was employed to evaluate the potential advantages of NMN administration for overall repair during the initial fracture stages in male mice through micro-CT analysis, histochemistry, and biomechanical testing. The pro-proliferative function of NMN on skeletal stem cells (SSCs) was investigated through flow cytometry, qRT-PCR, NAD content measurement, and cell proliferation assay. Results: In this study, we observed that the administration of NMN during the initial phase of fracture in mice led to a larger callus and corresponding improvement in micro-CT parameters. NMN enhances the cartilaginous component of the callus by elevating the NAD content, consequently accelerating subsequent endochondral ossification and the fracture healing process. Subsequent analyses elucidated that NMN was beneficial in promoting the expansion of diverse stem cells in vivo and in vitro potentially via modulation of the Notch signaling pathway. Moreover, the depletion of macrophages profoundly obstructs the proliferation of SSCs. Conclusion: Our discoveries provide a potential strategy for enhancing fracture healing through stimulation of callus SSC proliferation at an early stage, shedding light on the translational value of NMN as an enhancer for skeletal regeneration and highlighting the pivotal role of macrophage-stem cell interactions in governing the regenerative influence of NMN on stem cells.

Exogenous Cytokinin Induces Callus and Protocorm-Like-Bodies Formation in In Vitro Root Tips of Vanilla planifolia Andrews.

Vanilla is a popular flavouring essence derived from the pods of vanilla orchid plants. Due to the high demand for vanilla flavour, high yielding vanilla plantlets are necessary for establishing vanilla plantations. Clonal micropropagation is a viable technique for the mass production of high yielding vanilla plantlets. This study reports an efficient regeneration protocol by using cytokinin as the sole plant growth regulator to regenerate plantlets from the root tips of a commercial vanilla orchid species, Vanilla planifolia. Most studies to date have reported using seeds and nodes as starting explants for in vitro micropropagation of vanilla orchids. So far, regeneration from roots has not been very successful. Previous studies favoured the use of auxins only or high auxin to cytokinin ratios to induce callus, and sole cytokinins were used for direct shoot regeneration. However, it was sporadically observed in plantlets regeneration of V. planifolia that multiple shoots were regenerated from the tips of intact aerial roots submerged in media. This study therefore investigated the regeneration of excised vanilla root tips through the application of most commonly used auxins (1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid) and cytokinins (6-benzylaminopurine and thidiazuron). High auxin presence is known to promote callusing in in vitro plants. However, in this study, auxin treatment inhibits callusing in root tips. While cytokinin treatments, even at low levels, has promoted high rate of callusing. These callus cells regenerate into protocorm-like-body (PLB) shoots when cytokinin levels are increased to 0.5 mg/mL 6-benzylaminopurine (BAP) under light conditions. The findings of the study have the potential of providing large quantity of high yielding vanilla plantlets through clonal micropropagation.

Preliminary establishment of genetic transformation system for embryogenic callus of Acer truncatum 'Lihong'.

Introduction: Acer truncatum Bunge, belonging to the Acer genus in the Aceraceae family, is a commonly planted afforestation species across China, Japan, Korea, Europe, and North America. Renowned for its vibrant fall colors, it holds significant ecological and ornamental value. Methods: In this study, Acer truncatum ' Lihong ' was used as the research object. Starting from the callus induction of explants, the embryogenic callus of Acer truncatum 'Lihong' was obtained by systematically optimizing the medium and culture conditions. Then, the candidate gene AtrGST894 screened by transcriptome sequencing was transformed into embryogenic callus by Agrobacterium-mediated transformation. The genetic transformation system of Acer truncatum 'Lihong' embryogenic callus was initially established by continuously adjusting the conditions of Agrobacterium tumefaciens infection receptor materials, thus laying a material foundation for the study of the molecular regulation mechanism of Acer truncatum 'Lihong' leaf color, and also preparing for the later molecular improvement breeding of Acer truncatum. Therefore, this study has important theoretical and practical significance. Results: The results showed that the best medium for callus induction of Acer truncatum was 1/2MS+2 mg/L 2,4-D+0.3 mg/L 6-BA+0.5 mg/L NAA; The embryogenic callus induction medium of Acer truncatum was 1/2MS+3.0mg/L 6-BA+2.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA3; The proliferation medium of embryogenic callus of Acer truncatum was WPM+1.0mg/L TDZ+0.5mg/L IBA+0.1mg/L GA3+3mg/L 6-BA+1.0mg/L KT; The infection experiment of Agrobacterium tumefaciens on the embryogenic callus of Acer truncatum showed that the best antibacterial medium was WPM+30g/L sucrose+8g/L agar+0.5g/L acid-hydrolyzed casein+0.2mg/L KT+1.0 mg/L TDZ+0.5 mg/L IBA+0.1 mg/L GA3+200mmol/L carboxybenzyl+200mg/L cephalosporin, and then WPM+30g/L sucrose+8g/L agar+0.5g/L acid-hydrolyzed casein+0.2mg/L KT+1.0 mg/L TDZ+0.5 mg/L IBA+0.1 mg/L GA3+300mmol/L carboxybenzyl+200mg/L cephalosporin+25mg/L hygromycin. Screening medium screening, The obtained embryogenic callus browning rate, pollution rate and mortality rate were the lowest, and maintained vigorous growth. Discussion: The embryogenic callus was used as the infection material to verify that we successfully transferred the target gene into the embryogenic callus, which means that the genetic transformation system of Acer truncatum embryogenic callus was partially completed, and the infection process could be effectively inhibited. Although there was partial browning, it could continue to proliferate. Therefore, in future experiments, the focus is still to continue to verify the optimal conditions for optimizing the genetic transformation of Acer truncatum embryogenic callus and to solve the problems of difficulty in embryonic callus germination.

Novel mechanism of MicroRNA408 in callus formation from rice mature embryo.

Mature embryos are the main explants of tissue culture used in rice transgenic technology. However, the mechanism of mature embryo callus formation remains unclear. In this study, a microRNA-mediated gene regulatory network of rice calli was established using degradome sequencing. We identified a microRNA, OsmiR408, that regulates the formation of the callus derived from the mature rice embryo. OsUCLACYANIN 30 (OsUCL 30), a target gene of OsmiR408, was the most abundant cleavage mRNA in rice callus. OsUCL17 was verified as a target gene of OsmiR408 using RNA ligase-mediated 5'-RACE. In analysis of the OsmiR408 promoter reporter line and pri-miR408 transcript level, the promoter activity and transcript level of MIR408 were increased dramatically during callus formation. In phenotypic observations, OsmiR408 knockout caused severe defects in mature embryo callus formation, whereas OsmiR408 overexpression promoted callus formation. Transcriptome analysis demonstrated that OsUCLs and certain genes related to the plant hormone signal transduction and phenylpropanoid-flavonoid biosynthesis pathway had different differential expression patterns between OsmiR408 knockout and overexpression calli. Thus, OsmiR408 may regulate callus formation mainly by affecting plant hormone signal transduction and phenylpropanoid-flavonoid biosynthesis pathway. Our findings provide insight into OsmiR408/UCLs module function in callus formation.

Rice (Oryza sativa) Stem Cells as a Novel Promising Active Ingredient with Anti-Proliferative Effects for Potential Skin Cancer Prevention and Skin Whitening Activity.

Rice is one of the plants proven to possess antioxidant, anti-inflammatory, anti-proliferative, and whitening properties, making it one of the most beneficial plants in this regard. This study aimed to introduce a novel natural cosmetic and pharmaceutical product based on rice callus as a source of active ingredients that can inhibit skin melanoma cell (B16F10) proliferation and brighten the skin. The 2,4-D hormone at concentrations of 1 µg/mL and 1.5 µg/mL was used to induce rice callus formation. Rice callus extracts were then prepared using aqueous and ethanolic solvents, with a concentration of 1 mg/mL used for characterization tests. To determine the optimal hormone concentration, the phenols/flavonoids, antioxidant activity, proteins, and carbohydrates in the extracts were measured. The optimal concentration of the hormone was found to be 1 µg/mL. Finally, the anti-melanocyte and skin-whitening activity of the extracts was assessed through measurements of their cytotoxicity and inhibition of melanin synthesis-related enzymes. Cellular cytotoxicity measurements revealed that the ethanolic extract induced more cytotoxicity than the aqueous extract, with IC50 values of 566.3 µg/mL for the ethanolic extract and 1327 µg/mL for the aqueous extract. Skin-whitening-related tests demonstrated that the extracts were 1.7 times more effective than arbutin in inhibiting factors that cause hyperpigmentation. The aqueous extract achieved 85% inhibition of melanin biosynthesis at a concentration of 3200 µg/mL, compared to 68% for the ethanolic extract and 50% for arbutin. Based on these findings, rice callus extract can be introduced as a new, effective substance for skin-lightening and anti-melanocyte products in cosmeceutical and pharmaceutical formulations.

In Vitro Germination and Organogenesis of Endangered Neo-Endemic Baltic Dunes Species Linaria loeselii Schweigg.

This study focuses on the endangered neo-endemic Baltic dunes species Linaria loeselii Schweigg. (Plantaginaceae), also known as Linaria odora (M. Bieb.). By utilizing in vitro cultures, we successfully germinated seeds collected in situ. Our method, which involved using media supplemented with 5 µmol/L 6-benzylaminopurine, led to the indirect regeneration of shoots after 60 days of culture in the dark, significantly increasing the number of progeny plants. Additionally, the medium supplemented with 2.85 µmol/L indole-3-acetic acid and 10.2 µmol/L paclobutrazol allowed rooting after 30 days of shoot fragments. This research provides a potential basis for developing Linaria loeselii introduction programs into the environment, thereby contributing to the conservation of this endangered species.

Induction and Suspension Culture of Panax japonicus Callus Tissue for the Production of Secondary Metabolic Active Substances.

Using Panax japonicus as research material, callus induction and culture were carried out, and high-yielding cell lines were screened to establish a suspension culture system that promotes callus growth and the accumulation of the "total saponins" (total content of triterpenoid glycosides or ginsenosides). Using the root as an explant, the medium for callus induction and proliferation was optimized by adjusting culture conditions (initial inoculation amount, carbon source, shaking speed, hormone concentration, culture time) and a high-yielding cell line with efficient proliferation and high total saponins content was screened out. The conditions of suspension culture were refined to find out the most suitable conditions for the suspension culture of callus, and finally, the suspension culture system was established. We found that the lowest (5%) contamination rate was achieved by disinfecting the fresh roots with 75% alcohol for 60 s, followed by soaking in 10% NaClO for 15 min. The highest induction rate (88.17%) of callus was obtained using the medium MS + 16.11 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 sucrose + 7.5 g·L-1 agar. The callus was loose when the callus subcultured on the proliferation medium (MS + 5.37 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 sucrose + 3.8 g·L-1 gellan gum) for 21 days. The callus growth was cultured in a liquid growth medium (MS + 5.37 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 sucrose) with an initial inoculation amount of 40 g·L-1, a shaking speed of 110 r/min and darkness. Cell growth was fastest with a culture period of 21 days. We replaced the growth medium with the production medium (MS + 5.37 µmol·L-1 NAA + 13.32 µmol·L-1 6-BA + 30.0 g·L-1 glucose) for maximum accumulation of total saponins. [Conclusion] A callus induction and suspension culture system for the root of P. japonicus was established. In this way, we can promote the accumulation of total saponins in callus cells and provide a basis for large-scale cell culture and industrial production of medicinal total saponins.

Generic Workflow of a Highly Effective and Easy Anther Culture Method for Both Japonica and Indica Rice.

As one of the most important staple crops in the world, rice plays a pivotal role in world food security. The creation of doubled haploids based on anther culture is an important technology for rice breeding. However, at present, rice anther culture technology still faces many problems, such as genotype dependency, especially genotypes of indica rice. In this study, fifteen rice genotypes, including twelve japonica rice genotypes and three indica rice genotypes, were randomly selected and used to study anther culture by using a modified M8 medium. The results showed that the total callus induction rates of these different rice genotypes ranged from 0.81 to 13.95%, with an average of 6.64%, while the callus induction rates calculated for the top ten highest callus inductions for each rice genotype ranged from 2.75 to 17.00%, with an average of 10.56%. There were varying gaps between the total callus induction rates and the callus induction rates in these different rice genotypes. The fact that the gaps for some rice genotypes were relatively large indicated that standard tiller or anther collection was not applicable to all rice genotypes and that there was still a lot of room for improvement in the callus induction rate of some rice genotypes through optimization of the sampling method. The plantlet regeneration rates ranged from 12.55 to 456.54%, with an average of 200.10%. Although there were many albinos from anther culture for some rice genotypes, these would still meet the requirement if the rice genotypes had higher callus induction rates or regeneration rates. The percentages of seed setting of regenerated green seedlings ranged from 14% to 84%, with an average of 48.73%. Genetic diversity analysis showed that the genetic background of these different rice genotypes was representative, and the phylogenetic tree and Principal Component Analysis (PCA) divided them into indica and japonica types. Therefore, in this study, an anther culture method suitable for both indica and japonica rice genotypes was established, which could improve doubled haploid breeding in rice.

Silver Nanoparticles as Catalysts of Foeniculum vulgare L. Callus Formation and Its Content of Vitamins and some Fatty Acids.

Background: Plants are a precious resource of a wide range of secondary metabolites, that are benefitted as flavours, pharmaceuticals, colours, colognes, food additives and also biopesticides. Objective: The current study tested the impact of silver nanoparticles (AgNPs) on Foeniculum vulgare. Materials and Methods: Foeniculum vulgare seeds were surface sterilized, Vital leaf pieces are grown on MS media including diverse mixtures of plant growth regulators, the special effects of AgNPs plus PGRs on callus propagation were evaluated, and separated compounds of fatty acid and vitamins were identified. Results: Outcomes revealed that several intensities of AgNPs expressively influenced the callus propagation and significantly raised the callus biomass with combination including the plant growth regulators. Highest fresh (7.32 g.L-1) biomass addition of callus was remarked on the media elevated in vitro at 20 ppm AgNPs combined with (2 mg.L-1 2,4-D) and results noted that the callus appeared compact and greenish in colour with 40 ppm AgNPs in combination with (2 mg.L-1 2,4-D). The results elucidated the amplification of the value of both fatty acids (stearic acid (47.85 %), oleic acid (189.28 %), Linoleic (6.34 %) and Linolenic (0.83 %)), and vitamins (Vitamin E (8.99 U.mg-1) and vitamin A (27.19 U.mg-1) by using MS + 2,4-D (2 mg.L-1) + AgNPs (20ppm). Conclusion: Application of a combination of AgNPs along with PGRs led to callus proliferation in Foeniculum vulgare L. In vitro. But, the unaccompanied use of AgNPs was originate inductive in the biosynthesis of greater quantities of special fatty acids and vitamin metabolites.

Effects of Gamma Irradiation on Changes in Chemical Composition and Antioxidant Activity of Euphorbia maculata Callus.

In this study, we investigated the effects of gamma irradiation on the antioxidant activity and metabolite profiles of Euphorbia maculata calli (PC3012). Gamma irradiation at various doses (0, 0.05, 0.5, and 10 kGy) significantly enhanced the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS+) radical scavenging activities of the callus extracts of PC3012 in a dose-dependent manner. High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) analyses revealed that irradiation increased the lysophospholipid content, although no new antioxidant compounds were formed. Furthermore, a PLS-DA analysis revealed evident metabolic differences between non-irradiated and irradiated samples, which were further verified by statistical validation. These findings suggest that gamma irradiation induces specific biochemical modifications that enhance the bioactive properties of PC3012 calli. This technology exhibits potential for utilization in the natural product and food sectors, particularly in the development of functional foods and nutraceuticals with improved health benefits.

A point mutation in the IAA14 promoter enhances callus formation and regeneration.

Callus formation induced by auxin accumulation is considered the first step of in vitro plant regeneration. In Arabidopsis, degradation of the Aux/IAA protein, IAA14, in response to auxin signaling, which activates the AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 along with a series of downstream transcription factors, also plays a critical role in this process. However, the specific mechanism by which auxin regulates callus formation remains unclear. By screening mutant library in the solitary root 1 (iaa14/slr) Arabidopsis background we obtained the callus formation related 2 (cfr2) mutant. The cfr2 mutant exhibited a stronger capacity for callus formation, as well as lateral root and adventitious root regeneration from leaf explants than wild type (WT) seedlings, but did not recover gravitropism capability. The auxin signal in cfr2 was significantly enhanced, and the expression of some downstream transcription factors was increased. Map-based cloning, whole genome resequencing, and phenotypic complementation experiments showed that the phenotypes observed in the cfr2 mutant were caused by a point mutation in the IAA14 promoter region. This mutation, which is predicted to disrupt the binding of LBD16, LBD19, and LBD30 to the IAA14 promoter, changed the expression pattern of IAA14 in cfr2. Taken together, our results identified a new mutation in the IAA14 promoter region, which affects the expression pattern of IAA14 and in turn its ability to control plant regeneration. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01493-y.

Construction and optimization of a genetic transformation system for efficient expression of human insulin-GFP fusion gene in flax.

The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon, and a fusion gene expression vector of insulin combined with green fluorescent protein (GFP) was constructed. The optimization of the flax callus culturing was undertaken, and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved. The critical concentration values of hygromycin on the flax hypocotyl development, as well as on its differentiated callus, were explored by the method of antibiotic gradient addition, and the application of antibiotic screening for the verification of positive calluses was assessed. The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed, as confirmed through polymerase chain reaction and Western blotting. In conclusion, we have established a flax callus culture system suitable for insulin expression. By optimizing the conditions of the flax callus induction, transformation, screening, and verification of a transgenic callus, we have provided an effective way to obtain insulin. Moreover, the herein-employed flax callus culture system could provide a feasible, cheap, and environmentally friendly platform for producing bioactive proteins.