Search Results (31)

The establishment of a transient expression system in petals is significant for elucidating gene functions in flowering trees characterized by a prolonged juvenile phase. Genetic improvements in Camellia japonica have been hindered due to the absence of a functional validation platform. In this study, we explored an Agrobacterium-mediated and readily observable transient expression system in camellia petals to systematically optimize four critical factors affecting transformation efficiency. As a result, the bud stage, ‘Banliuxiang’ genotype, OD600 of 1.0, and 1-day co-cultivation achieved the highest intensity of transient expression, and overexpression of the Ruby1 reporter gene induced substantial anthocyanin synthesis, manifested as distinct red pigmentation. Furthermore, the optimized transient expression system revealed that the R2R3-MYB transcription factor CjMYB5, which interacted with CjGL3, promoted anthocyanin biosynthesis in camellia petals by transactivating key DFR structural genes. This transient expression platform not only advances functional genomics studies in ornamental woody species but also lays a foundation for molecular breeding programs in C. japonica. 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

The Silvermines Pb-Zn-Ag-Ba orebodies comprise vein, replacement, cross-cutting and stratiform mineralization mostly hosted in Lower Carboniferous limestones in the vicinity of a major ENE and E-W trending normal fault array and represent a classic example of Irish-Type Zn-Pb mineralization. Historically the deposits have been exploited at various times, but the major limestone-hosted Zn-Pb-Ba mineralization was not discovered until the 1960s. Structurally controlled crosscutting vein and breccia mineralization represent pathways of hydrothermal fluids escaping from the Silvermines fault at depth that exhaled and replaced shallowly buried Waulsortian limestones creating the larger stratiform orebodies such as the Upper G and B-Zones. The B-Zone, comprising a pre-mining resource of 4.64 Mt of 4.53% Zn, 3.58% Pb, 30 g/t Ag has a locally highly variable host mineralogy dominated by pyrite, barite, siderite, within dolomitic and limestone breccias with local silica-haematite alteration. A small, highly unusual pod of very high-grade Ag-rich mineralization in the B-Zone, the 4611 Pod, discovered in 1978, has not been previously documented. Unpublished records, field notes, and mineralogical and chemical data from consultant reports have been assimilated to document this interesting and unusual occurrence. The pod, representing an irregular lens of mineralization ca 2 m thick and representing 500 t, occurs within the B-Zone orebody and comprises high grade Zn and Pb sulfides with significant patches of proustite-pyrargyrite (ruby silvers) and a host of associated Pb, Ag, Sb, As, Cu, Ge sulfide minerals, including significant argyrodite. Although evidence of any distinct feeder below the pod is lacking, the nature of the pod, its unusual mineralogy and its paragenesis suggests that it represents a small, possibly late source of exotic hydrothermal fluid where it entered the B-Zone stratiform mineralizing system. Full article

Plukenetia volubilis L., a woody oilseed plant rich in α-linolenic acid, represents a promising source of polyunsaturated fatty acids. However, the lack of an efficient genetic transformation system has significantly hindered gene function research and molecular breeding in P. volubilis. In this study, we developed a highly efficient Agrobacterium rhizogenes-mediated hairy root transformation system for P. volubilis via the use of Agrobacterium gel in combination with the visually detectable RUBY reporter for gene function analysis in roots. The results indicate that the optimal transformation method involves infecting P. volubilis seedlings with Agrobacterium gel containing acetosyringone and inducing hairy root formation in perlite. This approach resulted in more than 18.97% of the seedlings producing positive hairy roots overexpressing the RUBY gene. Using this genetic transformation system, we successfully overexpressed the antimicrobial peptide-encoding gene CEMA in hairy roots, which enhanced the resistance of P. volubilis to Fusarium oxysporum. Furthermore, by combining this transformation system with the CRISPR-Cas9 tool, we validated the regulatory role of PvoSHR in the development of root epidermal cells in P. volubilis. Unexpectedly, a 123-bp DNA fragment from the T-DNA region of the A. rhizogenes Ri plasmid was found to be knocked in to the P. volubilis genome, replacing a 110-bp fragment of PvoSHR at CRISPR-Cas9 induced double-strand DNA breaks. Conclusively, this system provides a powerful tool for gene function research in P. volubilis and provides novel insights into the development of transformation and gene editing systems for other woody plants. Full article

Crassulaceae plants are valued for their horticultural, ecological, and economic significance, but their genetic improvement is hindered by the absence of efficient and stable genetic transformation methods. Therefore, the development of a tailored genetic transformation method is crucial for enhancing the progress of the genetic improvement of Crassulaceae plants. The results indicate that, in the transformation experiments conducted on Kalanchoe tetraphylla, the K599 strain exhibited the highest transformation efficiency (76.67%), while C58C1 was least efficient (21.43%). An acetosyringone concentration of 100 μM was optimal for the hairy root transformation, and the immersion method yielded the highest efficiency. Additionally, the Silwet L-77 concentration significantly influenced the transformation efficiency, with 0.05% leading to a decrease. Upon four Crassulaceae species, notable differences were observed, with K. tetraphylla exhibiting the highest efficiency of 100%, and Sedum alfredii displaying the lowest efficiency of 5%. The RUBY reporter gene offers a more distinct advantage over GFP in observing the transformation effects. This study developed a simple, feasible, and cost-effective method for obtaining transgenic roots from leaves of Crassulaceae. The methodology provides technical support for the genetic improvement and gene function research of Crassulaceae plants. Full article

Filamentous fungi associated with disease symptoms in non-native giant sequoia (Sequoiadendron giganteum) in Germany were investigated in ten cases of disease in Northwest Germany. During the study period from 2018 to 2023, a total of 81 species of Dikaria were isolated from woody tissue and needles of giant sequoia and morphotyped. Morphotypes were assigned to species designations based on ITS-sequence comparison and, in part, multi-locus phylogenetic analyses. Nine species were recognised as new reports for Germany or on giant sequoia: Amycosphaerella africana, Botryosphaeria parva, Coniochaeta acaciae, C. velutina, Muriformistrickeria rubi, Pestalotiopsis australis, P. monochaeta, Phacidiopycnis washingtonensis, and Rhizosphaera minteri. The threat posed to giant sequoia and other forest trees in Germany by certain, especially newly reported, fungal species is being discussed. The detection of a considerable number of new fungal records in the trees studied suggests that giant sequoia cultivation may be a gateway for alien fungal species in Germany. Full article

The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans. Full article

Lotus corniculatus is an important perennial legume forage species and has been widely used to study the relationships between plants and rhizobia or arbuscular mycorrhizal fungi. Composite plants (wild-type shoots with transgenic hairy roots) can be produced via genetic transformation mediated by Agrobacterium rhizogenes. The A. rhizogenes puncture method and a method of cutting embryonic roots and coating the hypocotyl incision with A. rhizogenes were used to induce hairy roots from seedlings. The identification of transgenic hairy roots from composite plants is also an important task. Here, young branches from adult plants were used as explants to produce composite plants instead of seedlings via the one-step cutting method in the natural environment. The results showed that the explants of young branches had higher transformation frequency than those of seedlings, and the composite plants obtained from the young branches had greater growth and were also more robust than the plants obtained from the seedlings. In this study, a system for the biosynthesis of betalains named RUBY was integrated into an expression vector as a reporter gene. It is very easy to distinguish transgenic roots from nontransgenic roots based on the red color of the hairy roots. Young branches were used as explants for hairy root transformation with higher transformation frequency than the seedlings. RUBY was used as a reporter gene to distinguish transgenic roots from nontransgenic roots based on the red color of the hairy roots. Full article

Insufficient endosomal escape presents a major hurdle for successful nucleic acid therapy. Here, for the first time, a chemical electron transfer (CET) system was integrated into small interfering RNA (siRNA) lipid nanoparticles (LNPs). The CET acceptor can be chemically excited using the generated energy between the donor and hydrogen peroxide, which triggers the generation of reactive oxygen species (ROS), promoting endosomal lipid membrane destabilization. Tetra-oleoyl tri-lysino succinoyl tetraethylene pentamine was included as an ionizable lipopeptide with a U-shaped topology for effective siRNA encapsulation and pH-induced endosomal escape. LNPs loaded with siRNA and CET components demonstrated a more efficient endosomal escape, as evidenced by a galectin-8-mRuby reporter; ROS significantly augmented galectin-8 recruitment by at least threefold compared with the control groups, with a p value of 0.03. Moreover, CET-enhanced LNPs achieved a 24% improvement in apoptosis level by knocking down the tumor-protective gene nuclear factor erythroid 2-related factor 2, boosting the CET-mediated ROS cell killing. Full article

This article reports on the recent mining and production status of ruby in Longido, Tanzania. Faceted-grade rubies and their matrix from Longido Area, Tanzania, were investigated by standard gemological testing, including FTIR, UV-VIS, Raman spectra, and LA-ICP-MS. Microscopic observations revealed dense needle-like and triangular inclusions, distinct growth lines, and color banding as typical inclusions. In agreement with the Raman results, the transmission FTIR spectrum confirmed the presence of aluminum hydroxide. The Raman spectra identified associated minerals and inclusions, including zoisite, parasites, feldspar within the matrix, rutile, and diaspore in the ruby host. The chemistry analysis revealed a high amount of Cr and relatively low iron as a good indicator of geographic origin. Full article

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO₂. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO₂ fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803. Full article

Rubi idaei fructus is a source of nutritionally important bioactive chemical compounds, mainly antioxidants, which strengthen the immune system and can be used in the prophylaxis and adjuvant therapies of many oxidative stress-induced diseases. There are no literature reports presenting a comprehensive comparative analysis of the antioxidant activity and nutritionally relevant metabolites contained in the fruits of repeat-fruiting raspberry cultivars, which are commonly grown in Europe. The aim of this study was to carry out a comparative analysis of the antioxidant potential (Folin–Ciocalteu, DPPH, FRAP), the content of selected primary and secondary metabolites, and the qualitative and quantitative composition of amino acids and fatty acids in the fruits of R. idaeus cv. ‘Pokusa’, ‘Polana’, and ‘Polka’. The fruits of the analyzed cultivars have a low caloric value (171–219 kcal/100 g); low content of available carbohydrates (6–6.6%) and total carbohydrates (3.4–4.8%); and high levels of dietary fiber (4.7–5.8%), vitamin C (22.8–27 mg/100 g), anthocyanins (25.1–29.6 mg/100 g), and flavonoids (0.5–2.6 mg/100 g). The fruits were found to contain valuable unsaturated fatty acids (35–60%), especially MUFAs with dominant oleic, elaidic, palmitic, and erucic acids and PUFAs (α-linolenic, eicosapentaenoic, and linoleic acids). MUFAs from the ω-9 group accounted for 12–18%, whereas the content of PUFAs from the ω-3 and ω-6 groups was in the range of 15–23 and 6–21%, respectively. Exogenous amino acids, accounting for 56–62%, were dominated by leucine, phenylalanine, and lysine. The following order of the total polyphenolic content was established in the fresh fruit juice from the analyzed cultivars: ‘Pokusa’ < ‘Polana’ < ‘Polka’. The different antioxidant capacity assays used in the study confirmed the high antioxidant potential of the fruits and fresh juice from the three R. idaeus cultivars. This indicates that raspberry fruits can serve as a source of nutrients and can be used as a valuable supplement in a healthy human diet and a raw material in the pharmaceutical and cosmetic industries. Full article

Water lentil, commonly known as duckweed, is an aquatic plant with great agronomic potential, as it can double its biomass in less than 24 h and contains up to 45% leaf proteins on a dry matter basis. However, extracting proteins from leaves is an arduous process due to the complexity of the matrix, which limits their uses in the food industry. In this study, water lentil protein extraction by solubilization was maximized using response surface methodology. By heating at 80 °C at pH 11 with a water lentil powder concentration of 2% or 4% for 2 h, up to 77.8% of total proteins were solubilized. Then, by precipitating the solubilized proteins at pH 4, a protein purity of 57.6% combined with a total protein yield of 60.0% was achieved. To the best of our knowledge, this is the highest leaf protein extraction yield reported in the literature with such protein purity. Proteomics analyses showed that the protein concentrate was composed of around 85.0% RubisCO, and protein structure analyses using ATR-FTIR and DSC were linked to a high protein solubility in water at pH 7. Moreover, a 1.5% protein solution of the protein concentrate at pH 7 showed excellent foaming properties compared to a 10.3% protein egg white solution. It had a superior foaming capacity (194% vs. 122%, respectively) for the same foaming stability after 60 min, which confirms water lentil proteins’ potential for human nutrition and food formulation. Full article

Seedlessness in grapes is much appreciated by consumers and especially in cultivars consumed either as table grapes or as raisins. In many parts of the world, low temperature is the main environmental stress limiting grape production. In this study, stenospermocarpic (seed abortion) cultivars were selected as the female parents while seeded cold-resistant cultivars were selected as the male parents to develop new cold-resistant seedless grapes using embryo rescue technology, which has previously been shown to be a highly efficient way of breeding seedless grapes. Here, we report optima in genotype, sampling time, and culture medium for the embryo rescue of 14 hybrid combinations. Our results indicate that the embryo development rate (39.9%) and the seedlings rate (21.5%) were highest among the 14 crosses when ‘Ruby Seedless’ was used as the female parent and ‘Beibinghong’ was used as the male parent. The best sampling times for ‘Yuehong Wuhe’, ‘Ruby Seedless’, and ‘Melissa seedless’ were 37, 55, and 52 days after flowering, respectively. Embryo rescue efficiency was highest when the sucrose concentration for seedlings was maintained at about 1.0%. Using molecular markers, we detected 91 hybrids with seedless traits and 18 hybrids with cold resistance traits. Full article

Betalains can be conveniently observed and quantified and, accordingly, have the potential as naked-eye visual screening reporters during plant transformation. RUBY is a new reporter system that uses “2A” peptides to fuse three key genes, CYP76AD1, DODA, and glucosyl transferase, for betalain biosynthesis, and has been successfully used for transformation of rice, Arabidopsis, and cotton, but its potential applications in the genetic transformation of various other plant species remain to be verified. In this study, RUBY was transferred into the hairy roots of Plukenetia volubilis and Nicotiana benthamiana, and was transferred into Arabidopsis by the floral-dip method. The expression levels of CYP76AD1, DODA, and glucosyl transferase were detected by RT−PCR and RT−qPCR, the relationship between the expression level of RUBY and red coloration was analyzed, and the genetic stability of RUBY in transgenic Arabidopsis was studied. The results showed that the expression of RUBY could reconstruct the betalain biosynthesis pathway in the hairy roots of P. volubilis, N. benthamiana, and Arabidopsis plants, indicating that it has the potential for versatile use across species. As a reporter, betalain did not affect callus induction, plant regeneration, development, or fertility. However, when used in plant transformation for observation and visual screening, it needed to accumulate to a certain extent to show red coloration, and it was positively correlated with gene expression. In general, RUBY is a convenient reporter for plant transformation, and has no obvious side effects during plant growth and development. However, the potential application of RUBY for visual screening is highly determined by the expression level, and further improvement is needed. Full article