Dark Culture Research Articles

Establishment of Agrobacterium-Mediated Transient Transformation System in Desert Legume Eremosparton songoricum (Litv.) Vass.

Eremosparton songoricum (Litv.) Vass. is a desert legume exhibiting extreme drought tolerance and the ability to withstand various harsh environments, making it a good candidate for investigating stress tolerance mechanisms and exploring valuable stress-resistant genes. However, the absence of a genetic transformation system for E. songoricum poses significant limitations for functionally validating these stress-resistant genes in situ. In this study, we developed an Agrobacterium-mediated transient transformation system for E. songoricum utilizing the β-glucuronidase (GUS) gene as a reporter. We investigated three types of explants (seedlings, assimilated branches and callus) and the effects of different Agrobacterium strains, seedling ages, OD600 values, acetosyringone (AS) concentrations, sucrose concentrations and infection times on the transformation efficiency. The results reveal that the optimal transformation system was infecting one-month-old regenerating assimilated branches with the Agrobacterium strain C58C1. The infection solution comprised 1/2 MS medium with 3% sucrose and 200 μM AS at an OD600 of 0.8, infection for 3 h and then followed by 2 days of dark cultivation, which achieving a maximum transformation rate of 97%. The maximum transformation rates of the seedlings and calluses were 57.17% and 39.51%, respectively. Moreover, we successfully utilized the assimilated branch transient transformation system to confirm the role of the previously reported transcription factor EsDREB2B in E. songoricum. The overexpression of EsDREB2B enhanced drought tolerance by increasing the plant's reactive oxygen species (ROS) scavenging capacity in situ. This study established the first transient transformation system for a desert legume woody plant, E. songoricum. This efficient system can be readily applied to investigate gene functions in E. songoricum. It will expedite the exploration of genetic resources and stress tolerance mechanisms in this species, offering valuable insights and serving as a reference for the transformation of other desert plants and woody legumes.

The Identification of Kabatiella zeae as a Causal Agent of Northern Anthracnose of Sorghum in China and Estimation of Host Resistance.

Sorghum northern anthracnose is a leaf disease affecting sorghum, which results in plant death and substantial yield loss. This study aimed to effectively understand the disease, clarify its biological characteristics, and evaluate the resistance of germplasm resources. A field sample was collected to isolate and purify the pathogen. The pathogen, identified as Kabatiella zeae Narita et Hiratsuka using both morphological and molecular techniques, was further confirmed as the causative agent of northern anthracnose of sorghum following Robert Koch's principles. The results revealed the optimal culture temperature to be 25 °C, preferred dark culture conditions, and the best growth on potato glucose agar medium with sucrose and L-leucine as the optimal carbon and nitrogen sources, respectively. A total of 138 sorghum germplasm resources were inoculated and evaluated using the isolated pathogen, with 20 lines (14.49%) exhibiting high resistance, 18 lines (13.04%) showing disease resistance, 27 lines (19.57%) demonstrating medium resistance, 37 lines (26.81%) being susceptible, and 36 lines (26.09%) classified as highly susceptible. The indoor fungicide screening was conducted through pathogen medium application, and enilconazole, pyraclostrobin, methylthiophanate, and flusilazole were screened for the best fungicide inhibition with a 100% inhibition rate compared with the control. This study provides reference for field pharmaceutical control in sorghum production.

Glycosyltransferase Slr1064 regulates carbon metabolism by modulating the levels of UDP-GlcNAc in Synechocystis sp. PCC 6803.

Glycosyltransferases (GTs) are enzymes that transfer sugars to various targets. They play important roles in diverse biological processes, including photosynthesis, cell motility, exopolysaccharide biosynthesis, and lipid metabolism; however, their involvement in regulating carbon metabolism in Synechocystis sp. PCC 6803 has not been reported. We identified a novel GT protein, Slr1064, involved in carbon metabolism. The effect of slr1064 deletion on the growth of Synechocystis cells and functional mechanisms of Slr1064 on carbon metabolism were thoroughly investigated through physiological, biochemistry, proteomic, and metabolic analyses. We found that this GT, which is mainly distributed in the membrane compartment, is essential for the growth of Synechocystis under heterotrophic and mixotrophic conditions, but not under autotrophic conditions. The deletion of slr1064 hampers the turnover rate of Gap2 under mixotrophic conditions and disrupts the assembly of the PRK/GAPDH/CP12 complex under dark culture conditions. Additionally, UDP-GlcNAc, the pivotal metabolite responsible for the O-GlcNAc modification of GAPDH, is downregulated in the Δslr1064. Our work provides new insights into the role of GTs in carbon metabolism in Synechocystis and elucidate the mechanism by which carbon metabolism is regulated in this important model organism.

Low temperature and dark culture inhibited explant browning and promoted in vitro regeneration of Juglans mandshurica

Low temperature and dark culture inhibited explant browning and promoted in vitro regeneration of Juglans mandshurica

Different wavelengths of LED irradiation promote secondary metabolite production in Pycnoporus sanguineus for antioxidant and immunomodulatory applications.

Pycnoporus sanguineus is a fungus of the phylum Basidiomycota that has many applications in traditional medicine, modern pharmaceuticals, and agricultural industries. Light plays an essential role in the metabolism, growth, and development of fungi. This study evaluated the mycelial growth and antioxidant and anti-inflammatory activities in P. sanguineus fermentation broth (PFB) cultured under different wavelengths of LED irradiation or in the dark. Compared to the dark cultures, the dry weight of mycelia in red- and yellow-light cultures decreased by 37 and 35% and the yields of pigments increased by 30.92 ± 2.18mg and 31.75 ± 3.06mg, respectively. Compared with the dark culture, the DPPH free radical scavenging ability, ABTS+ free radical scavenging capacity, and reducing power of yellow-light cultures increased significantly, and their total phenolic content peaked at 180.0 ± 8.34μg/mL. However, the reducing power in blue-light cultures was significantly reduced, though the total phenol content did not vary with that of dark cultures. In LPS- and IFN-γ-stimulated RAW 264.7 cells, nitrite release was significantly reduced in the red and yellow light-irradiated PFB compared with the dark culture. In the dark, yellow-, and green-light cultures, TNF-α production in the inflamed RAW 264.7 cells was inhibited by 62, 46, and 14%, respectively. With red-, blue-, and white-light irradiation, TNF-α production was significantly enhanced. Based on these results, we propose that by adjusting the wavelength of the light source during culture, one can effectively modulate the growth, development, and metabolism of P. sanguineus.

Transcriptomic Analysis Reveals the Potential Mechanisms for Improving Carotenoid Production in Rhodosporidium toruloides Z11 under Light Stress.

Carotenoids, as a type of tetraterpene compound, have been widely used in food, medical, and health areas owing to their antioxidant, immune enhancement, and disease risk reduction effects. Rhodosporidium toruloides is a promising oleaginous red yeast that can industrially synthesize carotenoids. In this study, the effects of different light exposure times and intervals on carotenoid production by R. toruloides Z11 were first investigated. Results showed that a higher carotenoid content (1.29 mg/g) can be achieved when R. toruloides Z11 was exposed to light for 12 h per day, which was increased by 1.98 times compared with that of dark cultivation. Transcriptome profiling revealed that light stress could effectively promote the gene expression levels of GGPS1 and AL1 in the carotenoid biosynthesis pathway and phr in the DNA photolysis pathway of R. toruloides. This work will provide a molecular foundation to further improve the production efficiency of carotenoids by genetic engineering.

High-intensity green light potentially activates the actinorhodin biosynthetic pathway in Streptomyces coelicolor A3(2).

The development of practices that enhance the potential of actinomycetes as major antibiotic producers is a challenge in discovering new secondary metabolites. Light, an essential external stimulus for most microorganisms, could be exploited to manipulate their physiological processes. However, the effects of monochromatic green light on the production of secondary metabolites in actinomycetes have not yet been reported. In this paper, we report a novel and simple method that uses high-intensity monochromatic green light to potentially induce the production of cryptic secondary metabolites in the model actinomycete Streptomyces coelicolor A3(2). Using actinorhodin (ACT), a blue-pigmented antibiotic, and undecylprodigiosin (RED), a red-pigmented antibiotic, as indicators, we found that irradiation with high-intensity monochromatic green light-emitting diodes promoted sporulation, significantly decreased RED production, and increased ACT production. Semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses revealed, for the first time, that stimulation with green light accelerated the expression of ActII-ORF4, a pathway-specific regulator of ACT biosynthesis in S. coelicolor A3(2). This approach of stimulating secondary metabolite biosynthesis pathways in actinomycetes by irradiation with high-intensity monochromatic green light is expected to facilitate the discovery of cryptic antibiotics that are not typically produced under conventional dark culture conditions. However, the effective intensity and duration of irradiation with green light that are required to activate these metabolite pathways may vary markedly among actinomycetes.

Unlocking microalgal host-exploring dark-growing microalgae transformation for sustainable high-value phytochemical production.

Microalgae have emerged as a promising, next-generation sustainable resource with versatile applications, particularly as expression platforms and green cell factories. They possess the ability to overcome the limitations of terrestrial plants, such non-arable land, water scarcity, time-intensive growth, and seasonal changes. However, the heterologous expression of interested genes in microalgae under heterotrophic cultivation (dark mode) remains a niche area within the field of engineering technologies. In this study, the green microalga, Chlorella sorokiniana AARL G015 was chosen as a potential candidate due to its remarkable capacity for rapid growth in complete darkness, its ability to utilize diverse carbon sources, and its potential for wastewater treatment in a circular bioeconomy model. The aims of this study were to advance microalgal genetic engineering via dark cultivation, thereby positioning the strain as promising dark-host for expressing heterologous genes to produce high-value phytochemicals and ingredients for food and feed. To facilitate comprehensive screening based on resistance, eleven common antibiotics were tested under heterotrophic condition. As the most effective selectable markers for this strain, G418, hygromycin, and streptomycin exhibited growth inhibition rates of 98%, 93%, and 92%, respectively, ensuring robust long-term transgenic growth. Successful transformation was achieved through microalgal cell cocultivation with Agrobacterium under complete darkness verified through the expression of green fluorescence protein and β-glucuronidase. In summary, this study pioneers an alternative dark-host microalgal platform, using, Chlorella, under dark mode, presenting an easy protocol for heterologous gene transformation for microalgal host, devoid of the need for expensive equipment and light for industrial production. Furthermore, the developed genetic transformation methodology presents a sustainable way for production of high-value nutrients, dietary supplements, nutraceuticals, proteins and pharmaceuticals using heterotrophic microalgae as an innovative host system.

Identification and evaluation of suitable reference genes for RT-qPCR analyses in Trichoderma atroviride under varying light conditions

BackgroundTrichoderma atroviride is a competitive soil-borne mycoparasitic fungus with extensive applications as a biocontrol agent in plant protection. Despite its importance and application potential, reference genes for RT-qPCR analysis in T. atroviride have not been evaluated. Light exerts profound effects on physiology, such as growth, conidiation, secondary metabolism, and stress response in T. atroviride, as well as in other fungi. In this study, we aimed to address this gap by identifying stable reference genes for RT-qPCR experiments in T. atroviride under different light conditions, thereby enhancing accurate and reliable gene expression analysis in this model mycoparasite. We measured and compared candidate reference genes using commonly applied statistical algorithms.ResultsUnder cyclic light–dark cultivation conditions, tbp and rho were identified as the most stably expressed genes, while act1, fis1, btl, and sar1 were found to be the least stable. Similar stability rankings were obtained for cultures grown under complete darkness, with tef1 and vma1 emerging as the most stable genes and act1, rho, fis1, and btl as the least stable genes. Combining the data from both cultivation conditions, gapdh and vma1 were identified as the most stable reference genes, while sar1 and fis1 were the least stable. The selection of different reference genes had a significant impact on the calculation of relative gene expression, as demonstrated by the expression patterns of target genes pks4 and lox1.ConclusionThe data emphasize the importance of validating reference genes for different cultivation conditions in fungi to ensure accurate interpretation of gene expression data.

Organic fertilizer facilitates the soil microplastic surface degradation and enriches the diversity of bacterial biofilm

The land-use of organic fertilizers is considered as an important sustainable method for resource utilization, which may have an impact on the microplastic behaviors in the soil. Here, a 240-d dark culture experiment was conducted to reveal the degradation and biofilm characteristics of degradable and refractory granule microplastics in soil and soil-fertilizer systems. The results indicated that microplastics generally exhibited a weak weight loss as well as a specific etiolation on the surface after the culture, especially polyvinyl-chloride and polyhydroxyalkanoates (PHA). Increase in carbon-oxygen functional groups and the changes of oxygen/carbon ratios were noticed, which implied that oxidation and degradation occurred on the surface of microplastics during the cultural process. The changes were more intense on the degradable PHA, and the fertilized-soil treatment than those of the refractory microplastics and the pure soil. Moreover, the addition of organic fertilizers enriched the community diversity of bacterial biofilm on multiple microplastic surfaces. In this regard, the animal fertilizers provided a stronger effect than the plant fertilizers. Overall, the soil, fertilizer and microplastic types affected the community structure and diversity of bacterial biofilm. The outcomes of this study would provide a theoretical basis for the utilization of organic matters for agricultural soil applications.

First Report of Fusarium oxysporum Causing Stem Rot on Mammillaria humboldtii in China.

Mammillaria humboldtii found in Mexico is a short-globose ornamental cactus species of the Cactaceae family, which has gained increasing popularity in China. It is characterized by tuberculate stems, dimorphic areoles, small pink flowers and pitted seed cell walls. The populations of wild M. humboldtii are critically endangered and are now of international conservation concern. In July 2021, stem rot symptoms were observed on M. humboldtii in a commercial greenhouse located in Zhangzhou (117°39'44.0064″E, 24°28'3.7236″N), Fujian Province (southern China). The typical symptoms were water-soaking, rotting and wilting on the stem, eventually leading to necrosis of the plants within 20 to 30 days. The vascular system of infected stems and roots showed a reddish-brown discolouration. The disease affected approximately 10% of 1000 plants. Fungi were isolated from the diseased stems of 26 samples, which were chopped into small pieces (5 × 5 mm), surface-sterilized with 75% ethanol for 40 s, and placed onto potato dextrose agar (PDA). After seven days of dark culture at 28°C, morphologically similar fungal isolates with whitish aerial mycelium and purple pigment were observed. On carnation leaf agar (CLA), isolates produced sickle and slightly curved macroconidia with three to four septa, measuring 12.8 to 27.9 × 1.9 to 3.8 μm (n = 15), and unicellular, ovoid to elliptical microconidia measuring 3.8 to 7.7 × 1.4 to 2.5 μm (n = 30). Smooth walled chlamydospores were terminal or intercalary, single or in pairs, measuring 9.2 to 13.1 μm (n = 15) in diameter. For molecular identification, the internal transcribed spacer (ITS) region of rDNA (Schoch et al. 2012), translation elongation factor-1α (EF1-α) (Maryani et al. 2019) and gene coding endopolygalacturonase 1 (PG1) (Hirano et al. 2006) of the representative isolate FJMH7 were amplified, purified and sequenced. BLASTn analysis of the ITS, EF1-α and pg1 sequences (GenBank accession numbers: ON832660, ON843495 and ON843496) showed 100%, 99.70% and 98.96% identity with F. oxysporum (GenBank accession numbers: KX611626, OM801797 and KF437345), respectively. Phylogenetic analysis based on the the concatenated ITS and EF1-α sequences and pg1 genes placed isolate FJMH7 with F. oxysporum reference strains in the phylogenetic trees. Based on morphological identification and sequence analysis, this isolate was identified as F. oxysporum. For the pathogenicity assay, six 6-month-old healthy plants of Mammillaria humboldtii were inoculated by dipping roots in a conidial suspension (106 conidia/mL) of isolate FJMH7 cultured in Bilai's medium for three days. Six noninoculated plants treated with Bilai's medium served as a control. Plants were transplanted into pots filled with sterilized soils and placed in a glasshouse at 25°C. After 15 days, all the inoculated plants exhibited rot symptoms on stems, which were similar to those observed in the commercial greenhouses. All inoculated plants were dead 30 days after inoculation. Control plants did not show any symptoms. F. oxysporum was reisolated and confirmed based on morphology and sequencing. No fungi were reisolated from control plants. To fulfil Koch's postulates, the pathogenicity assay was repeated twice with the same results. To date, F. oxysporum isolates have been reported on golden barrel cactus (Echinocactus grusonii) (Polizzi et al. 2004), night-blooming cereus (Hylocereus undatus) (Wright et al. 2007), apple cactus (Cereus peruvianus monstruosus) (Garibaldi et al. 2011), Schlumbergera truncate (Lops et al. 2013), Astrophytum ornatum (Quezada-Salinas et al. 2017) and Nopalea cochenillifera (Santiago et al. 2018). To our knowledge, this is the first report of F. oxysporum on M. humboldtii in China, indicating that this pathogen could cause wilt and rot disease on different cactus hosts.

Effects of Genotype, Light, and Plant Growth Regulators on Rhizome Browning, Proliferation, and Sprouting in Cymbidium

Chinese cymbidiums are important flowering ornamental plants. Traditional propagation via seed or division cannot satisfy growers’ demand for commercialization of new cultivars, and in vitro propagation has a low micropropagation efficiency due to the browning of rhizomes. In this study, rhizomes of Cymbidium ‘14-16-13’ and ‘14-16-5’ were cultured on half-strength Murashige and Skoog (MS) medium supplemented with 6-benzyl aminopurine (BAP), NAA (α-napthaleneacetic acid), or BAP with NAA under either the dark or light. The degree of browning was read, and rhizome proliferation or sprouting (sprout numbers) was evaluated. Results showed that there was significant difference in browning grade of rhizomes between ‘14-16-13’ and ‘14-16-5’ regardless of dark and light culture. Dark culture induced rhizome proliferation but failed to induce sprouts. Light culture slightly elevated the degree of browning but induced sprouting. Among the growth regulators evaluated, BAP was more effective for sprout induction. As rhizome browning appeared to be inevitable in micropropagation of the cymbidiums, a compromise between browning and sprout production could be a realistic approach. Our study showed that rhizomes cultured on half-strength MS medium supplemented with 1.5 mg·L−1 BAP were able to produce more than 16 sprouts per vessel even though browning occurred in the rhizomes. Thus, culturing rhizomes in this medium could be a practical solution for in vitro propagation of Chinese cymbidiums.

Identification and biological characterization of pathogen causing black spot of Pseudostellaria heterophylla in Fujian province

In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.

Establishment of a Highly Efficient In Vitro Propagation System of Diospyros lotus

Persimmon (Diospyros) is an economically important tree widely cultivated for woody grain production in China, and Diospyros lotus is mainly used as the grafting stock of persimmon. However, the breeding of stress-resistant rootstocks of D. lotus using molecular means has yet to be achieved; in particular, an efficient blade-regeneration system has not been perfected to date. This study examined the effects of different plant-growth regulators and concentrations on the primary culture of stems with buds, the induction of leaf callus, the differentiation of adventitious shoots, and rooting culture of D. lotus. The optimal formula for inducing axillary buds from stems with buds was 1/2 Murashige and Skoog (MS) medium containing 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L naphthaleneacetic acid (NAA), in which the induction rate of axillary buds approached 67.1%. The best medium formula for leaf callus induction was 1/2 MS medium containing 2.0 mg/L 6-BA and 0.5 mg/L NAA. Then callus was transferred to 1/2 MS medium containing 2.0 mg/L 6-(γ,γ-dimethylallylamino)purine (2iP), 2.0 mg/L thidizuron (TDZ), and 40 g/L sucrose to induce adventitious shoots after dark culture for 48 h, resulting in 7.9 shoots per explant and a 75.2% induction frequency of adventitious shoots. In addition, it was difficult to induce adventitious shoots from callus after six times of continuous transfer and differentiation. The adventitious shoots were transferred to 1/2 MS medium containing 2.0 mg/L zeatin (ZT) and 2.0 mg/L 2iP for proliferation culture, in which the multiplication coefficient approached 7.6. The adventitious shoots after multiplication were inoculated into 1/2 MS + 1.0 mg/L IBA + 0.5 mg/L NAA medium, the rooting rate was 70.2%, and the average number of heels was 9.6. Thus, studies in this area are expected to facilitate rapid and excellent growth, as well as theoretical support for factory saplings’ care and molecular breeding.

Programmed Cooling and Vitrification Method Applied to the Germplasm Preservation of Eriobotrya Plants

Long-term cryopreservation of germplasm is of great importance to maintaining genetic resources. A cryopreservation technology system has not yet been established for Eriobotrya, an important fruit tree in the Rosaceae. In this study, Eriobotrya plants were used as test materials for the following purposes: to study different types of vitrification solution, compare different methods involving programmed cooling vitrification and rapid freezing vitrification; and evaluate the effects of vitrification solution loading time, low-temperature acclimation culture, dark culture, and cryoprotectant use in preculture medium for ultra-low-temperature preservation of Eriobotrya germplasm resources. Plant vitrification solution 1 had the best comprehensive effect on different vitrification solution tests. The programmed cooling and vitrification method yielded a certain survival rate with different vitrification solutions and materials, but the survival rate under the rapid freezing method was 0%. The longer the vitrification solution was loaded, the higher the survival rate. The most suitable time for dark culture of Eriobotrya plants after cryopreservation was 20 days. The preculture medium supplemented with 5% dimethyl sulfoxide resulted in significantly higher preservation than the control medium, and the optimal sucrose concentration was 0.3 mol⋅L−1. A stable, high-survival, and novel cryopreservation procedure for loquat plants was established. The operating procedures are described in a procedural and standardized manner. These findings provide a theoretical basis for research of the cryopreservation of germplasm resources of Eriobotrya plants.

Mechanism of visible light enhances microbial degradation of Bisphenol A.

Bisphenol A (BPA) is a toxic endocrine disruptor detected in various environments. Microbial metabolic/enzymatic degradation has been thought to be the main pathway for BPA attenuation in natural environments. In this study, we found that under visible light conditions, superoxide produced by bacteria was the main reason for the rapid removal of BPA, accounting for 57% of the total removal rate. With visible light, the bacteria degraded BPA at a rate of 0.22mg/L/d, and the total removal within 8 days reached 85%, which is 4.7 times compared with that of dark culture. The intermediate product 4-iso-propenylphenol, which was considered as an end-product of microbial degradation of BPA in previous reports, was detected in large quantities at 24h in culture but gradually decreased in our experiment. Community analysis suggested bacteria with aromatic hydrocarbon degradation ability were more enriched under light incubation. Moreover, the bacteria showed well degradation ability to various pharmaceutically active but nonbiodegradable compounds including diclofenac and fluoxetine, with a removal rate of 88 % and 20%, respectively. Our study revealed the organic pollutant transformation pathway under the combined action of light and microorganisms, providing new insights into the microbial treatment of aromatic hydrocarbon pollutants.

Establishing a high-efficiency <i>in vitro</i> regeneration system for <i>Agrobacterium</i>-mediated transformation in <i>Fragaria nilgerrensis</i>

<italic>Fragaria nilgerrensis</italic> Schlechtendal ex J. Gay, a diploid wild strawberry, has many excellent characteristics such as fruit with white color and peach fragrance, and strong disease resistance. However, the lack of an efficient and stable regeneration and genetic transformation system for <italic>F. nilgerrensis</italic> has largely limited the functional studies of related genes governing excellent traits. In this study, a regeneration system for <italic>F. nilgerrensis</italic> leaf discs was developed by optimizing factors such as different hormone combinations, dark culture times and casein hydrolysate (CH) concentrations, with an average regeneration rate of 97.3% at 45 d of culture. By paraffin section observation of callus with different colors induced from the leaf disc regeneration, light yellow callus was determined to be embryogenic and the regenerative pathway was identified as indirect organogenesis. Based on this, an average transformation percentage of 8.67% was achieved by screening kanamycin concentration and referring to transformation procedures described by predecessors. PCR-positive transformants were obtained within 4-5 months by confirmation of PCR and histochemical GUS, and transgene integration was identified by transformants regeneration. The establishment of efficient regeneration system provided a feasible platform for genetic transformation, which laid the foundation for further gene functional studies in <italic>F. nilgerrensis</italic>.

Filosofi Hindu Narasi Banalisme Filosofi Hindu Dalam Konten Wonderland Indonesia 2 Karya Alffy Rev

This study raises the topic of Hindu pghilosophy contained in the Wonderland Indonesia 2 video by Alffy Rev. The method is narrative analysis with secondary text data in the form of videos on the youtube channel. The results show the: the prologue narration describes the historical symbol constructs the were excavated in the garuda mythology, the Majapahit symbol and depict the under or dark culture of the dragon and the light culture of the garuda. The content narration describes the relationship between the historicity of the mythological wolrd of garuda, Majapahit and national values combined with traditionl art of folk songs and supporting costume elements between various dimensions of banality. The element of banality constructed by the text produser describes a narrative about the integration and unification of the dimensions of nature, creation manajement and idependence after destruction in the trisula philosophy wich is integrated in Hindu culture and teachings. In the final naarative, it is described how the relationship between history and the younger generation of Indonesia is as a form of reconstruction of the values struggle and multiculturalism as a reading of the history of mythology, the history of Majapahit, the history of the independence of the Indonesian nation.&#x0D;

Efficient Co-production of Docosahexaenoic Acid Oil and Carotenoids in Aurantiochytrium sp. Using a Light Intensity Gradient Strategy.

Aurantiochytrium is a promising source of docosahexaenoic acid (DHA) and carotenoids, but their synthesis is influenced by environmental stress factors. In this study, the effect of different light intensities on the fermentation of DHA oil and carotenoids using Aurantiochytrium sp. TZ209 was investigated. The results showed that dark culture and low light intensity conditions did not affect the normal growth of cells, but were not conducive to the accumulation of carotenoids. High light intensity promoted the synthesis of DHA and carotenoids, but caused cell damage, resulting in a decrease of oil yield. To solve this issue, a light intensity gradient strategy was developed, which markedly improved the DHA and carotenoid content without reducing the oil yield. This strategy produced 30.16g/L of microalgal oil with 15.11g/L DHA, 221µg/g astaxanthin, and 386µg/g β-carotene. This work demonstrates that strain TZ209 is a promising DHA producer and provides an efficient strategy for the co-production of DHA oil together with carotenoids.

Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.