Anoectochilus roxburghii is a wild edible species and has been traditionally used for a wide range of diseases in many countries. Our research aims to find the optimal light-emitting diode (LED) lighting conditions to improve the growth and development of A. roxburghii seedling at the acclimation stage. Two-month-old explants were cultured under the various lighting conditions including red (R), blue (B), BR (one blue: four red), BRW151 (one blue: five red: one white), BRW142 (one blue: four red: two white), and fluorescent lamp (FL). The results showed that the lighting conditions not only affect the growth and morphology of plants but also the accumulation of total flavonoids. Single wavelengths (B or R LED) inhibited the growth and secondary biosynthesis of A. roxburghii, while the BR LED showed an enhancement in both growth and biomass accumulation. A. roxburghii plants were grown under BR LED light has average plant height (7.18 cm), stem diameter (17.6mm), number of leaves (5.78 leaves/tree), leaf area (4.67 cm²), fresh weight (0.459 g/tree), dry matter percentages (11.69%), and total flavonoid (1.811 mg/g FW) is considered to be superior to FL lamps and other LEDs in the experiment. This indicates that both blue and red wavelengths are required for the normal growth of A. roxburghii. To learn more about how light affects flavonoid biosynthesis, we evaluated the expression of genes involved in this process (pal, chs, chi, and fls) and found that BR LED light enhances the expression level of chi and fls genes compared to fluorescent lamps (1.18 and 1.21 times, respectively), leading to an increase in the flavonoid content of plant. Therefore, applying BR LED during in vitro propagation of A. roxburghii could be a feasible way to improve the medicinal value of this plant.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.

Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, Rhizobium rhizogenes mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber (Cucumis sativus L.). Under the optimized conditions including OD₆₅₀ at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other cucumis plants.

Exopolysaccharides (EPSs) produced by Bacillus species have recently emerged as promising commercial antioxidants in various industries, such as pharmaceutics and biomedicine. However, little is known about EPS production and function from Bacillus velezensis so far. In the present study, the effect of sugar sources on EPS production by B. velezensis VTX20 and the genetic biosynthesis, characteristics, and antioxidant activity of the resulting EPS were evaluated. The strain VTX20 produced the maximum EPS yield of 75.5 ± 4.8 g/L from an initial 200 g/L of sucrose after a 48-h cultivation. Through genomic analysis, ls-levB operon was found, for the first time, to be responsible for the levan-type EPS production in B. velezensis. Biochemical and structural characterization further confirmed the majority of levan, followed by an extremely low level of dextran biopolymer. The water solubility index and water holding capacity of the EPSs were 81.9 ± 3.4% and 100.2 ± 3.4%, respectively. In vitro antioxidant activity analyses showed strong scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radical values of 40.1–64.0% and 16.0–40%, respectively. These findings shed light on the EPS biosynthesis of B. velezensis at both structural and genetic levels and the potential application of EPS as a natural antioxidant for pharmaceutical and biomedical industries.