Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

Trace Identification and Visualization of Multiple Benzimidazole Pesticide Residues on Toona sinensis Leaves Using Terahertz Imaging Combined with Deep Learning.

Molecular spectroscopy has been widely used to identify pesticides. The main limitation of this approach is the difficulty of identifying pesticides with similar molecular structures. When these pesticide residues are in trace and mixed states in plants, it poses great challenges for practical identification. This study proposed a state-of-the-art method for the rapid identification of trace (10 mg·L-1) and multiple similar benzimidazole pesticide residues on the surface of Toona sinensis leaves, mainly including benzoyl (BNL), carbendazim (BCM), thiabendazole (TBZ), and their mixtures. The new method combines high-throughput terahertz (THz) imaging technology with a deep learning framework. To further improve the model reliability beyond the THz fingerprint peaks (BNL: 0.70, 1.07, 2.20 THz; BCM: 1.16, 1.35, 2.32 THz; TBZ: 0.92, 1.24, 1.66, 1.95, 2.58 THz), we extracted the absorption spectra in frequencies of 0.2-2.2 THz from images as the input to the deep convolution neural network (DCNN). Compared with fuzzy Sammon clustering and four back-propagation neural network (BPNN) models (TrainCGB, TrainCGF, TrainCGP, and TrainRP), DCNN achieved the highest prediction accuracies of 100%, 94.51%, 96.26%, 94.64%, 98.81%, 94.90%, 96.17%, and 96.99% for the control check group, BNL, BCM, TBZ, BNL + BCM, BNL + TBZ, BCM + TBZ, and BNL + BCM + TBZ, respectively. Taking advantage of THz imaging and DCNN, the image visualization of pesticide distribution and residue types on leaves was realized simultaneously. The results demonstrated that THz imaging and deep learning can be potentially adopted for rapid-sensing detection of trace multi-residues on leaf surfaces, which is of great significance for agriculture and food safety.

Feasibility of Laser-Induced Breakdown Spectroscopy and Hyperspectral Imaging for Rapid Detection of Thiophanate-Methyl Residue on Mulberry Fruit.

An effective and rapid way to detect thiophanate-methyl residue on mulberry fruit is important for providing consumers with quality and safe of mulberry fruit. Chemical methods are complex, time-consuming, and costly, and can result in sample contamination. Rapid detection of thiophanate-methyl residue on mulberry fruit was studied using laser-induced breakdown spectroscopy (LIBS) and hyperspectral imaging (HSI) techniques. Principal component analysis (PCA) and partial least square regression (PLSR) were used to qualitatively and quantitatively analyze the data obtained by using LIBS and HSI on mulberry fruit samples with different thiophanate-methyl residues. The competitive adaptive reweighted sampling algorithm was used to select optimal variables. The results of model calibration were compared. The best result was given by the PLSR model that used the optimal preprocessed LIBS-HSI variables, with a correlation coefficient of 0.921 for the prediction set. The results of this research confirmed the feasibility of using LIBS and HSI for the rapid detection of thiophanate-methyl residue on mulberry fruit.

Potential of Visible and Near-Infrared Hyperspectral Imaging for Detection of Diaphania pyloalis Larvae and Damage on Mulberry Leaves.

Mulberry trees are an important crop for sericulture. Pests can affect the yield and quality of mulberry leaves. This study aims to develop a hyperspectral imaging system in visible and near-infrared (NIR) region (400⁻1700 nm) for the rapid identification of Diaphania pyloalis larvae and its damage. The extracted spectra of five region of interests (ROI), namely leaf vein, healthy mesophyll, slight damage, serious damage, and Diaphania pyloalis larva at 400⁻1000 nm (visible range) and 900⁻1700 nm (NIR range), were used to establish a partial least squares discriminant analysis (PLS-DA) and least-squares support vector machines (LS-SVM) models. Successive projections algorithm (SPA), uninformation variable elimination (UVE), UVE-SPA, and competitive adaptive reweighted sampling were used for variable selection. The best models in distinguishing between leaf vein, healthy mesophyll, slight damage and serious damage, leaf vein, healthy mesophyll, and larva, slight damage, serious damage, and larva were all the SPA-LS-SVM models, based on the NIR range data, and their correct rate of prediction (CRP) were all 100.00%. The best model for the identification of all five ROIs was the UVE-SPA-LS-SVM model, based on visible range data, which had the CRP value of 97.30%. In summary, visible and near infrared hyperspectral imaging could distinguish Diaphania pyloalis larvae and their damage from leaf vein and healthy mesophyll in a rapid and non-destructive way.

Cas9-Guide RNA Directed Genome Editing in Soybean.

Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.

[Measurement of Soil Total Nitrogen Using Near Infrared Spectroscopy Combined with RCA and SPA].

Visible near spectra tecnnology was adopted to detect soil total nitrogen content. 394 soil samples were collected from Wencheng, Zhejiang province to be used for calibration model (n=263) and independent prediction set (n=131). Raw spectra and wavelength-reduced spectra with five different pretreatment methods (SG smoothing, SNV, MSC, 1st-D and 2nd-D) were compared to determine the optimal wavelength range and pretreatment method for analysis. The results with 5 different pretreatment methods were not improved compared to that both of full spectra PLS model and wavelength reduction spectra model. Spectral variable selection is an important strategy in spectrum modeling analysis, because it tends to parsimonious data representation and can lead to multivariate models with better performance. In order to simply calibration models, the wavelength variables selected by two different variable selection methods (i. e. regression coefficient analysis (RCA) and successive projections algorithm (SPA) were proposed to be the inputs of calibration methods of PLS, MLR and LS-SVM models separately. These calibration models were also compared to select the best model to predict soil TN. In total, 9 different models were built ahd the best results indicated that PLS, MLR and LS-SVM obtained the highest precision with determination coefficient of prediction R2(pre) =0. 81, RMSEP=0. 0031 and RPD=2. 26 based on wavelength variables selected by RCA (0. 0002) and SPA as inputs of models. SPA-MLR model and other three models based on 7 sensitive variables selected by RC using 0. 0002 regression coefficient threshold value obtained the best result with R2(pre), RMSEP and RPD as 0. 81, 0. 0031 and 2. 26. This prediction accuracy is classied to be very good. For all the models, it could be concluded that RCA and SPA could be very useful ways to selected sensitive wavelengths, and the selected wavelengths were effective to estimate soil TN. It is recommended to adopt SPA variable selection or RCA variable selection method with both linear and nonlinear calibration models for measurement of the soil TN using Vis-NIR spectroscopy technology, and wavelengths selection could be very useful to reduce collinearity and redundancies of spectra.

[Review of Crop Canopy Spectral Information Detection Technology and Methods].

Compared with the traditional chemical methods and the subjective visual ways for measuring plant physiology information indicators, the assessments of crop canopy information through spectral radiometer are more simple, rapid and accurate. The applications of different types of spectral radiometer, especially for international general used Cropscan multispectral radiometer, for predicting crop canopy leaf area index under different growth stage, biomass, nitrogen, chlorophyll and yield, and monitoring plant diseases and insect pests were summarized based on crop group information acquisition methods in recent years. The varity of vegetation indices (VIs) were concluded after comparing regression coefficients of related models among different crops. In general, the correlation coefficients of mathematical models were high and it can realize the crop detection of various kinds of physiological information. Besides, the combination of multispectral radiometer and other sensors can provide useful information to evaluate the status of crops growth, which is very important in practice.

Antibacterial mechanism of vanillin against Escherichia coli O157: H7.

Vanillin, a plant-derived antimicrobial volatile substance, has potential microbial control applications in the food industry. However, the effect of vanillin on the food-borne pathogen Escherichia coli (E. coli) O157:H7 has not been well studied. This study aims to explore the antibacterial mechanism of vanillin against E. coli O157:H7. The minimum inhibitory concentration (MIC) and antibacterial effect of vanillin were determined by microdilution. Scanning electron microscopy (SEM) was used to observe the damage of vanillin to the cell membrane, while cell membrane potential and the leakage of nucleic acid protein were measured to explore the effect of vanillin on the membrane system. Confocal laser scanning and intracellular adenosine triphosphate (ATP) concentration determination were utilized to investigate the effects of vanillin on the energy, life, and death of E. coli. Finally, transcriptome sequencing was conducted to investigate the gene expression differences induced by vanillin treatment. The results showed that vanillin treatment effectively controlled E. coli O157:H7 with an MIC of 2 mg/mL. After treatment, damage to the membrane system, depolarization of the membrane, and leakage of nucleic acid and protein were observed. Meanwhile, vanillin treatment caused decreased ATP content and cell death. Transcriptome analysis showed that vanillin treatment significantly affected the expression of genes involved in cell membrane formation, tricarboxylic acid (TCA) cycling pathway, and oxidative phosphorylation pathway in E. coli O157:H7. In conclusion, membrane damage and energy metabolism disruption are important mechanisms of vanillin's inhibitory effect on E. coli O157:H7. This study provides new insights into the molecular reaction mechanism of vanillin against E. coli O157:H7, highlighting its potential as an antibacterial substance for preventing E. coli contamination in the food industry.

Detection and classification of volatile compounds emitted by three fungi-infected citrus fruit using gas chromatography-mass spectrometry.

Citrus fruit produced some characteristic volatile compounds when infected by fungi compared with the healthy fruit. In the present study, volatile metabolites of postharvest citrus fruit with three different diseases including stem-end rot, blue mold and green mold were detected. Multivariate analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to classify the volatile compounds between the infected and non-infected citrus fruit. The results indicated that volatile compounds of unrotten, unrotten-rotten junction, and rotten tissues were successfully classified. Importantly, eight volatile compounds as biomarkers for stem-end rot and one biomarker for green mold of citrus were screened to discriminate the infected citrus fruit. This study offers the application potential of odor profiling of volatile compounds for detecting the fungi infection in postharvest citrus fruit.

Protective Application of Morus and Its Extracts in Animal Production.

Different components of the mulberry tree (fruits, leaves, twigs, and roots) are rich in active compounds, and have been reported to possess potent beneficial properties, including antioxidative, anti-inflammatory, antimicrobial, anticancer, anti-allergenic, antihypertensive, and neuroprotective. The mulberry and its extracts can effectively improve the growth performance and fitness of animals. They not only possess the properties of being safe and purely natural, but also they are not prone to drug resistance. According to the literature, the supplemental level of the mulberry and its extracts in animal diets varies with different species, physiological status, age, and the purpose of the addition. It has been observed that the mulberry and its extracts enhanced the growth performance, the quality of animal products (meat, egg, and milk), the antioxidant and the anti-inflammatory responses of animals. Furthermore, the mulberry and its extracts have antibacterial properties and can effectively moderate the relative abundance of the microbial populations in the rumen and intestines, thus improving the immunity function of animals and reducing the enteric methane (CH4) production in ruminants. Furthermore, the mulberry and its extracts have the potential to depurate tissues of heavy metals. Collectively, this review summarizes the nutrients, active compounds, and biological functions of mulberry tree products, as well as the application in livestock production with an aim to provide a reference for the utilization of the mulberry and its extracts in animal production.

Identification and Antioxidant Capacity of Free and Bound Phenolics in Six Varieties of Mulberry Seeds Using UPLC-ESI-QTOF-MS/MS.

Mulberry seeds are a byproduct of juice processing and may be an important resource for its abundant compounds. In this study, we analyzed the qualitative composition of free and bound phenolics from six varieties of mulberry seeds using UPLC-ESI-QTOF-MS/MS. Free phenolics (FPs) and bound phenolics (BPs) were measured using the Folin-Ciocalteu method; antioxidant capacity was determined by measuring 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, using the ferric reducing antioxidant power assay. A total of 28 free and 11 bound phenolics were extracted and identified, wherein five free phenolics were found in mulberry matrices for the first time. The six varieties of mulberry seeds exhibited higher content of FPs than BPs, and there was a correlation between the phenolic content and antioxidant capacity. Consequently, three varieties were selected for their high phenolic content and antioxidant capacity. This study might offer a theoretical basis for the utilization of mulberry seed.

Application of essential oils in packaging films for the preservation of fruits and vegetables: A review.

Fruits and vegetables are highly perishable in nature. Several factors could affect the quality and shelf life of fruits and vegetables. Packaging materials (usually made up of polymers, proteins, lipids, polysaccharides, etc.,) are incorporated with essential oil (EO) which is high in antimicrobial and antioxidant compounds that can enhance the shelf life of fruits and vegetables without affecting their quality. However, the use of EO for postharvest preservation can alter the organoleptic properties of fresh produce. Exploiting synergistic interactions between several EOs, encapsulation of EO, or combining EO with non-thermal techniques such as irradiation, UV-C, cold plasma, ultrasound, etc., may help in preventing the spoilage of food products at lower concentrations without altering their organoleptic properties. This review aims to discuss the overview and current scenario of packaging film with EO for the preservation of fruit and vegetables. We have also discussed the spoilage mechanism of fruits and vegetables, mode of action of EOs, and the effect of EO with packaging film on antimicrobial and sensory properties of fruits and vegetables.

Nondestructive measurement of pectin polysaccharides using hyperspectral imaging in mulberry fruit.

Pectin polysaccharide is an important phytochemical with potential biomedical applications. It is commonly measured by time-consuming destructive chemical methods. This work demonstrates the feasibility of using visible and near-infrared hyperspectral imaging (HSI) techniques to rapidly measure pectin polysaccharides in intact mulberry fruits. Based on spatial information provided by HSI images, the representative spectrum of each whole mulberry was accurately extracted without background. The effects of storage temperature on two varieties of mulberries for model establishment were studied. The performances of two spectral ranges obtained by Si and InGaAs CCD detectors for pectin prediction were compared. The best predictions were obtained from dilute alkali soluble pectin and total soluble pectin in Dashi mulberry fruit stored at room temperature, with residual predictive deviation values of 2.317 and 1.935, respectively. Our results show that HSI is a promising alternative to the chemical method to rapidly and nondestructively measure the pectin content.

Recent innovations of ultrasound green technology in herbal phytochemistry: A review.

Ultrasound (US) has become one of the most important techniques in green chemistry and emerging technologies. Many research investigations documented the usefulness of US in a wide range of applications in food science, nanotechnology, and complementary medicine, where effective extraction of natural products is important. However, as with all novel technologies, US has advantages and limitations that require clarification for full adaptation at an industrial scale. The present review discusses recent applications of US in herbal phytochemistry with the emphasis on US effects on chemical structures of bioactive compounds extracted from herbs and their bioactivities. The impact of different US processing conditions such as frequency, intensity, duration, temperature, and pressure on the effectiveness of the extraction process and the properties of the extracted materials are also discussed. Different frequencies and intensities of US have demonstrated its potential applications in modifying, determining, and predicting the physicochemical properties of herbs and their extracts. US has important applications in nanotechnology where it supports the fabrication of inexpensive and eco-friendly herbal nanostructures, as well as acoustic-based biosensors for chemical imaging of the herbal tissues. The application of US enhances the rates of chemical processes such as hydrolysis of herbal fibers, which reduces the time and energy consumed without affecting the quality of the final products. Overall, the use of US in herbal science has great potential to create novel chemical constructions and to be used as an innovative diagnostic system in various biomedical, food, and analytical applications.

Building a stable and accurate model for heavy metal detection in mulberry leaves based on a proposed analysis framework and laser-induced breakdown spectroscopy.

Laser-induced breakdown spectroscopy (LIBS) was used to rapidly detect heavy metals in mulberry leaves. For the purpose of increasing detection stability and accuracy, a novel analysis framework consisting of a Kohonen self-organizing map (SOM), a variable selection method using the successive projection algorithm (SPA) and uninformative variable elimination (UVE), and a consensus modeling strategy was proposed for processing LIBS data to determine copper (Cu) and chromium (Cr) content. Results showed that the best regression model for Cu and Cr content achieved the residual predictive deviation (RPD) values of 10.0494 and 8.3874, respectively, and root mean square error of prediction (RMSEP) values of 110.4550 and 41.4561, respectively. The proposed strategy provides a high-accuracy and rapid alternative to the traditional method for monitoring heavy metals in mulberry leaves, which could guarantee the quality of mulberry leaves and potentially be used in food-related industries.

Ultrasound-mediated molecular self-assemble of thymol with 2-hydroxypropyl-ß-cyclodextrin for fruit preservation.

In this study, the inclusion complex (IC) of thymol with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) was fast synthetized by ultrasonic technology and its antifungal activities were evaluated. The thymol/HPßCD-IC was characterized by UV-vis absorption spectroscopy, fluorescence emission spectroscopy, powder X-ray diffraction, FT-IR, 1H-NMR, TGA and DSC. The phase solubility studies proved that the aqueous solubility of thymol was significantly improved by forming the inclusion complex with HPßCD, and the thermal stability analysis showed that thymol/HPßCD-IC had a better thermal stability than pure thymol. The in vitro antifungal activities of thymol/HPßCD-IC against Botrytis cinerea, Penicillium digitatum and Alternaria alternata were significantly improved compared with pure thymol. Furthermore, the gray mold rot of tomatoes was evidently inhibited by thymol/HPßCD-IC treatment in vivo study. Therefore, the complexation with HPßCD assisted by ultrasound is a promising approach to solubilize and stabilize thymol for application as an antifungal agent in fruit preservation.

Heavy metal detection in mulberry leaves: Laser-induced breakdown spectroscopy data.

Five copper or chromium stress levels were carried out on mulberry leaf, and 20 samples were collected for each metal stress level. A total of 100 samples (copper or chromium) were processed into uniform pressed pellet. The mulberry leaf pellet was placed on a sample platform of laser-induced breakdown spectroscopy (LIBS) system. A laser was used to ablate the sample pellet and generate the emission lines, the intensity and delay time of laser ablation were 80 mJ and 4 µs respectively. To reduce the acquisition errors, 16 different positions of each sample were ablated for 5 accumulation. Then, 80 spectra were collected per sample and the average of them was considered as the sample spectrum for subsequent analysis. Finally, a total of 200 spectra of copper and chromium in mulberry leaves with a wavelength range of 219-877 nm were obtained for calibration analysis [1].

[Research on the sugar content measurement of grape and berries by using Vis/NIR spectroscopy technique].

Aiming at the nonlinear correlation characteristic of Vis/NIR spectra and the corresponding sugar content of grape and berries, the Vis/NIR spectra of grape and berries were obtained by diffusion reflectance. A mixed algorithm was presented to predict sugar content of grape and berries. The original spectral data were processed using partial least squares (PLS), and three best principal factors were selected based on the reliabilities. The scores of these 3 principal factors would be taken as the input of the three-layer back-propagation artificial neural network (BP-ANN). Trained with the samples in calibration collection, the BP-ANN predicted the samples in prediction collection. The values of decision coefficient (r2), the root mean squared error of prediction (RMSEP), and bias were used to estimate the mixed model. The observed results using PLS-ANN (r2 = 0.908, RMSEP = 0.112 and Bias = 0.013) were better than those obtained by PLS (r2 = 0.863, RMSEP = 0.171, Bias = 0.024). The result indicted that the detection of internal quality of grape and berries such as sugar content by nondestructive determination method was very feasible and laid a solid foundation for setting up the sugar content forecasting model for grape and berries.

Comparison of different CCD detectors and chemometrics for predicting total anthocyanin content and antioxidant activity of mulberry fruit using visible and near infrared hyperspectral imaging technique.

This study investigated the potential of using hyperspectral imaging technique in tandem with chemometrics for rapid and invasive predicting total anthocyanin content and antioxidant activity of mulberry fruit. Two calibration methods of partial least square regression and least-squares support vector machines and three wavelength selection algorithms of successive projections algorithm, uninformation variable elimination, and competitive adaptive reweighted sampling were applied. The best prediction models for the analysis of total anthocyanin content and antioxidant activity had Rval2 of 0.959 and 0.995 respectively. The performances of two CCD detectors named silicon (Si) and indium gallium arsenide (InGaAs) were compared. The results show that hyperspectral imaging has a great potential for the assessment of total anthocyanin content and antioxidant activity of mulberry fruit.

[Emergy analysis of mulberry-silkworm ecosystem in China].

Mulberry-silkworm ecosystem is one of the important agro-ecosystems in China. Based on the principles and methods of emergy analysis, this paper studied the interior structure of mulberry-silkworm ecosystem and its relationship with exterior environment and economy. Some emergy indices for this ecosystem were quantitatively calculated, and compared with those of the agro-ecosystem in China. The results showed that the emergy investment ratio, emergy yield ratio, environmental loading ratio and emergy sustainability index was 3.78, 4.68, 0.18 and 26.0, respectively, suggesting the low environmental pressure and good ecological benefit in mulberry-silkworm ecosystem in China. Hi-technology was required to further decrease the labor force input and enhance the comprehensive utilization of sericultural resources.