Development of a novel precision applicator for spot treatment of granular agrochemical in wild blueberry.

While spot spraying has gained increasing popularity in recent years, spot application of granule agrochemical has seen little development. Despite the potential for the technology, there currently exists no commercially available granular applicators capable of spot application. Therefore, the goal of this study was to design, build, and lab evaluate a precision applicator for spot applying granular agrochemical in wild blueberry. The design incorporated a John Deere RC2000 with a custom control box, recirculation system, and electrically actuated valves. All components were modified to fit a Valmar 1255 Twin-Roller. The system receives inputs from a predeveloped prescription map and can actuate each of the twelve valves separately to provide individual orifice control. Casoron® G4 was used as the testing agrochemical and in cycling the product pneumatically for 1 hour incurred no significant product degradation (p = 0.110). In lab evaluations, the applicator encountered zero errors in reading prescription maps and actuating the correct valves accordingly. Further, the granule recycling system had zero instances where product built up in the lines or jammed the valves. In all, this project represents the first successful development of a precision granular spot applicator for any cropping system.

Enhancing surface drainage mapping in eastern Canada with deep learning applied to LiDAR-derived elevation data.

Agricultural dykelands in Nova Scotia rely heavily on a surface drainage technique called land forming, which is used to alter the topography of fields to improve drainage. The presence of land-formed fields provides useful information to better understand land utilization on these lands vulnerable to rising sea levels. Current field boundaries delineation and classification methods, such as manual digitalization and traditional segmentation techniques, are labour-intensive and often require manual and time-consuming parameter selection. In recent years, deep learning (DL) techniques, including convolutional neural networks and Mask R-CNN, have shown promising results in object recognition, image classification, and segmentation tasks. However, there is a gap in applying these techniques to detecting surface drainage patterns on agricultural fields. This paper develops and tests a Mask R-CNN model for detecting land-formed fields on agricultural dykelands using LiDAR-derived elevation data. Specifically, our approach focuses on identifying groups of pixels as cohesive objects within the imagery, a method that represents a significant advancement over pixel-by-pixel classification techniques. The DL model developed in this study demonstrated a strong overall performance, with a mean Average Precision (mAP) of 0.89 across Intersection over Union (IoU) thresholds from 0.5 to 0.95, indicating its effectiveness in detecting land-formed fields. Results also revealed that 53% of Nova Scotia's dykelands are being used for agricultural purposes and approximately 75% (6924 hectares) of these fields were land-formed. By applying deep learning techniques to LiDAR-derived elevation data, this study offers novel insights into surface drainage mapping, enhancing the capability for precise and efficient agricultural land management in regions vulnerable to environmental changes.

Functional characterisation of Dof gene family and expression analysis under abiotic stresses and melatonin-mediated tolerance in pitaya (Selenicereus undatus).

DNA binding proteins with one finger (Dof ) transcription factors are essential for seed development and defence against various biotic and abiotic stresses in plants. Genomic analysis of Dof has not been determined yet in pitaya (Selenicereus undatus ). In this study, we have identified 26 Dof gene family members, renamed as HuDof-1 to HuDof-26 , and clustered them into seven subfamilies based on conserved motifs, domains, and phylogenetic analysis. The gene pairs of Dof family members were duplicated by segmental duplications that faced purifying selection, as indicated by the K a /K s ratio values. Promoter regions of HuDof genes contain many cis -acting elements related to phytohormones including abscisic acid, jasmonic acid, gibberellin, temperature, and light. We exposed pitaya plants to different environmental stresses and examined melatonin's influence on Dof gene expression levels. Signifcant expression of HuDof -2 and HuDof -6 were observed in different developmental stages of flower buds, flowers, pericarp, and pulp. Pitaya plants were subjected to abiotic stresses, and transcriptome analysis was carried out to identify the role of Dof gene family members. RNA-sequencing data and reverse transcription quantitative PCR-based expression analysis revealed three putative candidate genes (HuDof -1, HuDof -2, and HuDof -8), which might have diverse roles against the abiotic stresses. Our study provides a theoretical foundation for functional analysis through traditional and modern biotechnological tools for pitaya trait improvement.

A Critical Review of Conventional and Modern Approaches to Develop Herbicide-Resistance in Rice.

Together with rice, weeds strive for nutrients and space in farmland, resulting in reduced rice yield and quality. Planting herbicide-resistant rice varieties is one of the effective ways to control weeds. In recent years, a series of breakthroughs have been made to generate herbicide-resistant germplasm, especially the emergence of biotechnological tools such as gene editing, which provides an inherent advantage for the knock-out or knock-in of the desired genes. In order to develop herbicide-resistant rice germplasm resources, gene manipulation has been conducted to enhance the herbicide tolerance of rice varieties through the utilization of techniques such as physical and chemical mutagenesis, as well as genome editing. Based on the current research and persisting problems in rice paddy fields, research on the generation of herbicide-resistant rice still needs to explore genetic mechanisms, stacking multiple resistant genes in a single genotype, and transgene-free genome editing using the CRISPR system. Current rapidly developing gene editing technologies can be used to mutate herbicide target genes, enabling targeted genes to maintain their biological functions, and reducing the binding ability of target gene encoded proteins to corresponding herbicides, ultimately resulting in herbicide-resistant crops. In this review article, we have summarized the utilization of conventional and modern approaches to develop herbicide-resistant cultivars in rice as an effective strategy for weed control in paddy fields, and discussed the technology and research directions for creating herbicide-resistant rice in the future.

Phylogenomic curation of Ovate Family Proteins (OFPs) in the U's Triangle of Brassica L. indicates stress-induced growth modulation.

The Ovate Family Proteins (OFPs) gene family houses a class of proteins that are involved in regulating plant growth and development. To date, there is no report of the simultaneous functional characterization of this gene family in all members of U's Triangle of Brassica. Here, we retrieved a combined total of 256 OFP protein sequences and analyzed their chromosomal localization, gene structure, conserved protein motif domains, and the pattern of cis-acting regulatory elements. The abundance of light-responsive elements like G-box, MRE, and GT1 motif suggests that OFPs are sensitive to the stimuli of light. The protein-protein interaction network analysis revealed that OFP05 and its orthologous genes were involved in regulating the process of transcriptional repression through their interaction with homeodomain transcription factors like KNAT and BLH. The presence of domains like DNA binding 2 and its superfamily speculated the involvement of OFPs in regulating gene expression. The biotic and abiotic stress, and the tissue-specific expression analysis of the RNA-seq datasets revealed that some of the genes such as BjuOFP30, and BnaOFP27, BolOFP11, and BolOFP10 were highly upregulated in seed coat at the mature stage and roots under various chemical stress conditions respectively which suggests their crucial role in plant growth and development processes. Experimental validation of prominent BnaOFPs such as BnaOFP27 confirmed their involvement in regulating gene expression under salinity, heavy metal, drought, heat, and cold stress. The GO and KEGG pathway enrichment analysis also sheds light on the involvement of OFPs in regulating plant growth and development. These findings have the potential to serve as a forerunner for future studies in terms of functionally diverse analysis of the OFP gene family in Brassica and other plant species.

Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops.

The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.

Targeted genome editing in polyploids: lessons from Brassica.

CRISPR-mediated genome editing has emerged as a powerful tool for creating targeted mutations in the genome for various applications, including studying gene functions, engineering resilience against biotic and abiotic stresses, and increasing yield and quality. However, its utilization is limited to model crops for which well-annotated genome sequences are available. Many crops of dietary and economic importance, such as wheat, cotton, rapeseed-mustard, and potato, are polyploids with complex genomes. Therefore, progress in these crops has been hampered due to genome complexity. Excellent work has been conducted on some species of Brassica for its improvement through genome editing. Although excellent work has been conducted on some species of Brassica for genome improvement through editing, work on polyploid crops, including U's triangle species, holds numerous implications for improving other polyploid crops. In this review, we summarize key examples from genome editing work done on Brassica and discuss important considerations for deploying CRISPR-mediated genome editing more efficiently in other polyploid crops for improvement.

Deep learning supported machine vision system to precisely automate the wild blueberry harvester header.

An operator of a wild blueberry harvester faces the fatigue of manually adjusting the height of the harvester's head, considering spatial variations in plant height, fruit zone, and field topography affecting fruit yield. For stress-free harvesting of wild blueberries, a deep learning-supported machine vision control system has been developed to detect the fruit height and precisely auto-adjust the header picking teeth rake position. The OpenCV AI Kit (OAK-D) was used with YOLOv4-tiny deep learning model with code developed in Python to solve the challenge of matching fruit heights with the harvester's head position. The system accuracy was statistically evaluated with R2 (coefficient of determination) and σ (standard deviation) measured on the difference in distances between the berries picking teeth and average fruit heights, which were 72, 43% and 2.1, 2.3 cm for the auto and manual head adjustment systems, respectively. This innovative system performed well in weed-free areas but requires further work to operate in weedy sections of the fields. Benefits of using this system include automated control of the harvester's head to match the header picking rake height to the level of the fruit height while reducing the operator's stress by creating safer working environments.

Putting CRISPR-Cas system in action: a golden window for efficient and precise genome editing for crop improvement.

The daunting task of feeding an ever-growing population is an immense challenge for the contemporary scientific community, especially in view of the rapidly changing climate throughout the world. Amidst these threatening crises, we witness rapid development in genome editing (GE) technologies, revolutionizing the field of applied genomics and molecular breeding. Various GE tools have been developed during the last two decades, but the CRISPR/Cas system has most recently made a significant impact on crop improvement. The major breakthroughs of this versatile toolbox are genomic modifications like single base-substitutions, multiplex GE, gene regulation, screening mutagenesis, and enhancing the breeding of wild crop plants. Previously, this toolbox was used to modify genes related to significant traits such as biotic/abiotic resistance/tolerance, post-harvest traits, nutritional regulation, and to address self-incompatibility analysis-related challenges. In the present review, we have demonstrated the functional dynamics of CRISPR-based GE and its applicability in targeting genes to accomplish novel editing of crops. The compiled knowledge will provide a solid foundation for highlighting the primary source for applying CRISPR/Cas as a toolbox for enhancing crops, to achieve food and nutritional security.

New possibilities for trait improvement via mobile CRISPR-RNA.

CRISPR/Cas9 gene technology is transported as RNA from transgenic roots to distal parts of unmodified grafted scion, where it is translated into proteins to induce heritable mutagenesis at desired loci. This technique has the potential to produce transgene-free and genetically stable plants in difficult-to-propagate and near-extinct species.

Computational Tools and Resources for CRISPR/Cas Genome Editing.

The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants, as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genome editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduce the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focus on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights into the limitations of available computational tools that would help researchers of this field for further optimization. Lastly, we suggest a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.

Using Exogenous Melatonin, Glutathione, Proline, and Glycine Betaine Treatments to Combat Abiotic Stresses in Crops.

Abiotic stresses, such as drought, salinity, heat, cold, and heavy metals, are associated with global climate change and hamper plant growth and development, affecting crop yields and quality. However, the negative effects of abiotic stresses can be mitigated through exogenous treatments using small biomolecules. For example, the foliar application of melatonin provides the following: it protects the photosynthetic apparatus; it increases the antioxidant defenses, osmoprotectant, and soluble sugar levels; it prevents tissue damage and reduces electrolyte leakage; it improves reactive oxygen species (ROS) scavenging; and it increases biomass, maintains the redox and ion homeostasis, and improves gaseous exchange. Glutathione spray upregulates the glyoxalase system, reduces methylglyoxal (MG) toxicity and oxidative stress, decreases hydrogen peroxide and malondialdehyde accumulation, improves the defense mechanisms, tissue repairs, and nitrogen fixation, and upregulates the phytochelatins. The exogenous application of proline enhances growth and other physiological characteristics, upregulates osmoprotection, protects the integrity of the plasma lemma, reduces lipid peroxidation, increases photosynthetic pigments, phenolic acids, flavonoids, and amino acids, and enhances stress tolerance, carbon fixation, and leaf nitrogen content. The foliar application of glycine betaine improves growth, upregulates osmoprotection and osmoregulation, increases relative water content, net photosynthetic rate, and catalase activity, decreases photorespiration, ion leakage, and lipid peroxidation, protects the oxygen-evolving complex, and prevents chlorosis. Chemical priming has various important advantages over transgenic technology as it is typically more affordable for farmers and safe for plants, people, and animals, while being considered environmentally acceptable. Chemical priming helps to improve the quality and quantity of the yield. This review summarizes and discusses how exogenous melatonin, glutathione, proline, and glycine betaine can help crops combat abiotic stresses.

Genome-Wide Identification and Expression Pattern of the GRAS Gene Family in Pitaya (Selenicereus undatus L.).

The GRAS gene family is one of the most important families of transcriptional factors that have diverse functions in plant growth and developmental processes including axillary meristem patterning, signal-transduction, cell maintenance, phytohormone and light signaling. Despite their importance, the function of GRAS genes in pitaya fruit (Selenicereus undatus L.) remains unknown. Here, 45 members of the HuGRAS gene family were identified in the pitaya genome, which was distributed on 11 chromosomes. All 45 members of HuGRAS were grouped into nine subfamilies using phylogenetic analysis with six other species: maize, rice, soybeans, tomatoes, Medicago truncatula and Arabidopsis. Among the 45 genes, 12 genes were selected from RNA-Seq data due to their higher expression in different plant tissues of pitaya. In order to verify the RNA-Seq data, these 12 HuGRAS genes were subjected for qRT-PCR validation. Nine HuGRAS genes exhibited higher relative expression in different tissues of the plant. These nine genes which were categorized into six subfamilies inlcuding DELLA (HuGRAS-1), SCL-3 (HuGRAS-7), PAT1 (HuGRAS-34, HuGRAS-35, HuGRAS-41), HAM (HuGRAS-37), SCR (HuGRAS-12) and LISCL (HuGRAS-18, HuGRAS-25) might regulate growth and development in the pitaya plant. The results of the present study provide valuable information to improve tropical pitaya through a molecular and conventional breeding program.

A Critical Review: Recent Advancements in the Use of CRISPR/Cas9 Technology to Enhance Crops and Alleviate Global Food Crises.

Genome editing (GE) has revolutionized the biological sciences by creating a novel approach for manipulating the genomes of living organisms. Many tools have been developed in recent years to enable the editing of complex genomes. Therefore, a reliable and rapid approach for increasing yield and tolerance to various environmental stresses is necessary to sustain agricultural crop production for global food security. This critical review elaborates the GE tools used for crop improvement. These tools include mega-nucleases (MNs), such as zinc-finger nucleases (ZFNs), and transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, this review addresses the latest advancements in the role of CRISPR/Cas9 for genome manipulation for major crop improvement, including yield and quality development of biotic stress- and abiotic stress-tolerant crops. Implementation of this technique will lead to the production of non-transgene crops with preferred characteristics that can result in enhanced yield capacity under various environmental stresses. The CRISPR/Cas9 technique can be combined with current and potential breeding methods (e.g., speed breeding and omics-assisted breeding) to enhance agricultural productivity to ensure food security. We have also discussed the challenges and limitations of CRISPR/Cas9. This information will be useful to plant breeders and researchers in the thorough investigation of the use of CRISPR/Cas9 to boost crops by targeting the gene of interest.

Characterization of SHATTERPROOF Homoeologs and CRISPR-Cas9-Mediated Genome Editing Enhances Pod-Shattering Resistance in Brassica napus L.

Brassica napus is the most important oil crop plant for edible oil and renewable energy source worldwide. Yield loss caused by pod shattering is a main problem during B. napus harvest. In this study, six BnSHP1 and two BnSHP2 homoeologs were targeted by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome editing system and multiple SHP1 and SHP2 mutated lines were identified for evaluating the contribution for pod-shattering resistance. Our data suggest that BnSHP1A09 is probably a promising homoeolog for controlling lignin contents at dehiscence zone. Simultaneous mutation of BnSHP1A09/C04-B/A04 and BnSHP2A05/C04-A exhibited reduced lignified layer and separation layer adjacent to valves and replum. The pod-shattering resistance index (SRI) subsequently increased to 0.31 in five homoeolog mutation lines compared with the wild type (SRI = 0.036), which provide the theoretical basis for breeding of commercial pod-shattering resistance variety.

Omics: The way forward to enhance abiotic stress tolerance in Brassica napus L.

Plant abiotic stresses negative affects growth and development, causing a massive reduction in global agricultural production. Rapeseed (Brassica napus L.) is a major oilseed crop because of its economic value and oilseed production. However, its productivity has been reduced by many environmental adversities. Therefore, it is a prime need to grow rapeseed cultivars, which can withstand numerous abiotic stresses. To understand the various molecular and cellular mechanisms underlying the abiotic stress tolerance and improvement in rapeseed, omics approaches have been extensively employed in recent years. This review summarized the recent advancement in genomics, transcriptomics, proteomics, metabolomics, and their imploration in abiotic stress regulation in rapeseed. Some persisting bottlenecks have been highlighted, demanding proper attention to fully explore the omics tools. Further, the potential prospects of the CRISPR/Cas9 system for genome editing to assist molecular breeding in developing abiotic stress-tolerant rapeseed genotypes have also been explained. In short, the combination of integrated omics, genome editing, and speed breeding can alter rapeseed production worldwide.

Insights on SNP types, detection methods and their utilization in Brassica species: Recent progress and future perspectives.

The genus Brassica, family Brassicaceae (Cruciferae), comprises many important species of oil crops, vegetables and medicinal plants including B. rapa, B. oleracea, B. nigra, B. napus, B. juncea, B. carinata. Genomic researches in Brassica species is constrained by polyploidization, mainly due to its complicated genomic structure. However, rapid development of methods for detecting single nucleotide polymorphisms (SNP), such as next generation sequencing and SNP microarray, has accelerated release of reference Brassica species genomes as well as discovery of large numbers and genome-wide SNPs, thus intensifying forward genetics in this genus. In this review, we summarize biological characteristics, classification and various methods for detecting SNPs, focusing on high-throughput techniques. Moreover, we describe the pivotal roles of SNPs in genetic diversity, linkage map construction and QTL mapping, comparative genomics, linkage disequilibrium and genome-wide association studies. These insights are expected to deepen our understanding and guide further advancements in Brassica species research.

QTL and Candidate Gene Identification for Silique Length Based on High-Dense Genetic Map in Brassica napus L.

Silique length (SL) is an important yield trait and positively correlates with seeds per silique and seed weight. In the present study, two double haploid (DH) populations, established from crosses Zhongshuang11 × R11 (ZR) and R1 × R2 (RR), containing 280 and 95 DH lines, respectively, were used to map quantitative trait loci (QTL) for SL. A high-dense genetic map from ZR population was constructed comprising 14,658 bins on 19 linkage groups, with map length of 2,198.85 cM and an average marker distance of 0.15 cM. Genetic linkage map from RR population was constructed by using 2,046 mapped markers anchored to 19 chromosomes with 2,217-cM map length and an average marker distance of 1.08 cM. Major QTL qSL_ZR_A09 and qSL_RR_A09b on A09 were identified from ZR and RR populations, respectively. Both QTL could be stably detected in four environments. QTL qSL_RR_A09b and qSL_ZR_A09 were located on 68.5-70.8 cM and 91.33-91.94 cM interval with R2 values of 14.99-39.07% and 15.00-20.36% in RR and ZR populations, respectively. Based on the physical positions of single nucleotide polymorphism (SNP) markers flanking qSL_ZR_A09 and gene annotation in Arabidopsis, 26 genes were identified with SNP/Indel variation between parents and two genes (BnaA09g41180D and BnaA09g41380D) were selected as the candidate genes. Expression analysis further revealed BnaA09g41180D, encoding homologs of Arabidopsis fasciclin-like arabinogalactan proteins (FLA3), as the most promising candidate gene for qSL_ZR_A09. The QTL identification and candidate gene analysis will provide new insight into the genomic regions controlling SL in Brassica napus as well as candidate genes underlying the QTL.

Organelle genome composition and candidate gene identification for Nsa cytoplasmic male sterility in Brassica napus.

BACKGROUND: Nsa cytoplasmic male sterility (CMS) is a novel alloplasmic male sterility system derived from somatic hybridization between Brassica napus and Sinapis arvensis. Identification of the CMS-associated gene is a prerequisite for a better understanding of the origin and molecular mechanism of this CMS. With the development of genome sequencing technology, organelle genomes of Nsa CMS line and its maintainer line were sequenced by pyro-sequencing technology, and comparative analysis of the organelle genomes was carried out to characterize the organelle genome composition of Nsa CMS as well as to identify the candidate Nsa CMS-associated genes. RESULTS: Nsa CMS mitochondrial genome showed a higher collinearity with that of S. arvensis than B. napus, indicating that Nsa CMS mitochondrial genome was mainly derived from S. arvensis. However, mitochondrial genome recombination of parental lines was clearly detected. In contrast, the chloroplast genome of Nsa CMS was highly collinear with its B. napus parent, without any evidence of recombination of the two parental chloroplast genomes or integration from S. arvensis. There were 16 open reading frames (ORFs) specifically existed in Nsa CMS mitochondrial genome, which could not be identified in the maintainer line. Among them, three ORFs (orf224, orf309, orf346) possessing chimeric and transmembrane structure are most likely to be the candidate CMS genes. Sequences of all three candidate CMS genes in Nsa CMS line were found to be 100% identical with those from S. arvensis mitochondrial genome. Phylogenetic and homologous analysis showed that all the mitochondrial genes were highly conserved during evolution. CONCLUSIONS: Nsa CMS contains a recombined mitochondrial genome of its two parental species with the majority form S. arvensis. Three candidate Nsa CMS genes were identified and proven to be derived from S. arvensis other than recombination of its two parental species. Further functional study of the candidate genes will help to identify the gene responsible for the CMS and the underlying molecular mechanism.