Introduction:Soybean agglutinin (SBA) is a major antinutritional factor in soybean seeds, reducing digestibility in monogastric animals. The Le1 gene encodes the primary lectin accumulated in seeds. Genome editing offers a direct strategy to eliminate this factor in elite cultivars. Methods:Two gRNAs targeting Le1 were inserted into a CRISPR/Cas9 binary vector and used for Agrobacterium tumefaciens-mediated transformation of the soybean cultivar BRS 537. Edited plants were screened by PCR, Sanger sequencing, protein electrophoresis (SDS-PAGE), hemagglutination assays, and segregating generations were tested to identify transgene-free progeny. Agronomic traits were evaluated under field conditions. Results:Twenty transformation events were generated, with an editing efficiency of 10%. Event AF12-13-1 carried a 4-bp deletion producing a truncated, unstable lectin protein. SDS-PAGE confirmed the absence of the ~30 kDa SBA band, and hemagglutination assays showed complete loss of lectin activity. Transgene-free T2 plants lacking Bar, Cas9, and AtU6 sequences were identified. Agronomic traits-including yield and thousand-seed weight-were comparable to the wild-type cultivar. Discussion:CRISPR/Cas9 editing of Le1 effectively eliminated SBA accumulation without compromising key agronomic traits. The resulting low-lectin soybean lines represent a promising approach to improve digestibility and feed efficiency for monogastric animals.

Genetic diversity studies and molecular characterization offer crucial insights into preserving germplasm and maximizing its utility effectively. Therefore, the present study aimed to verify the genetic diversity, population structure and linkage disequilibrium in a set of maize (Zea mays L.) germplasm adapted to South Brazil composed of 351 inbred lines of field corn, popcorn and sweet corn. Through genotyping-by-sequencing (GBS), we identified 291,633 high-quality single nucleotide polymorphism (SNP), with 57% common across the three maize types. Unique SNPs were also discovered: field corn (24,618 or 8%), popcorn (17,000 or 5%) and sweet corn (479 or 0.16%). Bayesian analysis revealed two distinct population groups: one encompassing popcorn lines and another combining field and sweet corn lines. The fixation index (Fst) indicated minimal differentiation between field and sweet corn. Conversely, field and sweet corn lines exhibited higher nucleotide diversity than popcorn. Linkage disequilibrium analysis showed quick decay, significantly affected by population structure and kinship. From this diverse germplasm, future genome-wide association studies (GWAS) can be used to identify genomic regions and genes of interest in maize.

Soybean is the main oilseed cultivated worldwide. Even though Brazil is the world's largest producer and exporter of soybean, its production is severely limited by biotic factors. Soil borne diseases are the most damaging biotic stressors since they significantly reduce yield and are challenging to manage. In this context, the present study aimed to evaluate the potential of a bacterial strain (Ag109) as a biocontrol agent for different soil pathogens (nematodes and fungi) of soybean. In addition, the genome of Ag109 was wholly sequenced and genes related to secondary metabolite production and plant growth promotion were mined. Ag109 showed nematode control in soybean and controlled 69 and 45% of the populations of Meloidogyne javanica and Pratylenchus brachyurus, respectively. Regarding antifungal activity, these strains showed activity against Macrophomia phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. For S. sclerotiorum, this strain increased the number of healthy plants and root dry mass compared to the control (with inoculation). Based on the average nucleotide identity and digital DNA-DNA hybridization, this strain was identified as Bacillus velezensis. Diverse clusters of specific genes related to secondary metabolite biosynthesis and root growth promotion were identified, highlighting the potential of this strain to be used as a multifunctional microbial inoculant that acts as a biological control agent while promoting plant growth in soybean.

Microsatellites have been widely used to genotype individuals and to address a myriad of biological questions in many research fields for decades. However, when implementing a microsatellite marker analysis routine from scratch, various problems can arise throughout the process from DNA extraction to allele scoring, including inputting errors in the database, decreasing the reliability of results and having profound negative impacts on the derived decisions. Therefore, correctly assigning a genotype to a sample is crucial and dependent on acquiring knowledge of the technique steps, such as the chemistry of reactions, software, and data curation. This study tested two previously constructed simple sequence repeat (SSR) sets containing ten primer pairs each (ten-plex) in 1142 maize genotypes. Here, we describe the challenges faced when implementing this microsatellite-based genotyping protocol in our laboratory and possible ways to overcome them, hopefully aiding other novice research teams in this field.

Producing double-stranded RNA (dsRNA) represents a bottleneck for the adoption of RNA interference technology in agriculture, and the main hurdles are related to increases in dsRNA yield, production efficiency, and purity. Therefore, this study aimed to optimize dsRNA production in E. coli HT115 (DE3) using an in vivo system. To this end, we designed a new vector, pCloneVR_2, which resulted in the efficient production of dsRNA in E. coli HT115 (DE3). We performed optimizations in the culture medium and expression inducer in the fermentation of E. coli HT115 (DE3) for the production of dsRNA. Notably, the variable that had the greatest effect on dsRNA yield was cultivation in TB medium, which resulted in a 118% increase in yield. Furthermore, lactose induction (6 g/L) yielded 10 times more than IPTG. Additionally, our optimized up-scaled protocol of the TRIzol (TM) extraction method was efficient for obtaining high-quality and pure dsRNA. Finally, our optimized protocol achieved an average yield of 53.3 mu g/mL after the production and purification of different dsRNAs, reducing production costs by 72%. Improved production of double-stranded RNA (dsRNA) in E. coli HT115 (DE3) bacteria resulted in significant yield increases and cost reductions through optimized methods. Graphical Abstract Improved production of double-stranded RNA (dsRNA) in E. coli HT115 (DE3) bacteria resulted in significant yield increases and cost reductions through optimized methods.

Phosphorus (P) is an essential macronutrient for maize (Zea mays L.) growth and development. Therefore, generating cultivars with upgraded P use efficiency (PUE) represents one of the main strategies to reduce the global agriculture dependence on phosphate fertilizers. In this work, genome-wide association studies (GWAS) were performed to detect quantitative trait nucleotide (QTN) and potential PUE-related candidate genes and associated traits in greenhouse and field trials under contrasting P conditions. The PUE and other agronomy traits of 132 maize inbred lines were assessed in low and normal P supply through the greenhouse and field experiments and Multi-locus GWAS was used to map the associated QTNs. Wide genetic variability was observed among the maize inbred lines under low and normal P supply. In addition, we confirm the complex and quantitative nature of PUE. A total of 306 QTNs were associated with the 24 traits evaluated using different multi-locus GWAS methods. A total of 186 potential candidate genes were identified, mainly involved with transcription regulator, transporter, and transference activity. Further studies are still needed to elucidate the functions and relevance of these genes regarding PUE. Nevertheless, pyramiding the favorable alleles pinpointed in the present study can be considered an efficient strategy for molecular improvement to increase maize PUE.

The objective of this work was to develop a method to create and validate CRISPR-Cas systems and different gRNAs in soybean (Glycine max) embryos. Two model genes were used for simple mutation with one gRNA or partial gene deletion with two guides. The gRNAs were inserted into the CRISPR transformation vectors by a type IIS restriction enzyme or by subcloning and inserting the promoter + gRNA2 in the final transformation vector using the classic restriction enzyme cloning method. The vectors were successfully constructed for one and two gRNAs. Agrobacterium-mediated transient transformation in soybean was carried out to test the quality of gRNAs and of the system itself (expression cassette). Simple mutation and gene deletion were detected in the embryos transformed after DNA enrichment by enzyme digestion followed by polymerase chain reaction and sequencing, which indicates that the CRISPR-Cas system and guides were working. This protocol can be used to accelerate CRISPR-based genome editing strategies for genetic transformation in soybean.

Soybean is the primary source of vegetable protein and is used for various purposes, mainly to feed animals. This crop can have diverse seed coat colors, varying from yellow, black, brown, and green to bicolor. Black seed coat cultivars have already been assigned as favorable for both seed and grain production. Thus, this work aimed to identify genes associated with soybean seed quality by comparing the transcriptomes of soybean seeds with contrasting seed coat colors. The results from RNA-seq analyses were validated with real-time PCR using the cultivar BRS 715A (black seed coat) and the cultivars BRS 413 RR and DM 6563 IPRO (yellow seed coat). We found 318 genes differentially expressed in all cultivars (freshly harvested seeds and seeds stored in cold chamber). From the in silico analysis of the transcriptomes, the following genes were selected and validated with RT-qPCR: ACS1, ACSF3, CYP90A1, CYP710A1, HCT, CBL, and SAHH. These genes are genes induced in the black seed coat cultivar and are part of pathways responsible for ethylene, lipid, brassinosteroid, lignin, and sulfur amino acid biosynthesis. The BRSMG 715A gene has almost 4times more lignin than the yellow seed coat cultivars. These attributes are related to the BRSMG 715A cultivar’s higher seed quality, which translates to more longevity and resistance to moisture and mechanical damage. Future silencing studies may evaluate the knockout of these genes to better understand the biology of soybean seeds with black seed coat.

Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH4 +), the sugarcane's preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH3/H+ cotransport at high availability of NH4 + and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of 15N-NH4 + in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH4 + transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (Km 270.9 µM; Vmax 209.3 µmol g-1 root DW h-1). We were able to infer that ScAMT3;3 plays a presumed role in NH4 + source-sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency.

Heirloom tomatoes are open-pollinated varieties bearing a wide diversity of colors and shapes that may be used by breeders aiming to improve physical and biochemical fruit traits. Hence, in this work heirloom tomato accessions were characterized, gathering information to genetic breeding programs focusing on human food. For that, 67 heirloom tomato accessions were evaluated for fruit size, fruit mass, fruit volume, color, vitamin C, titratable acidity, soluble solids content, phenolic compounds content, total flavonoid content, and antioxidant activity. The experiment was conducted in a randomized complete block design with three repetitions. Linear mixed model, Pearson's correlation and hierarchical clustering were applied to data. Five groups were formed by Ward's clustering method. The accession UEL 300 constituted group A, which had the greatest mass and volume fruit. Eight accessions formed group B and showed mostly yellow fruits. Group C was comprised of 13 accessions, which had the highest levels of carotenoids, vitamin C, and antioxidant activity. Thirty-three accessions that constituted group D did not stand out for any of the attributes, while 12 accessions into group E had the highest content of phenolic compounds and flavonoids, along with the highest ratio of soluble solids and acidity. Five accessions in this collection (UEL 296, UEL 146, UEL 238, UEL 231, and UEL 217) stood out for their biochemical traits. The wide diversity for physical and biochemical fruit traits can be explored in tomato breeding programs, seeking to develop new cultivars, and strengthening family farming.

AMMONIUM TRANSPORTER/METHYLAMMONIUM PERMEASE/RHESUS (AMT) family members transport ammonium across membranes in all life domains. Plant AMTs can be categorized into AMT1 and AMT2 subfamilies. Functional studies of AMTs, particularly AMT1-type, have been conducted using model plants but little is known about the function of AMTs from crops. Sugarcane (Saccharum spp.) is a major bioenergy crop that requires heavy nitrogen fertilization but depends on a low carbon-footprint for competitive sustainability. Here, we identified and functionally characterized sugarcane ScAMT2;1 by complementing ammonium uptake-defective mutants of Saccharomyces cerevisiae and Arabidopsis thaliana. Reporter gene driven by the ScAMT2;1 promoter in A. thaliana revealed preferential expression in the shoot vasculature and root endodermis/pericycle according to nitrogen availability and source. Arabidopsis quadruple mutant plants expressing ScAMT2;1 driven by the CaMV35S promoter or by a sugarcane endogenous promoter produced significantly more biomass than mutant plants when grown in NH4+ and showed more 15N-ammonium uptake by roots and nitrogen translocation to shoots. In A. thaliana, ScAMT2;1 displayed a Km of 90.17 µM and Vmax of 338.99 µmoles h-1 g-1 root DW. Altogether, our results suggest that ScAMT2;1 is a functional high-affinity ammonium transporter that might contribute to ammonium uptake and presumably to root-to-shoot translocation under high NH4+ conditions.

The interaction of mitochondria with cellular components evolved differently in plants and mammals; in plants, the organelle contains proteins such as ALTERNATIVE OXIDASES (AOXs), which, in conjunction with internal and external ALTERNATIVE NAD(P)H DEHYDROGENASES, allow canonical oxidative phosphorylation (OXPHOS) to be bypassed. Plant mitochondria also contain UNCOUPLING PROTEINS (UCPs) that bypass OXPHOS. Recent work revealed that OXPHOS bypass performed by AOXs and UCPs is linked with new mechanisms of mitochondrial retrograde signaling. AOX is functionally associated with the NO APICAL MERISTEM transcription factors, which mediate mitochondrial retrograde signaling, while UCP1 can regulate the plant oxygen-sensing mechanism via the PRT6 N-Degron. Here, we discuss the crosstalk or the independent action of AOXs and UCPs on mitochondrial retrograde signaling associated with abiotic stress responses. We also discuss how mitochondrial function and retrograde signaling mechanisms affect chloroplast function. Additionally, we discuss how mitochondrial inner membrane transporters can mediate mitochondrial communication with other organelles. Lastly, we review how mitochondrial metabolism can be used to improve crop resilience to environmental stresses. In this respect, we particularly focus on the contribution of Brazilian research groups to advances in the topic of mitochondrial metabolism and signaling.

Water stress drastically hinders crop yield, including soybean - one of the world's most relevant feeding crops - threatening the food security of an ever-growing global population. Hemoglobins (GLBs) are involved in water stress tolerance; however, the role they effectively play in soybean remains underexplored. In this study, in silico and in vivo analyses were performed to identify soybean GLBs, capture their transcriptional profile under water stress, and overexpress promising members to assess how soybean cope with waterlogging. Seven GLBs were found, two GLB1 (non-symbiotic) and five GLB2 (symbiotic or leghemoglobins). Three out of the seven GLBs were differentially expressed in soybean RNA-seq libraries of water stress and were evaluated by real-time PCR. Consistently, GmGLB1-1 and GmGLB1-2 were moderately and highly expressed under waterlogging, respectively. Composite plants with roots overexpressing GmGLB1-1 or GmGLB1-2 (mostly) showed higher transcript abundance of stress-defensive genes involved in anaerobic, nitrogen, carbon, and antioxidant metabolism when subjected to waterlogging. In addition, soybean bearing p35S:GmGLB1-2 had lower H2O2 root content, a reactive oxygen species (ROS), under water excess compared with the control condition. Altogether these results suggest that GmGLB1-2 is a strong candidate for soybean genetic engineering to generate waterlogging-tolerant soybean cultivars.

Climate change and population size records threaten food security. Therefore, the call for a more sustainable and efficient crop production has never been more urgent. Traditional plant breeding was one of the first successful approaches to expand cultivation areas and crop yield. Later, biotechnological tools and their products, such as genetically modified organisms containing exogenous DNA, further broadened the limits of agricultural results, yet bringing huge financial, bureaucratic, and public rejection hurdles. In the 90s, scientific advances brought the opportunity to drive mutations using engineered nucleases, and since 2013 CRISPR-Cas has emerged as the most practical toolkit to edit genomes. One of the most striking possibilities is to generate edited and non-transgenic plants. In this review, we present the working mechanism behind CRISPR-induced mutations and pinpoint the latest techniques developed, as well as its myriad of applications in agriculture. The enhancing scope of CRISPR ranges from introducing traits of agronomic interest – such as herbicide resistance, resistance/tolerance to biotic and abiotic stresses, and quality and durability of products – to accelerating plant breeding processes, including haploid induction, generating male-sterile lines, fixating hybrid vigor, and overcoming self-incompatibility. We also discuss regulatory issues surrounding edited plants and derived products around the world, challenges that must be overcome, and future prospects to harness all the potential of this amazing tool to guarantee the new crop production revolution.

The objective of this study was to determine the genetic diversity among Brazilian carioca common bean (Phaseolus vulgaris L.) cultivars for traits related to nitrogen use efficiency (NUsE). An additional objective was to identify potential parents and promising parental combinations to assist common bean breeding programs in the development of cultivars with improved NUsE. The experiments were carried out at the Experimental Stations of the Instituto de Desenvolvimento Rural do Parana (IDR-Parana) in Londrina and Ponta Grossa, Brazil, during the rainy season in 2017. Thirty-nine common bean cultivars were evaluated under high (40 kg N ha(-1)) and low N (0 kg N ha(-1)) applied as top-dressing. The cultivars were genotyped using the genotyping-by-sequencing (GBS) approach with restriction enzyme CviAII. A total of 23,823 single nucleotide polymorphism (SNP) markers were identified throughout the common bean genome. A wide genetic variability was observed for NUsE-related traits under high and low N conditions. In addition, correlations were found between shoot dry biomass and NUsE, N uptake efficiency (NUpE), and N utilization efficiency (NUtE) under low N, indicating the possibility of indirect selection of these traits by the plant shoot dry biomass. Interestingly, the most low-N-tolerant cultivars were not necessarily the most responsive and/or NUsE, suggesting that different morphophysiological mechanisms control these traits. The cultivars that stood out positively in this study can be used as parents in breeding programs aimed at developing cultivars that are adapted to N limitation.

The choice of promising parents represents a crucial step in developing improved cultivars in breeding programs for mini-tomatoes— highly demanded miniature vegetables. The association of non-sensory and sensory features of fruit greatly enhances the generation of cultivars that meet the expectations of the productive and commercial chain of tomatoes, focusing on diverse market niches. Thus, in this study, five genotypes of mini-tomatoes were characterized based on physical, biochemical, and sensory attributes. The genetic material encompasses four cultivars (BRS Iracema, BRS Zamir, Iraí, and Sweet Heaven) and one landrace (UEL 238). The fruit were characterized by their dimension, color, firmness, soluble solids content, acidity, vitamin C, carotenoids, phenolic compounds, flavonoids, and antioxidant activity. In the sensorial test, 109 participants, including 27 chefs, evaluated the shape, size, color, aroma, flavor, texture, and overall liking of the fruit. The cultivar Sweet Heaven, with an oblong red fruit, brought together the main desirable physical traits, such as greater mass, pericarp thickness, firmness, and soluble solids content; while BRS Zamir presented superior values for biochemical characteristics, such as total phenolic compounds, total flavonoids, and antioxidant activity. Despite the lesser appreciation of the landrace UEL 238, all genotypes were accepted by consumers, especially BRS Iracema (round red fruit), followed by Iraí (oblong yellow fruit). The characterized mini-tomatoes may be explored in breeding programs as promising parents to combine desirable sensory attributes with the highest nutraceutical quality, resulting in superior cultivars that have been increasingly demanded on the market.

Water deficit is one of the major limitations to soybean production worldwide, yet the genetic basis of drought-responsive mechanisms in crops remains poorly understood. In order to study the gene expression patterns in leaves and roots of soybean, two contrasting genotypes, Embrapa 48 (drought-tolerant) and BR 16 (drought-sensitive), were evaluated under moderate and severe water deficit. Transcription factors from the AP2/EREBP and WRKY families were investigated. Embrapa 48 showed 770 more up-regulated genes than BR 16, in eight categories. In general, leaves presented more differentially expressed genes (DEGs) than roots. Embrapa 48 responded to water deficit faster than BR 16, presenting a greater number of DEGs since the first signs of drought. Embrapa 48 exhibited initial modulation of genes associated with stress, while maintaining the level of the ones related to basic functions. The genes expressed exclusively in the drought-tolerant cultivar, belonging to the category of dehydration responsive genes, and the ones with a contrasting expression pattern between the genotypes are examples of important candidates to confer tolerance to plants. Finally, this study identified genes of the AP2/EREBP and WRKY families related to drought tolerance.

An optimal condition for extraction of soluble sugars from green coffee using water and a validated chromatographic method for its separation and quantification were proposed in this research. An orbital incubator shaker (OIS) and microwave-assisted extraction (MAE) were the 2 techniques used to extract soluble sugars. In such experiments, the variables: sample amount (300, 400, and 500 mg), time (30, 60, and 90 min), and temperature (30, 45, and 60 °C) were tested. The separation of sugars was performed in a chromatographic system (high-performance liquid chromatography refractive index detector [HPLC-RID]), which presented the selectivity for the analytes, a limit of detection of 0.020 g/L, a limit of quantification of 0.0625 g/L, and recovery rates greater than 95%. The repeatability and inter-day precision had low dispersion, RSD < 2.0% and < 3.0%, respectively. Sucrose content ranged from 0.65 to 2.39 g/L using an OIS and from 1.19 to 2.72 g/L by MAE, while glucose and fructose concentration varied from 0.08 to 0.12 g/L using both methods. The OIS technique is preferably indicated for extraction of soluble sugars at the following conditions: 500 mg of grounded green coffee, 90 min, and 60 °C. The proposed method for soluble sugar extraction and quantification may be applied in research laboratories and food industries since it is a low-cost and environment-friendly technique.

Drought is one of the main limiting factors of soybean production. The great deal of time and effort that current available phenotyping methods demand hampers the selection of tolerant genotypes. Therefore, the development of techniques capable of determining the water status of plants in a fast and practical way may improve the ability to distinguish genotypes under water deficit conditions. The aim of this study was to correlate physiological variables such as relative water content and gas exchange measurements, with vegetation indices (VIs) and spectral bands in order to optimize tools for plant phenotyping. Two trials were carried out, one in a climatic chamber and one in the field. The soybean genotypes were submitted to water deficit and control (irrigated) conditions. The variables measured were relative water content, leaf temperature, photosynthesis, transpiration, stomatal conductance and internal CO2 content. The VIs NDWI(1000–1600), NDWI(1000–2300), NMDI, MSI and the spectral bands SWIR1600, SWIR2300, ρ1440, ρ1920, ρ1440+ρ1920, ρ1920−ρ1440 and SWIR−ρ1440 were obtained using a hyperspectral sensor. According to the results, the physiological measurements, the VIs and the spectral bands were able to differentiate the water conditions to which the genotypes were submitted and, in some cases, the indices and bands were more sensitive than the physiological measures to detect genotype effect. All indices and bands were efficient in determining the water status of soybean plants. However, the SWIR indices were the most sensitive, allowing the differentiation of a greater number of genotypes with high accuracy.