CRISPR-transient expression in soybean for simplified gRNA screening in planta

O objetivo deste trabalho foi desenvolver um método para criar e validar sistemas CRISPR-Cas e diferentes gRNAs em embriões de soja (Glycine max). Dois genes modelo foram usados para mutação simples com um gRNA ou deleção parcial do gene com dois guias. Os gRNAs foram inseridos nos vetores de transformação CRISPR por uma enzima de restrição do tipo IIS ou por subclonagem e inserção do promotor + gRNA2 no vetor de transformação final, com uso do método clássico de clonagem por enzimas de restrição. Os vetores foram construídos com sucesso para um e dois gRNAs. A transformação transiente de soja por Agrobacterium foi realizada para testar a qualidade dos gRNAs e do próprio sistema (cassete de expressão). Detectaram-se mutação simples e deleção gênica nos embriões transformados após o enriquecimento do DNA por digestão seguida de reação em cadeia da polimerase e sequenciamento, o que indica que o sistema CRISPR-Cas e os guias estavam funcionando. Este protocolo pode ser usado para acelerar as estratégias de edição de genoma baseadas em CRISPR, para transformação genética em soja.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

Tolerância à seca em cultivares-elite de soja com a introgressão do transgene AtAREB1

The objective of this work was to verify if the introgression of the AtAREB1 gene in the 'LS93-0375' and 'BMX Desafio RR' elite soybean germplasms increases the tolerance of these plants to water deficit. The F4 progenies of these two elite cultivars and of the AtAREB1 transgenic line (BR16-AtAREB1) and its background ('BR16') were subjected to water deficit assays. The water deficit bioassays were performed in a greenhouse using the following six soybean lines: the genetically modified BR16-AtAREB1 and its background 'BR16'; 'LS93' and its F4 progeny, LS93-AtAREB1; and 'BMX Desafio RR' and its F4 progeny, Desafio-AtAREB1. A randomized complete block experimental design was carried out in a 6x2 factorial arrangement, with  the six soybean genotypes and two water conditions – control (C) and water deficit (WD) treatments – with nine replicates. Soybean genotypes containing the AtAREB1 gene showed better physiological performances under drought stress and altered expressions of drought-responsive genes. The intogression of AtAREB1 in soybean increases the plant drought tolerance, regardless of the genetic background in which the gene was introduced.O objetivo deste trabalho foi verificar se a introgressão do gene AtAREB1 em dois germoplasmas-elite de soja, 'LS93-0375' e 'BMX Desafio RR', aumenta a tolerância dessas plantas ao deficit hídrico. As progênies F4 das duas cultivares-elite e da linhagem transgênica AtAREB1 (BR16-AtAREB1) e de seu background ('BR16') foram submetidas a deficit hídrico. Os bioensaios de deficit hídrico foram realizados em casa de vegetação, tendo-se utilizado as seis seguintes linhagens de soja: a geneticamente modificada BR16-AtAREB1 e seu background 'BR16'; 'LS93' e sua progênie F4, LS93-AtAREB1; e 'BMX Desafio RR' e sua progênie F4, Desafio-AtAREB1. Utilizou-se delineamento experimental de blocos completos com tratamentos casualizados, em arranjo fatorial 6x2, com os seis genótipos de soja e duas condições hídricas – controle (C) e tratamentos de deficit hídrico (WD) –, com nove repetições. Os genótipos de soja que contêm o gene AtAREB1 exibiram melhor desempenho fisiológico sob estresse hídrico e expressão alterada de genes responsivos à seca. A introgressão de AtAREB1 na soja aumenta a tolerância à seca, independentemente do background genético em que o gene foi introduzido

Análise de transcriptoma de experimentos de RNA- Seq com e sem repetições biológicas: revisão.

The discovery of nucleic acids opened new frontiers of knowledge, enablingresearchers to access an enormous amount of data, through large-scale sequencing methodologiesand bioinformatics tools. Amongst these new possibilities, RNA-Seq has been used to identify andquantify RNA molecules. To obtain more accurate biological responses from RNA-Seq data somequestions should be considered such as experimental design, type ofsynthesized library, size ofthefragments generated, number ofbiological replicates, depth, and coverage ofthe sequencing, speciesgenome availability and, the choice of software to properly perform the computational analyzes.Accurate bioinformatics analyzes allow the selection ofgenes with a lower error rate, increasing thevalidation assertiveness via RT-qPCR and thus, reducing costs. The objective of this review was topresent the analysis stages of RNA-Seq data, from experimental design to systems biology,considering relevant points, as well as to pointed out some software currently available to carry theseanalyzes out. Besides, with this review, we aimed to help the academic community to understand allsteps and biases involved in RNA-Seq data analysis, from experiments with or without biologicalreplicates.A descoberta de ácidos nucléicos abriu novas fronteiras de conhecimento, permitindoque os pesquisadores acessassem uma enorme quantidade de dados, através de metodologias desequenciamento em larga escala e ferramentas de bioinformática. Entre essas novas possibilidades,o RNA-Seq (sequenciamento de RNA) tem sido usado para identificar e quantificar moléculas deRNA. Para obter respostas biológicas mais precisas a partir dos dados de RNA-Seq, algumasquestões devem ser consideradas, como o desenho experimental, o tipo de biblioteca sintetizada, otamanho dos fragmentos gerados, o número de repetições biológicas, a profundidade e cobertura dosequenciamento, a disponibilidade do genoma da espécie e, a escolha dos softwares para executaradequadamente as análises computacionais. Análises bioinformáticas precisas permitem a seleçãode genes com menor taxa de erro, aumentando a assertividade da validação via RT-qPCR e, assim,reduzindo custos. O objetivo desta revisão foi apresentar as etapas de análise de dados de RNA-Seq,desde o projeto experimental até a biologia dos sistemas, considerando pontos relevantes, bemcomo apontar alguns softwares atualmente disponíveis para realizar essas análises. Além disso, comesta revisão, objetivamos ajudar a comunidade acadêmica a compreender todas as etapas e viesesenvolvidos na análise de dados de RNA-Seq, a partir de experimentos com ou sem réplicasbiológicas

Alterações fisiológicas de soja cultivada com remineralizador de solo no Cerrado sob regimes hídricos variáveis

The objective of this work was to evaluate the influence of the soil remineralizer fine-graded mica schist (FMS) on soybean (Glycine max) physiology, yield, and grain quality under different water regimes (WRs) in the Brazilian Cerrado. The experiment was conducted under field conditions for two years, using four WRs and three treatments: mica schist, conventional fertilization, and control. In 2017 and 2018, the following WRs were evaluated: WR1, WR2, WR3, and WR4, corresponding to a mean value of 100, 65, 44, and 28% of crop evapotranspiration replacement, respectively. Photosynthesis, stomatal conductance, transpiration, internal CO2 concentration, effective quantum yield of photosystem II (PSII) (Fv’/Fm’), quantum yield (PSII) (ᶲFSII), and electron transport rate reduced as a function of the advanced phenological stage of soybean and the reduction in WR. Grain quality was only affected by the WR. The mica schist was statistically similar to conventional fertilization and the control in 2017 and 2018. Yield decreased due to the anticipation of soybean phenological age and WR, but there were no differences between the three treatments in 2017 and 2018. The reduction in soybean yield is attributed to stomatal closure, loss of photoprotective capacity, and damage to the photosynthetic machinery caused by drought.O objetivo deste trabalho foi avaliar a influência do remineralizador de solo finos de mica xisto (FMS) na fisiologia, na produtividade e na qualidade de grãos de soja (Glycine max) em diferentes regimes hídricos (RHs), no Cerrado brasileiro. O experimento foi conduzido em condições de campo durante dois anos, tendo-se utilizado quatro RHs e três tratamentos: mica xisto, adubação convencional e controle. Em 2017 e 2018, foram avaliados os seguintes RHs: RH1, RH2, RH3 e RH4, que correspondiam ao valor médio de 100, 65, 44 e 28% da reposição da evapotranspiração da cultura, respectivamente. A fotossíntese, a condutância estomática, a transpiração, a concentração interna de CO2, o rendimento quântico efetivo do fotossistema II (FSII) (Fv’/Fm’), o rendimento quântico (FSII) (ᶲFSII) e a taxa de transporte de elétrons reduziram em função do avanço da fase fenológica da soja e da redução do RH. A qualidade dos grãos foi afetada apenas pelo RH. O mica xisto foi estatisticamente semelhante à adubação convencional e ao controle em 2017 e 2018. A produtividade reduziu devido à antecipação da idade fenológica da soja e do RH, mas não houve diferenças entre os três tratamentos em 2017 e 2018. A redução da produtividade da soja é atribuída ao fechamento estomático, à perda da capacidade fotoprotetora e aos danos ao maquinário fotossintético causados pela seca

Análise estrutural de vagens e sementes de soja submetidas à deterioração por umidade em pré-colheita

The objective of this work was to analyze structurally the pods and seed coats of soybean (Glycine max) cultivars, as well as to determine the quality of seeds when subjected to deterioration by weathering in pre-harvest. A 7×3 factorial arrangement was used – seven cultivars and three volumes of simulated rainfall (0, 54, and 162 mm). Exposure to rainfall was simulated in the R8 phenological stage. Then, the plants were taken to a greenhouse, where they were kept until the time of collection of pods and seeds. After collection, the following evaluations were carried out: tetrazolium test, lignin content in pods and seed coats, and structural analysis of pods (exocarp, mesocarp, and endocarp thickness) and seed coats (epidermis, hypodermis, and parenchymal-cell thickness). Pre-harvest rainfall of 54 and 162 mm reduces the quality of soybean seeds; however, the response to deterioration by weathering differs according to the cultivar. The tolerance to all simulated rainfall was greater for BRSMT Pintado, BRS Jiripoca, and M8210IPRO and lower for BRS 1010IPRO. Pods with a greater thickness of the exocarp, mesocarp, and endocarp and a high lignin content show greater resistance to weathering deterioration and seeds with greater vigor and viability. The thickness of the hypodermis of the testa is related to resistance to weathering deterioration and to the obtainment of high-quality seeds.O objetivo deste trabalho foi analisar estruturalmente as vagens e os tegumentos de cultivares de soja (Glycine max), bem com determinar a qualidade das sementes quando submetidas à deterioração por umidade em pré-colheita. Utilizou-se arranjo fatorial 7×3 – sete cultivares e três volumes de precipitações pluviais simuladas (0, 54 e 162 mm). A exposição à chuva foi simulada no estádio fenológico R8. Em seguida, as plantas foram levadas à casa de vegetação, onde foram mantidas até a coleta das vagens e das sementes. Após a coleta, realizaram-se as seguintes avaliações: teste de tetrazólio, teor de lignina nas vagens e nos tegumentos, e análise estrutural de vagens (espessura do exocarpo, do mesocarpo e do endocarpo) e tegumentos (espessura da epiderme, da hipoderme e das células parenquimatosas). Precipitações de 54 e 162 mm em pré-colheita reduzem a qualidade das sementes de soja; entretanto, a resposta à deterioração por umidade difere de acordo com a cultivar. A tolerância a todas as precipitações pluviais simuladas foi maior para BRSMT Pintado, BRS Jiripoca e M8210IPRO e menor para BRS 1010IPRO. Vagens com maior espessura do exocarpo, do mesocarpo e do endocarpo e elevados teores de lignina apresentam maior tolerância à deterioração por umidade e sementes com maior vigor e viabilidade. A espessura da hipoderme da testa de tegumentos está relacionada à tolerância à deterioração por umidade e à obtenção de sementes de elevada qualidade

Physiological and sanitary quality of soybean seeds under different chemical treatments during storage

ABSTRACT The purpose of this study was to evaluate the effect of the chemical treatment with fungicide, insecticide, micronutrient and polymer on physiological and sanitary quality of soybean seeds during storage. The treatments were arranged in a 3 x 5 x 5 factorial scheme (cultivars x seeds treatment x storage period), in completely randomized design with four replicates. Three cultivars were used: NA 4823RG, BMX TurboRR and Fundacep 62RR. The treatments were: T1: no chemical treatment, T2: fungicide, insecticide and micronutrient; T3: fungicide, insecticide, micronutrient and polymer, T4: fungicide; T5: insecticide. After the chemical treatment, the seeds were stored under environmental conditions from May to December 2012, and seed quality was evaluated at 0, 2, 4, 6 and 8 months of storage. Seeds water content and physiological quality were determined through tests of germination, accelerated aging, seedling length, seedling dry weight and sanity. The treatment with fungicides, insecticides, micronutrients and polymer did not affect seed quality over eight months of storage and promoted the control fungi associated with the seeds

Susceptibility to weathering damage and oxidative stress on soybean seeds with different lignin contents in the seed coat

Abstract: This study aimed at evaluating the relationship among lignin content in the seed coat of soybean seeds with tolerance to weathering damage and oxidative stress. Four soybean cultivars (CD 208, CD 215, CD 202 and BRS 184), with lignin contents in the seed coat of 4.2, 2.6, 2.0 and 1.7%, respectively, were used. Seeds were subjected to deterioration by moisture, through artificially simulated rains. Pods of all cultivars were subjected to irrigation by constant sprinkling during 4 hours, and then dried in oven, set at 35 °C, for 37 hours; repeating twice such procedure. After these treatments seeds were subjected to tests and then compared to the control. Tests performed were: germination, accelerated aging and tetrazolium test; thereby quantifying the occurrence of damages by weathering damage. The activity of enzymes superoxide dismutase and guaiacol peroxidase was measured; and the lipid peroxidation was assessed. Soybean cultivars CD 208 and CD 215, with higher lignin content in the seed coat, were less susceptible to weathering damage. Seeds of cultivar CD 208 have also presented lower oxidative stress, when subjected to weathering damage stress

Cytogenetic analysis of wheat seeds submitted to artificial aging stress

Several studies have been developed to understand the physiology of seed deterioration, however, the initial steps of the deterioration process are not completely clear. Seed vigor and viability losses are also associated with genetic material damage. This study was carried out to detect cytogenetic and physiological alterations occurring during the deterioration process of wheat seeds, due to artificial aging stress. Two cultivars, Ônix and Fundacep Campo Real, were submitted to six different treatments: control (T1), 24 (T2), 48 (T3), 72 (T4), 96 (T5) and 120 (T6) hours under artificial aging conditions: 42 °C and 100% relative humidity. Germination and vigor tests (First Count of Germination, Electrical Conductivity, Seedling Emergence in the Field and Emergence Speed Index) were performed. Cytogenetic evaluations were performed through smear technique for root tips, stained with 2% acetic orcein. Total number of cells under division and mitotic index (MI) were determined. According to results, vigor and mitotic index were affected due to deterioration process induced by artificial aging stress. A reduced number of dividing cells were observed in seeds submitted to prolonged period of aging. Chromosomal abnormalities in mitotic cells of aged seeds were detected with the occurrence of micronuclei, chromosome breaks and bridges

Transcriptional Profile of Soybean Seeds with Contrasting Seed Coat Color

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