Resumo 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. CRISPRCas Cas Glycine max. max . max) final cassete expressão. expressão expressão) Detectaramse Detectaram se sequenciamento funcionando

Abstract 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. CRISPRCas Cas Glycine max Agrobacteriummediated Agrobacterium mediated expression cassette. cassette . cassette) sequencing working CRISPRbased based

Resumo 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.

Abstract 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.

Resumo 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.

Abstract 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.

ABSTRACT Differences in seed storage tolerance between soybean cultivars have been frequently observed. Thus, identifying compounds found in them that may be related to these differences is necessary to support the selection of cultivars with seeds with better storage potential. Therefore, this study aimed to evaluate changes in viability and vigor of soybean seeds during storage in two environments, relating them to antioxidant activity, production of reactive oxygen species, and metabolites of the phenylpropanoid pathway. The experimental design was completely randomized in a 4 × 5 factorial scheme (storage periods × cultivars). Cultivars with contrasting characteristics of lignin content, seed coat color, and isoflavone content in the seed were used, stored for six months in a cold and dry chamber and an uncontrolled environment. Every two months, viability and vigor, enzymatic activity (superoxide dismutase and catalase), and hydrogen peroxide content were evaluated. Storage in a cold and dry chamber maintains seed viability of cultivars A, C, and D; it is reduced in all cultivars in an uncontrolled environment. Seed vigor is reduced during storage. There is no association between the seeds’ physiological quality and the superoxide dismutase and catalase activities. The increase in the hydrogen peroxide content in the seed coat is an indicator of the reduction in the seed physiological quality when stored in an uncontrolled environment. The difference in deterioration tolerance during storage is associated with the lignin content in the seed coat.

Abstract Soybean is a key crop in many countries, being used from human food to the animal industry due to its nutritional properties. Financially, the grain chain moves large sums of money into the economy of producing countries. However, like other agricultural commodities around the world, it can have its final yield seriously compromised by abiotic environmental stressors, like drought. As flowers imply in pods and in grains inside it to minimize damages caused by water restriction, researchers have focused on understanding flowering-process related genes and their interactions. Here a review dedicated to the soybean flowering process and gene network involved in it is presented, describing gene interactions and how genes act in this complex mechanism, also ruled by environmental triggers such as day-light and circadian cycle. The objective was to gather information and insights on the soybean flowering process, aiming to provide knowledge useful to assist in the development of drought-tolerant soybean lines, minimizing losses due to delays or anticipation of flowering and, consequently, restraining financial and productivity losses.

Abstract 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.

Abstract Water deficit is an important climatic problem that can impair agriculture yield and economy. Genetically modified soybean plants containing the AtNCED3 gene were obtained aiming drought-tolerance improvement. The NCED3 gene encodes a 9-cis-epoxycarotenoid dioxygenase (NCED, EC 1.13.11.51), an important enzyme in abscisic acid biosynthesis. ABA activates the expression of drought-responsive genes, in water-deficit conditions, targeting defense mechanisms and enabling plants to survive under low water availability. Results from greenhouse experiments showed that the transgene AtNCED3 and the endogenous genes GmAREB1, GmPP2C, GmSnRK2 and GmAAO3 presented higher expression under water deficit (WD) in the event 2Ha11 than in WT-plants. No significant correlation was observed between the plant materials and WD conditions for growth parameters; however, gas exchange measurements decreased in the GM event, which also showed 80% higher intrinsic water use when compared to WT plants. In crop season 2015/16, event 2Ha11 showed higher total number of pods, higher number of pods with seeds and yield than WT plants. ABA concentration was also higher in GM plants under WD. These results obtained in field screenings suggest that AtNCED3 soybean plants might outperform under drought, reducing economic and yield losses, thus being a good candidate line to be incorporated in the soybean-breeding program to develop drought-tolerant cultivars.

RESUMO O grande desafio para garantir a segurança alimentar global está na necessidade de aumentar a produção agrícola. Neste contexto, a biotecnologia destaca-se como uma ferramenta importante para gerar plantas geneticamente melhoradas, com maior resistência/tolerância a pragas, doenças e condições climáticas adversas, que utilizem de forma eficiente os recursos naturais. Uma abordagem avançada para a geração de variabilidade genética de maneira precisa e direcionada tem chamado a atenção dos melhoristas, a técnica de edição genômica CRISPR/Cas (repetições palindrômicas curtas agrupadas e regularmente interespaçadas/proteínas associadas ao CRISPR). O sistema de edição genômica CRISPR/Cas é formado por um RNA guia que direciona uma nuclease para gerar cortes específicos no DNA alvo. Esta ruptura ativa os sistemas de reparo celular e, eventualmente, leva a deleções ou inserções de nucleotídeos. Assim, a tecnologia CRISPR/Cas pode ser aplicada tanto na pesquisa científica básica, quanto no desenvolvimento de culturas com características agronômicas melhoradas, com o potencial de trazer soluções inovadoras para a agricultura. Esta técnica tem sido aplicada em um grande número de culturas agrícolas, incluindo algumas hortaliças. Nesta revisão, apresentamos detalhes do sistema natural e artificial CRISPR/Cas, suas possibilidades como ferramenta biotecnológica, vantagens sobre outras abordagens de melhoramento genético, questões regulatórias e sua aplicabilidade nas hortaliças, bem como desafios futuros.

ABSTRACT The burden of the current global challenge involving food security lies in the need to improve crop production. In this regard, biotechnology stands out as an essential tool to generate plants able to cope with pests, diseases, and harsh climatic conditions, and more efficient in the use of natural resources. An advanced approach to create genetic variability in a precise and targeted way, the genome-editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins), has drawn the attention of breeders. The genome editing CRISPR/Cas system relies on a guiding RNA that directs a nuclease to generate a double-strand break (DSB) at a target DNA, activating the cell repair systems and eventually leading to deletions or insertions of nucleotides. Therefore, CRISPR/Cas is a toolbox to achieve many goals, from basic science investigations to the development of crops with improved agronomic traits, with potential to bring innovative solutions to food production. The CRISPR/Cas system has been applied in a large number of plants, including some horticultural species. In this review, we present details of the CRISPR/Cas natural and artificial systems, its possibilities as a biotechnological tool, advantages over other breeding techniques, regulatory issues, and its applicability in horticultural crops, as well as future challenges.

Resumo: O estudo teve como objetivo avaliar a relação entre o teor de lignina no tegumento de sementes de soja com tolerância à deterioração por umidade e ao estresse oxidativo. Foram utilizadas quatro cultivares de soja: CD 208, CD 215, CD 202 e BRS 184, com teores de lignina no tegumento de 4,2; 2,6; 2,0 e 1,7%, respectivamente. As sementes foram submetidas a uma deterioração por umidade através de chuvas simuladas (4 horas), e, posteriormente, secas em estufa (35 °C), por 37 horas, totalizando dois ciclos de umedecimento e secagem. Foram realizados os testes: germinação, envelhecimento acelerado e teste de tetrazólio. Foi medida a atividade das enzimas antioxidantes: Superóxido Dismutase e Guaiacol Peroxidase; e a peroxidação lipídica. As avaliações foram realizadas no delineamento inteiramente casualizado, em esquema fatorial 2x4, com quatro repetições. As cultivares de soja CD 208 e CD 215, com maior teor de lignina no tegumento, são menos suscetíveis ao dano por umidade. As sementes da cultivar CD 208 também apresentaram menor estresse oxidativo, quando submetidas a deterioração por umidade.

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.