Effects of fungicides on the ultrastructure of the hypopharyngeal glands and the strength of the hives of Apis mellifera Linnaeus, 1758 (Hymenoptera: Apidae).

Several crops of agronomic interest depend on bees' pollination, and Apis mellifera L (Hymanoptera: Apidae) is the most studied direct pollinator. Nevertheless, the use of pesticides in agricultural environments is common, including fungicides. Studies that seek to evaluate the effects of fungicides on the hypopharyngeal glands of bees, the site of royal jelly synthesis, are lacking. Thus, this work aimed to evaluate the effect of field doses of fungicides (Captan SC® and Zignal®), alone or in mixture, on the hypopharyngeal glands and their subsequent effect on the strength of hives. The evaluations were carried out under field conditions in three hives per treatment. For a period of one month, bee hives received feed containing sugar syrup, pollen and 1.2 mL of Zignal® and 3 mL of Captan SC® in the isolated treatments and 4.2 mL in the mixture. The action of fungicides on the hypopharyngeal glands was determined by transmission electron microscopy analysis in bees 7 and 15 days old, collected in the hives one month after exposure to fungicides. The strength of the hives was evaluated for six months based on the number of frames with adult bees, open and closed brood, and stored food. The results indicate that fungicides promote early degeneration of the rough endoplasmic reticulum and morphological and structural changes in mitochondria. In addition, a reduction in adult population, open and closed breeding and food stock was observed. More pronounced damage occurred when bees were exposed to the mixture of fungicides. Overall, it can be concluded that the presence of fungicides in bee diets promotes harm accentuated over time and compromises the survival of hives. It will be worth estimating the fungicide effects of the queen development and on the colony heath.

Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations.

The term new genomic techniques (NGTs) is an umbrella term used to describe a variety of techniques that can alter the genetic material of an organism and that have emerged or have been developed since 2001, when the existing genetically modified organism (GMO) legislation was adopted. The analytical framework used to detect GMOs in Europe is an established single harmonized procedure that is mandatory for the authorization of GM food and feed, thus generating a reliable, transparent, and effective labeling scheme for GMO products. However, NGT products can challenge the implementation and enforcement of the current regulatory system in the EU, relating in particular to the detection of NGT products that contain no foreign genetic material. Consequently, the current detection methods might fail to meet the minimum performance requirements. Although existing detection methods may be able to detect and quantify even small alterations in the genome, this does not necessarily confirm the distinction between products resulting from NGTs subject to the GMO legislation and other products. Therefore, this study provides a stepwise approach for the in silico prediction of PCR systems' specificity by testing a bioinformatics pipeline for amplicon and primer set searches in current genomic databases. In addition, it also empirically tested the PCR system evaluated during the in silico analysis. Two mutant genotypes produced by CRISPR-Cas9 in Arabidopsis thaliana were used as a case study. Overall, our results demonstrate that the single PCR system developed for identifying a nucleotide insertion in the grf1-3 genotype has multiple matches in the databases, which do not enable the discrimination of this mutated event. Empirical assays further support this demonstration. In contrast, the second mutated genotype, grf8-61, which contains a -3 bp deletion, did not yield any matches in the sequence variant database. However, the primer sequences were not efficient during the empirical assay. Our approach represents a first step in decision making for analytical methods for NGT detection, identification, and quantification in light of the European labeling regulations.

Critical analysis of the extensive aerial application of pesticides and its implications for human health

Agriculture is one of the most important commercial activities worldwide since it contributes to a great amount of the nation's gross domestic product, labor opportunities, and food production. However, on the other hand, current industrial agriculture is extremely dependent on chemicals, both pesticides and fertilizers that are a serious threat to the health of people and the environment. Despite the advent of new technologies like unmanned aerial spraying systems (UASS), regulations surrounding the aerial spraying of pesticides to accommodate the benefits and limitations necessary to ensure the protection of humans and ecosystems are still scarce. High concentrations of chemical substances released by drones in a spray solution at improper altitudes, inappropriate ambient temperatures, or with incorrect droplet sizes increase the risk of phytotoxicity effects and spreading to non-target areas, potentially contaminating non-resistant neighboring crops, agricultural workers, and surrounding communities. Following the increase in the number of aircraft, the contamination events due to the drift events of pesticides increased parallelly. Research points out that “technical drift” may reach up to 19% of the sprayed volume, which does not reach the target, but goes to the soil, water, air, nearby plantations, and communities. Exposure to pesticides in smaller and regular doses can lead to chronic health conditions, which is much more difficult to study and prove. In some cases, illnesses develop years or decades after exposure but still are of great concern since the use of pesticides, notably in highly agricultural countries, has increased greatly to reach food and commodities demand.

Structural and evolutive features of the Plinia phitrantha and P. cauliflora plastid genomes and evolutionary relationships within tribe Myrteae (Myrtaceae).

Plinia phitrantha and P. cauliflora are Myrtaceae species with recognized horticultural and pharmacological potential. Nevertheless, studies on molecular genetics and the evolution of these species are absent in the literature. In this study, we report the complete plastid genome sequence of these species and an analysis of structural and evolutive features of the plastid genome within the tribe Myrteae. The two plastid genomes present the conserved quadripartite structure and are similar to already reported plastid genomes of Myrteae species concerning the size, number, and order of the genes. A total of 69-70 SSR loci, 353 single nucleotide polymorphisms, and 574 indels were identified in P. phitrantha and P. caulifora. Observed evolutive features of the plastid genomes support the development of programs for the conservation and breeding of Plinia. The phylogenomic analysis based on the complete plastid genome sequence of 15 Myrteae species presented a robust phylogenetic signal and evolutive traits of the tribe. Ten hotspots of nucleotide diversity were identified, evidence of purifying selection was observed in 27 genes, and relative conservation of the plastid genomes was confirmed for Myrteae. Altogether, the outcomes of the present study provide support for planning conservation, breeding, and biotechnological programs for Plinia species.

Phenological development of fruits in cultivars of feijoa (Acca sellowiana) and its relationship with south american fruit fly infestation / Desenvolvimento fenológico de frutos em cultivares de goiabeira-serrana (Acca sellowiana) e sua relação com infestação da mosca-das-frutas sul americana

The present study aimed to evaluate the developmental phases of feijoa fruits associated with infestation by Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) and the nonpreference of fruit flies for ripe fruits of feijoa. Two tests were carried out. In the first trial, we evaluated the association between the developmental stages of feijoa fruits and infestation by A. fraterculus. To accomplish this, fruits at five different stages of development, from green to ripe, were examined from the Alcântara cultivar. In the second trial, we evaluated the nonpreference of A. fraterculus for ripe fruits of Alcântara, Helena, Mattos and access 2316 cultivars. Physicochemical analyses were performed on the fruits of both trials. In the second test, analyses of polyphenol index and tannin concentration were also performed. Feijoa fruits in stage I are the likely targets of attack by A. fraterculus. The fruits of Alcântara cultivar from stage II, with 39 mm of transversal diameter and soluble solids, presented four puparium / fruit. The fruits of Alcântara and access 2316 cultivars showed greater and lesser infestation, respectively. The fruits of access 2316 also showed a higher concentration of tannins compared to the three commercial cultivars tested, which may have conferred the greatest protection against infestation by A. fraterculus. Feijoa fruits from Alcântara and Mattos cultivars showed high susceptibility to infestation by A. fraterculus, requiring management practices to guarantee commercial production.

O presente estudo teve como objetivos avaliar as fases de desenvolvimento de frutos de feijoa associadas à infestação por Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) e a não preferência por frutos maduros. No primeiro ensaio, foi avaliado a influência dos cinco estágios de desenvolvimento dos frutos de feijoa na infestação por A. fraterculus na cultivar Alcântara. No segundo ensaio, foi avaliado a não preferência de A. fraterculus por frutos maduros das cultivares Alcântara, Helena, Mattos e acesso 2316. Foram realizadas análises físico-químicas nos frutos de ambos os ensaios. No segundo ensaio, também foram realizadas análises do índice de polifenóis e concentração de taninos. Frutos de feijoa no estágio I são os prováveis alvos de ataque por A fraterculus. Os frutos do cultivar Alcântara, a partir do estádio II, quando apresentavam 39 mm de diâmetro transversal e sólidos solúveis, apresentaram quatro pupários/fruto. Os frutos da cultivar Alcântara e do acesso 2316 apresentaram maior e menor infestação, respectivamente. Os frutos do acesso 2316 também apresentaram a maior concentração de taninos do que nas três cultivares comerciais testadas, o que pode ter conferido a maior proteção contra infestação por A. fraterculus. Frutos de feijoa das cultivares Alcântara e Mattos apresentaram alta suscetibilidade à infestação por A. fraterculus, o que requer práticas de manejo para garantir a produção comercial.

Reflections on proposed modifications to the regulation of genetically modified food labeling in Brazil. / Reflexões sobre a proposta de modificação da regulamentação de rotulagem de alimentos transgênicos no Brasil.

Given the uncertainty surrounding the safety of genetically modified organisms (GMOs), the precautionary principle and constitution provide that consumers should have the right to access adequate information on the presence of transgenics through food labelling. This article discusses the implications of proposed modifications to GM food labelling in Brazil. Current labelling legislation and the government agencies involved in labelling do not guarantee that food products not bearing GMO labels are free of transgenics. The approval of Chamber of Deputies Bill No. 34/2015 goes against the Consumer Protection Code by undermining consumer autonomy and choice. In addition, it is likely to weaken the country's biosurveillance capabilities to identify and seize products that have a harmful effect on the health of humans, animals and the environment. The proposed changes constitute a retrograde step in the regulation of food labelling in Brazil and violate the individual and collective rights enshrined in the Federal Constitution, Consumer Protection Code, and international agreements signed by Brazil.

Diante da existência de incertezas científicas em relação à segurança dos transgênicos para a saúde humana e considerando o Princípio da Precaução e preceitos constitucionais em vigor, o consumidor deve ter o direito de ser informado de maneira adequada sobre a presença de transgênicos nos alimentos, por meio da rotulagem. Este ensaio tem por objetivo apresentar as implicações acerca da nova proposta de rotulagem de transgênicos no Brasil. A atual legislação brasileira de rotulagem de alimentos transgênicos e agências governamentais envolvidas não garantem que os produtos não identificados como tal sejam livres de transgênicos. A aprovação do PLC nº 34/2015 contraria dispositivos do Código de Defesa do Consumidor, indo na contramão da escolha e autonomia do consumidor. Além disso, a biovigilância será mais ainda inepta a executar uma atividade de identificação e apreensão de produtos que venham causar danos à saúde humana, animal e ao meio ambiente. A mudança proposta representa um retrocesso na regulamentação de rotulagem de transgênicos vigente no Brasil e um desrespeito aos direitos individuais e coletivos previstos na Constituição Federal, no Código de Defesa do Consumidor e em acordos internacionais assinados pelo Brasil.

Reflexões sobre a proposta de modificação da regulamentação de rotulagem de alimentos transgênicos no Brasil / Reflections on proposed modifications to the regulation of genetically modified food labeling in Brazil

Resumo Diante da existência de incertezas científicas em relação à segurança dos transgênicos para a saúde humana e considerando o Princípio da Precaução e preceitos constitucionais em vigor, o consumidor deve ter o direito de ser informado de maneira adequada sobre a presença de transgênicos nos alimentos, por meio da rotulagem. Este ensaio tem por objetivo apresentar as implicações acerca da nova proposta de rotulagem de transgênicos no Brasil. A atual legislação brasileira de rotulagem de alimentos transgênicos e agências governamentais envolvidas não garantem que os produtos não identificados como tal sejam livres de transgênicos. A aprovação do PLC nº 34/2015 contraria dispositivos do Código de Defesa do Consumidor, indo na contramão da escolha e autonomia do consumidor. Além disso, a biovigilância será mais ainda inepta a executar uma atividade de identificação e apreensão de produtos que venham causar danos à saúde humana, animal e ao meio ambiente. A mudança proposta representa um retrocesso na regulamentação de rotulagem de transgênicos vigente no Brasil e um desrespeito aos direitos individuais e coletivos previstos na Constituição Federal, no Código de Defesa do Consumidor e em acordos internacionais assinados pelo Brasil.

Abstract Given the uncertainty surrounding the safety of genetically modified organisms (GMOs), the precautionary principle and constitution provide that consumers should have the right to access adequate information on the presence of transgenics through food labelling. This article discusses the implications of proposed modifications to GM food labelling in Brazil. Current labelling legislation and the government agencies involved in labelling do not guarantee that food products not bearing GMO labels are free of transgenics. The approval of Chamber of Deputies Bill No. 34/2015 goes against the Consumer Protection Code by undermining consumer autonomy and choice. In addition, it is likely to weaken the country's biosurveillance capabilities to identify and seize products that have a harmful effect on the health of humans, animals and the environment. The proposed changes constitute a retrograde step in the regulation of food labelling in Brazil and violate the individual and collective rights enshrined in the Federal Constitution, Consumer Protection Code, and international agreements signed by Brazil.

Proteomic Profile of Glyphosate-Resistant Soybean under Combined Herbicide and Drought Stress Conditions.

While some genetically modified (GM) plants have been targeted to confer tolerance to abiotic stressors, transgenes are impacted by abiotic stressors, causing adverse effects on plant physiology and yield. However, routine safety analyses do not assess the response of GM plants under different environmental stress conditions. In the context of climate change, the combination of abiotic stressors is a reality in agroecosystems. Therefore, the aim of this study was to analyze the metabolic cost by assessing the proteomic profiles of GM soybean varieties under glyphosate spraying and water deficit conditions compared to their non-transgenic conventional counterparts. We found evidence of cumulative adverse effects that resulted in the reduction of enzymes involved in carbohydrate metabolism, along with the expression of amino acids and nitrogen metabolic enzymes. Ribosomal metabolism was significantly enriched, particularly the protein families associated with ribosomal complexes L5 and L18. The interaction network map showed that the affected module representing the ribosome pathway interacts strongly with other important proteins, such as the chloro-plastic gamma ATP synthase subunit. Combined, these findings provide clear evidence for increasing the metabolic costs of GM soybean plants in response to the accumulation of stress factors. First, alterations in the ribosome pathway indicate that the GM plant itself carries a metabolic burden associated with the biosynthesis of proteins as effects of genetic transformation. GM plants also showed an imbalance in energy demand and production under controlled conditions, which was increased under drought conditions. Identifying the consequences of altered metabolism related to the interaction between plant transgene stress responses allows us to understand the possible effects on the ecology and evolution of plants in the medium and long term and the potential interactions with other organisms when these organisms are released in the environment.

Effect of sowing time and density for vegetative and reproductive traits of genotypes of maize landrace in an agroecological system / Efeito de época e densidade de semeadura nas características vegetativas e reprodutivas em genótipos de variedades crioulas de milho em sistema de base agroecológica

Family farmers preserve the tradition of cultivating maize landrace varieties because these plants have characteristics that hybrids lack. The greatest challenge in conserving in situ on-farm genetic diversity is avoiding gene flow and genetic introgression of transgenes to landrace varieties. Thus, farmers are obliged to change sowing times to guarantee temporal isolation since most farms are small, making spatial isolation impossible. The objective of the present work was to evaluate the behavior of maize landrace varieties submitted to different sowing times and densities in an agroecological system. The test was conducted in sub-sub-divided plots, where the main plot was represented by the sowing time, the sub-plot by the genotype, and the sub-sub-plot by the density, with three replications, for two consecutive years. The results demonstrate the effect of sowing time and density on the characteristics evaluated. In western Santa Catarina, the best time to sow seeds of maize landrace varieties in an agroecological system is in September, which is when the varieties expressed the greatest potential at densities from 45,000 to 50,000 pl.ha-1 , but it will not avoid transgene contamination.(AU)

Famílias camponesas preservaram a tradição do cultivo das variedades crioulas de milho estimuladas por características que os híbridos não apresentam. Sendo, o maior desafio para a conservação da diversidade genética in situ on farm evitar o fluxo gênico e a introgressão genética de trangenes nas variedades crioulas. Nesse sentido, os agricultores são obrigados a alterar épocas de semeadura para garantir isolamento temporal uma vez que a maioria das áreas é pequena inviabilizando o isolamento no espaço. O objetivo do presente trabalho foi avaliar o comportamento de variedades crioulas de milho em sistema de base agroecológica submetidas às diferentes épocas e densidades de semeadura. No ensaio conduzido em parcelas sub-sub-divididas, a parcela principal foi representada pela época, a sub-parcela pelo genótipo e a sub-sub-parcela pela densidade, com três repetições, por dois anos consecutivos. Os resultados demostraram efeito de época e densidade de semeadura nas características avaliadas. É possível indicar que a época mais adequada para semeadura de variedades crioulas de milho em sistema de base agroecológica é em setembro para o oeste de Santa Catarina, sendo que para essa época as variedades expressaram maior potencial de produtividade de grãos na faixa de densidade de 45000 a 50000 pl.ha-1 , mas não impedirá a contaminação por transgene.(AU)

Production of Honeydew by Scale Insects Associated with Bracatinga (Mimosa scabrella Benth) in Serra Catarinense, Southern Brazil

ABSTRACT Scale insects Stigmacoccus paranaensis (Foldi, 2006) associated with bracatinga trees (Mimosa scabrella) in Santa Catarina, Brazil, can play an important economic role and guarantee the production of "bracatinga honeydew honey". This scale insect has the ability to excrete large amounts of honeydew, of economic importance for beekeepers, especially due to the natural occurrence of host plants at high density, known as "bracatingais". In this sense, the objective of this study was to evaluate the honeydew produced by scale insects associated with M. scabrella in the Bom Retiro, Santa Catarina, Brazil. We determined the density of the host plant in the studied area, the intensity of infestation by scale insects, and the volume and sugar concentration of excreted honeydew. The average density of bracatinga was 437.5 individuals ha-1, and the average density of scale insects per plant was 8,287. During 10 minutes, the average rate of honeydew excretion by scale insects was 0.45 µl and 0.34 µl, respectively, in 2002 and 2004. The estimated volume of honeydew production was 0.54 L in 2002 and 0.41 L in 2004 tree-1 day-1. The highest sugar concentration was observed in 2004 (28.13%). The excretion rate was affected by climatological factors and the size of the insects. This is the first study presenting a systematic evaluation of honeydew production by scale insects in Southern Brazil. The results of the present study support production of honeydew honey in the Serra Catarinense region as an important sustainable economic and traditional activity.

Agrotóxicos: críticas à regulação que permite o envenenamento do país / Pesticides: criticisms of the regulation that allows poisoning the country

The article contains an analysis of the marketing authorizations and the consumption of pesticides in the last decade, as well as their effects on human health and the environment. In the period between 2010 and 2018, the amount of pesticides sold in Brazil increased more than twice as much as the cultivated area in the country. The permissiveness of pesticide governance in Brazil is illustrated by the fact that, of the total chemical active ingredients with register in the country in July 2020, 121 (30.0%) had no use allowed in the European Union (EU). The reason for the ban on the use in the EU of products authorized in Brazil, in the referred period, is associated with the adverse effects on humans and other organisms resulting from exposure to chemical ingredients of pesticides, already described in the scientific literature. Soybean, corn, cotton and sugar cane are among those that had the largest number of new pesticides authorized for use in Brazil between January 2019 and June 2020. Another lack of rigor in pesticide governance is illustrated by the drift, which causes acute and chronic damage to third parties. Another weakness of the Brazilian standard refers to the establishment of a dose value, below which, they would be safe. However, experimental results from studies have shown, for example, that pesticides based on glyphosate, melation and pyriproxifem caused the death of fish embryos in just 24 hours of exposure, regardless of the concentration used, challenging the hypotheses that there is safe dose for exposure to such pesticides. Thus, it is urgent that there is a need not only to increase the scientific rigor of the studies on the subject, but also to promote the public participation in decisionmaking process ensuring the access to all information contained in the dossiers accompanying requests for registration and authorization

artigo contém uma análise das autorizações para a comercialização e o consumo de agrotóxicos no país na última década, bem como os seus efeitos na saúde humana e no meio ambiente. No período entre 2010 e 2018, a quantidade de agrotóxicos comercializados no Brasil aumentou mais do que o dobro do que cresceu a área cultivada no país. A permissividade da gestão de agrotóxicos no Brasil é ilustrada pelo fato de que, do total de ingredientes ativos químicos com registro no país em julho de 2020, 121 (30%) não tinham uso permitido na União Europeia (EU). A razão da proibição do uso na EU dos produtos autorizados no Brasil, no período referido, está associada aos efeitos adversos a humanos e a outros organismos resultantes da exposição aos ingredientes químicos de agrotóxicos, já descritos na literatura científica. Soja, milho, algodão e cana-de-açúcar constam entre as culturas que tiveram o maior número de novos agrotóxicos com uso autorizado no Brasil entre janeiro de 2019 e junho de 2020. Outra falta de rigor na gestão de agrotóxicos é ilustrada pela deriva, que causa danos agudos e crônicos a terceiros. Adicionalmente, a fragilidade da norma brasileira refere-se ao estabelecimento de um valor de dose, abaixo da qual eles seriam seguros. Contudo, resultados experimentais de estudos mostraram, por exemplo, que os agrotóxicos à base de glifosato, melation e piriproxifem causaram a morte de embriões de peixes em apenas 24 horas de exposição, independentemente da concentração utilizada, desafiando assim a hipótese de que existe dose segura para a exposição a tais produtos. Dessa forma, é urgente não só aumentar o rigor científico dos estudos sobre o tema, mas também promover a participação pública nas tomadas de decisão, garantindo o acesso a todas as informações contidas nos dossiês que acompanham as solicitações de registro e de autorização dos agrotóxicos.

Effect of sowing time and density for vegetative and reproductive traits of genotypes of maize landrace in an agroecological system / Efeito de época e densidade de semeadura nas características vegetativas e reprodutivas em genótipos de variedades crioulas de milho em sistema de base agroecológica

ABSTRACT: Family farmers preserve the tradition of cultivating maize landrace varieties because these plants have characteristics that hybrids lack. The greatest challenge in conserving in situ on-farm genetic diversity is avoiding gene flow and genetic introgression of transgenes to landrace varieties. Thus, farmers are obliged to change sowing times to guarantee temporal isolation since most farms are small, making spatial isolation impossible. The objective of the present work was to evaluate the behavior of maize landrace varieties submitted to different sowing times and densities in an agroecological system. The test was conducted in sub-sub-divided plots, where the main plot was represented by the sowing time, the sub-plot by the genotype, and the sub-sub-plot by the density, with three replications, for two consecutive years. The results demonstrate the effect of sowing time and density on the characteristics evaluated. In western Santa Catarina, the best time to sow seeds of maize landrace varieties in an agroecological system is in September, which is when the varieties expressed the greatest potential at densities from 45,000 to 50,000 pl.ha-1 , but it will not avoid transgene contamination.

RESUMO: Famílias camponesas preservaram a tradição do cultivo das variedades crioulas de milho estimuladas por características que os híbridos não apresentam. Sendo, o maior desafio para a conservação da diversidade genética in situ on farm evitar o fluxo gênico e a introgressão genética de trangenes nas variedades crioulas. Nesse sentido, os agricultores são obrigados a alterar épocas de semeadura para garantir isolamento temporal uma vez que a maioria das áreas é pequena inviabilizando o isolamento no espaço. O objetivo do presente trabalho foi avaliar o comportamento de variedades crioulas de milho em sistema de base agroecológica submetidas às diferentes épocas e densidades de semeadura. No ensaio conduzido em parcelas sub-sub-divididas, a parcela principal foi representada pela época, a sub-parcela pelo genótipo e a sub-sub-parcela pela densidade, com três repetições, por dois anos consecutivos. Os resultados demostraram efeito de época e densidade de semeadura nas características avaliadas. É possível indicar que a época mais adequada para semeadura de variedades crioulas de milho em sistema de base agroecológica é em setembro para o oeste de Santa Catarina, sendo que para essa época as variedades expressaram maior potencial de produtividade de grãos na faixa de densidade de 45000 a 50000 pl.ha-1 , mas não impedirá a contaminação por transgene.

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.

Root colonization and arbuscular mycorrhizal fungal community composition in a genetically modified maize, its non-modified isoline, and a landrace.

The use of genetically modified (GM) plants has increased in recent decades, but there are uncertainties about their effects on soil microbial communities. Aiming to quantify root colonization and characterize arbuscular mycorrhizal fungi (AMF) communities associated with roots and rhizosphere soil of different maize genotypes, a field trial was carried out in Southern Brazil with three maize genotypes as follows: a GM hybrid (DKB 240 VTPRO), its non-modified isoline (DKB 240), and a landrace (Pixurum). Soil samples were collected to evaluate the occurrence of AMF during the growth of corn genotypes at sowing and V3 (vegetative), R1 (flowering), and R3 (grain formation) stages of the crop. The occurrence of AMF was determined by the morphological identification of spores, and by analyzing AMF community composition in soil and roots of maize, using PCR-DGGE. The GM genotype of maize promoted lower mycorrhizal colonization in the vegetative stage and had lower sporulation at grain development than the conventional hybrid and the landrace maize. Twenty AMF morphotypes were identified and 13 were associated with all maize genotypes. The genera Acaulospora, Glomus, and Dentiscutata had the largest numbers of species. There were no differences in AMF community composition due to maize genotypes or genetic modification, but crop phenological stages affected AMF communities associated with maize roots.

Molecular relationships of Campomanesia xanthocarpa within Myrtaceae based on the complete plastome sequence and on the plastid ycf2 gene.

Plastomes are very informative structures for comparative phylogenetic and evolutionary analyses. We sequenced and analyzed the complete plastome of Campomanesia xanthocarpa and compared its gene order, structure, and evolutionary characteristics within Myrtaceae. Analyzing 48 species of Myrtaceae, we identified six genes representing 'hotspots' of variability within the plastomes (ycf2, atpA, rpoC2, pcbE, ndhH and rps16), and performed phylogenetic analyses based on: (i) the ycf2 gene, (ii) all the six genes identified as 'hotspots' of variability, and (iii) the genes identified as 'hotspots' of variability, except the ycf2 gene. The structure, gene order, and gene content of the C. xanthocarpa plastome are similar to other Myrtaceae species. Phylogenetic analyses revealed the ycf2 gene as a promissing region for barcoding within this family, having also a robust phylogenetic signal. The synonymous and nonsynonymous substitution rates and the Ka/Ks ratio revealed low values for the ycf2 gene among C. xanthocarpa and the other 47 analyzed species of Myrtaceae, with moderate purifying selection acting on this gene. The average nucleotide identity (ANI) analysis of the whole plastomes produced phylogenetic trees supporting the monophyly of three Myrtaceae tribes. The findings of this study provide support for planning conservation, breeding, and biotechnological programs for this species.

Phylogeography of plastid DNA sequences suggests post-glacial southward demographic expansion and the existence of several glacial refugia for Araucaria angustifolia.

Despite the high diversity of the Southern South American environments, the patterns and processes driving both their species diversity and demographic history are still poorly known and are a challenging task. In this study, we evaluate plastid DNA sequences of the conifer species Araucaria angustifolia aiming to (i) assess the species genetic structure within its main range of occurrence, (ii) infer its population demographic history, looking for evidence of southward expansion, (iii) search for evidence of glacial refugia within the species distribution area and (iv) discuss some conservation and management strategies for this species. Twenty haplotypes were identified, revealing the presence of three distinct genetic groups across the geographic range of the species and structuring the populations into Northern, Central and Southern groups. Our results suggest the occurrence of post-glacial expansion of A. angustifolia towards the south, as well as the existence of at least three refugia within the species occurrence area. Testing the occurrence of historical demographic expansion, we suggest that genetic groups experienced fluctuations in effective size, associated to a structured distribution of populations. The identification of three genetic groups in this study corroborates the proposition of using the geographic distribution of A. angustifolia for selecting in situ conservation areas, for planning seed collection for ex situ conservation, as well as for the delineation of seed zones.

Post-seminal structure and development of the hemiparasitic plant Escobedia grandiflora (Orobanchaceae)

Escobedia grandiflora(L.f.) Kuntze is a wild hemiparasitic plant with orange roots. Little is known about the development of initial parasitism with the host, despite the significant value of roots for Central and South American communities. Therefore, this study aimed to characterize post-seminal structure and development of E. grandiflorain Pennisetum purpureumhost. To analyze the structure and development of E. grandiflora, seedlings, stems and roots samples were processed and examined underlight, confocal and scanning electron microscopy. Escobedia grandifloraseeds are composed of seed coat, perisperm, and embryo. Emergence of the radicle began eleven days after imbibition. Seedlings showed a root hair collar encircling the axis at the root-hypocotyl junction with elongation of internal cortical cells. Seedlings formed haustoria and successfully reached of the host roots 22 days following root emergence. In the root many starch grains were observed, albeit more scarce in the hypocotyl. After 43 days of root emergence, the seedling stage was finished with the formation of the definitive leaves, and star of the plant stage. After 64 days, root ramification, amount of starch, and orange pigmentation increased with formation of haustoria. The developmental pattern of E. grandiflora plants was slow, but the roots grew faster than the stem. Escobedia grandifloraseeds were not endospermic and have limited nutritional value. After root emergence, the young seedling must develop roots and starch storage towards to haustorium formation and attachment to host roots.

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

MAIN CONCLUSION: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

The Crambe abyssinica plastome: Brassicaceae phylogenomic analysis, evolution of RNA editing sites, hotspot and microsatellite characterization of the tribe Brassiceae.

Crambe abyssinica is an important oilseed crop that accumulates high levels of erucic acid, which is being recognized as a potential oil platform for several industrial purposes. It belongs to the family Brassicaceae, assigned within the tribe Brassiceae. Both family and tribe have been the subject of several phylogenetic studies, but the relationship between some lineages and genera remains unclear. Here, we report the complete sequencing and characterization of the C. abyssinica plastome. Plastome structure, gene order, and gene content of C. abyssinica are similar to other species of the family Brassicaceae. The only exception is the rps16 gene, which is absent in many genera within the family Brassicaceae, but seems to be functional in the tribe Brassiceae, including C. abyssinica. However, the analysis of gene divergence shows that the rps16 is the most divergent gene in C. abyssinica and within the tribe Brassiceae. In addition, species of the tribe Brassiceae also show similar SSR loci distribution, with some regions containing a high number of SSRs, which are located mainly at the single copy regions. Six hotspots of nucleotide divergence among Brassiceae species were located in the single copy regions by sliding window analysis. Brassicaceae phylogenomic analysis, based on the complete plastomes of 72 taxa, resulted in a well-supported and well-resolved tree. The genus Crambe is positioned within the Brassiceae clade together with the genera Brassica, Raphanus, Sinapis, Cakile, Orychophragmus and Sinalliaria. Moreover, we report several losses and gains of RNA editing sites that occurred in plastomes of Brassiceae species during evolution.