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Background: Sugarcane (Saccharum spp.) covers vast areas of land (around 25 million ha worldwide), and its processing is already linked into infrastructure for producing bioethanol in many countries. This makes it an ideal candidate for improving composition of its residues (mostly cell walls), making them more suitable for cellulosic ethanol production. In this paper, we report an approach to improving saccharification of sugarcane straw by RNAi silencing of the recently discovered BAHD01 gene responsible for feruloylation of grass cell walls. Results: We identified six BAHD genes in the sugarcane genome (SacBAHDs) and generated five lines with substantially decreased SacBAHD01 expression. To find optimal conditions for determining saccharification of sugarcane straw, we tried multiple combinations of solvent and temperature pretreatment conditions, devising a predictive model for finding their effects on glucose release. Under optimal conditions, demonstrated by Organosolv pretreatment using 30% ethanol for 240 min, transgenic lines showed increases in saccharification efficiency of up to 24%. The three lines with improved saccharification efficiency had lower cell-wall ferulate content but unchanged monosaccharide and lignin compositions. Conclusions: The silencing of SacBAHD01 gene and subsequent decrease of cell-wall ferulate contents indicate a promising novel biotechnological approach for improving the suitability of sugarcane residues for cellulosic ethanol production. In addition, the Organosolv pretreatment of the genetically modified biomass and the optimal conditions for the enzymatic hydrolysis presented here might be incorporated in the sugarcane industry for bioethanol production. Keywords: Sugarcane, Made available in DSpace on 2019-06-22T00:35:25Z (GMT). No. of bitstreams: 1 SilencingofaBAHDacyltransferase.pdf: 1991226 bytes, checksum: 2c98f01c74be1c72d0b268e90455a2cb (MD5) Previous issue date: 2019

BackgroundA major limiting factor for plant growth is the aluminum (Al) toxicity in acidic soils, especially in tropical regions. The exclusion of Al from the root apex through root exudation of organic acids such as malate and citrate are both the most ubiquitous tolerance mechanisms in the plant kingdom. Two families of anion channels that confer Al tolerance are well described in the literature, ALMT and MATE family. ResultsIn this study, sugarcane plants constitutively overexpressing the Sorghum bicolor MATE gene (SbMATE) showed improved tolerance to Al when compared to non-transgenic (NT) plants, characterized by sustained root growth and exclusion of aluminum from the root apex based on the result obtained with hematoxylin staining. In addition, genome-wide analysis of the recently released sugarcane genome identified 11 ALMT genes and molecular studies showed potential new targets for aluminum tolerance. ConclusionsOur results indicate that the transgenic plants overexpressing the Sorghum bicolor MATE has an improved tolerance to Al. The expression profile of ALMT genes revels potential candidate genes to be used has an alternative for agricultural expansion in Brazil and other areas with aluminum toxicity in poor and acid soils.

Grass cell walls have hydroxycinnamic acids attached to arabinosyl residues of arabinoxylan (AX), and certain BAHD acyltransferases are involved in their addition. In this study, we characterized one of these BAHD genes in the cell wall of the model grass Setaria viridis. RNAi silenced lines of S. viridis (SvBAHD05) presented a decrease of up to 42% of ester-linked p-coumarate (pCA) and 50% of pCA-arabinofuranosyl, across three generations. Biomass from SvBAHD05 silenced plants exhibited up to 32% increase in biomass saccharification after acid pre-treatment, with no change in total lignin. Molecular dynamics simulations suggested that SvBAHD05 is a p-coumaroyl coenzyme A transferase (PAT) mainly involved in the addition of pCA to the arabinofuranosyl residues of AX in Setaria. Thus, our results provide evidence of p-coumaroylation of AX promoted by SvBAHD05 acyltransferase in the cell wall of the model grass S. viridis. Furthermore, SvBAHD05 is a promising biotechnological target to engineer crops for improved biomass digestibility for biofuels, biorefineries and animal feeding.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

As técnicas tradicionais de fenotipagem de plantas usam características facilmente mensuráveis, com base em medidas relacionadas, por exemplo, ao peso e ao tamanho das plantas. Tais medidas podem ser realizadas em qualquer horário, sem protocolo, preparação prévia da amostra ou conhecimentos profundos para a obtenção e interpretação dos dados. Por isso, são as mais usadas na experimentação a campo, embora envolvam métodos destrutivos e sejam altamente demandadoras de tempo, recursos financeiros e mão-de-obra. Em razão dos desafios atuais relacionados à competitividade do mercado de cultivares, à sustentabilidade do agronegócio e às mudanças climáticas globais, os cientistas de plantas têm buscado agregar modernas técnicas de fenotipagem ao processo de desenvolvimento de novas cultivares. Tais técnicas devem permitir uma análise ampla, detalhada e precisa da manifestação das informações genéticas do indivíduo e sua interação com o meio ambiente, aumentando assim a eficiência e eficácia do processo de seleção. Consequentemente, as novas técnicas de fenotipagem têm avançado em escala, precisão, reprodutibilidade e complexidade em relação às técnicas tradicionais, principalmente pelo uso de imagens geradas em plataformas automatizadas. Tais plataformas são constituídas de estações para a geração de imagens que utilizam diferentes técnicas espectroscópicas, para cobrir diferentes regiões do espectro eletromagnético. As estações mais comuns capturam imagens digitais RGB, de fluorescência da clorofila, UV/VIS, termográficas e na região do NIR, as quais possibilitam a avaliação do crescimento das plantas, do aparato fotoquímico, dos teores de pigmentos, da temperatura da copa e de diferentes metabólitos encontrado nas plantas. Traditional techniques of plant phenotyping use easily measurable traits, based on measures related for instance to plant weight and size. Such measurements can be made at any time, without protocol, prior sample preparation or deep knowledge for obtaining and interpreting the data. However, they require field experimentation, financial resources and manpower, use destructive methods and are time-consuming. Due to the current challenges related to the competitiveness of the cultivars market, the sustainability of agribusiness and global climate change, plant scientists have sought more specific and dynamics features, which are used to assist in explaining the responses of plants to the environment. To evaluate these characteristics, plant phenotyping has advanced in scale, precision, reproducibility and complexity compared to traditional techniques, especially by using automated platforms images. Such platforms consist of automated imaging stations that use different techniques to cover different regions of the electromagnetic spectrum. The most common imaging stations capture digital RGB, chlorophyll fluorescence, UV/VIS, thermographic and NIR images, which enable the evaluation of plant growth, photochemical apparatus, pigments content, canopy temperature and several metabolites found in plants. Keywords: plant evaluation, spectroscopy, phenotyping platforms, sensors

Setaria viridis is an emerging model for C4 species, and it is an important model to validate some genes for further C4 crop transformation, such as sugarcane, maize, and wheat. Here, we describe two protocols for stable transformation of S. viridis mediated by Agrobacterium tumefaciens with three different reporter genes and two selectable markers. Routine transformation efficiency reaching 29% was achieved using embryogenic callus in S. viridis (accession A10.1). Alternatively, we developed a transformation method by floral dip with 0.6% efficiency. The developed protocols could be useful for genetic and genomics studies of important food-feed-fiber-fuel C4 crops.

Setaria viridis is an emerging model for C4 species, and it is an important model to validate some genes for further C4 crop transformation, such as sugarcane, maize, and wheat. Here, we describe two protocols for stable transformation of S. viridis mediated by Agrobacterium tumefaciens with three different reporter genes and two selectable markers. Routine transformation efficiency reaching 29% was achieved using embryogenic callus in S. viridis (accession A10.1). Alternatively, we developed a transformation method by floral dip with 0.6% efficiency. The developed protocols could be useful for genetic and genomics studies of important food-feed-fiber-fuel C4 crops.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty.

After the publication of the article [1], it was brought to our attention that the GenBank Accession number for the EF1 is missing in Methods section. Please find the accession number in the erratum below.

Background Second-generation ethanol (2G-bioethanol) uses lignocellulosic feedstocks for ethanol production. Sugarcane is one among the most suitable crops for biofuel production. Its juice is extracted for sugar production, while sugarcane bagasse, straw, and senescing leaves are considered industrial waste. Senescence is the age-dependent deterioration of plant cells, ultimately leading to cell death and completion of the plant life cycle. Because senescing leaves may also be used for biofuel production, understanding the process of natural senescence, including remobilization of nutrients and its effect on cell walls can provide useful information for 2G-bioethanol production from sugarcane leaves. Results The natural senescence process in leaves of the commercial sugarcane cultivar RB867515 was investigated. Senescence was characterized by strong reduction in photosynthetic pigments content, remobilization of the nutrients N, P, K, B, Cu, Fe, and Zn, and accumulation of Ca, S, Mg, B, Mn, and Al. No significant changes in the cell-wall composition occurred, and only small changes in the expression of cell wall-related genes were observed, suggesting that cell walls are preserved during senescence. Senescence-marker genes, such as SAG12-like and XET-like genes, were also identified in sugarcane and found to be highly expressed. Conclusions Our study on nutrient remobilization under senescence in a vigorous sugarcane cultivar can contribute to the understanding on how nutrient balance in a high-yielding crop is achieved. In general, neutral monosaccharide profile did not change significantly with leaf senescence, suggesting that senescing leaves of sugarcane can be as a feedstock for biofuel production using pretreatments established for non-senescing leaves without additional efforts. Based on our findings, the potential biotechnological applications for the improvement of sugarcane cultivars are discussed. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0568-0) contains supplementary material, which is available to authorized users.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.

The potential use of laser-induced fluorescence imaging to monitor citrus greening disease in sweet orange (Citrus sinensis) plants was investigated. For this purpose, an experiment using healthy plants as the control and plants of the same variety inoculated with Candidatus Liberibacter asiaticus (CLas) was performed to verify the fluorescence response through 8months after bacterial inoculation. A system using laser-induced fluorescence at 473nm of wavelength excitation was configured. Color descriptors from the collected fluorescence images were determined using the average values for red (R); green (G); blue (B); hue (H); saturation (S); value (V, or Intensity); relative red (rR); relative green (rG); relative blue (rB) and luminosity (L). The data were analyzed using a paired Student's t-test by means of comparison between diseased and healthy plants. The results demonstrate a potential method to identify citrus greening at an early stage of the disease (first month), prior to the onset of visual symptoms at confidence level of 95%.

This study investigated the organic and inorganic constituents of healthy leaves and Candidatus Liberibacter asiaticus (CLas)-inoculated leaves of citrus plants. The bacteria CLas are one of the causal agents of citrus greening (or Huanglongbing) and its effect on citrus leaves was investigated using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics. The information obtained from the LIBS spectra profiles with chemometrics analysis was promising for the construction of predictive models to identify healthy and infected plants. The major, macro- and microconstituents were relevant for differentiation of the sample conditions. The models were then applied to different inoculation times (from 1 to 8 months). The models were effective in the classification of 82-97% of the diseased samples with a 95% significance level. The novelty of this method was in the fingerprinting of healthy and diseased plants based on their organic and inorganic contents.

Drought cause serious yield losses in soybean (Glycine max), roots being the first plant organ to detect the water-stress signals triggering defense mechanisms. We used two drought induction systems to identify genes differentially expressed in the roots of the drought-tolerant soybean cultivar MG/BR46 (Conquista) and characterize their expression levels during water deficit. Soybean plants grown in nutrient solution hydroponically and in sand-pots were submitted to water stress and gene expression analysis was conducted using the differential display (DD) and real time polymerase chain reaction (PCR) techniques. Three differentially expressed mRNA transcripts showed homology to the Antirrhinum majus basic helix-loop-helix transcription factor bHLH, the Arabidopsis thaliana phosphatidylinositol transfer protein PITP and the auxin-independent growth regulator 1 (axi 1). The hydroponic experiments showed that after 100 min outside the nutrient solution photosynthesis completely stopped, stomata closed and leaf temperature rose. Both stress induction treatments produced significant decrease in the mitotic indices of root cells. Axi 1, PITP and bHLH were not only differentially expressed during dehydration in the hydroponics experiments but also during induced drought in the pot experiments. Although, there were differences between the two sets of experiments in the time at which up or down regulation occurred, the expression pattern of all three transcripts was related. Similar gene expression and cytological analysis results occurred in both systems, suggesting that hydroponics could be used to simulate drought detection by roots growing in soil and thus facilitate rapid and easy root sampling.

Background The citrus industry worldwide has faced major economic losses due to the occurrence of Huanglongbing (HLB), a disease caused by Candidatus Liberibacter spp., Gramnegative bacteria restricted to the phloem of their hosts. Due to the lack of commercial varieties that could resist to these bacteria, genetic transformation comes as an important alternative for improving these varieties and managing the disease. Most of the work with citrus genetic transformation uses constitutive promoters, but recently studies have intensified the search for tissue-specific promoters, making possible to direct the transgene expression to the phloem and reduce it in other undesired parts of the plant, such as the fruits. It is possible that tissue-specific promoter sequences obtained from the same plant species have better acceptance by the customers and this was the objective of our work: prospect phloem-specific promoters from the citrus genome that can be used for citrus transformation with the objective to deliver transgene expression directly to the tissue of Ca. Liberibacter spp. accumulation.

Background Citrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam. Results The microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas. Conclusions Many gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB., BMC Genomics, 14, ISSN:1471-2164

The first step in plant transformation using somatic embryogenesis or organogenesis for plant regeneration in vitro has been the identification of genotypes with the best totipotency. Thus, the objective of this experiment was to evaluate the totipotency of ten cultivars and one soybean line from the Coodetec Gene Bank. The somatic embryos developed from immature cotyledons measuring from 4 to 6 mm in length, and the organogenesis was evaluated from mature soybean seeds. The cultivars CD 201, CD 216 and CD 220 were the most promising genotypes to produce somatic embryos, whereas the cultivar CD 217 had the highest quantity of differentiated explants, which represents the best regeneration efficiency for participating in organogenic systems. In the experiment with somatic embryogenesis, time to plant regeneration was ten months, but the plants were regenerated after only four months in the experiment with organogenesis. A identificação de genótipos que possuam maior totipotência quando submetidos à embriogênese somática e organogênese é a primeira etapa para a condução de um programa de transformação genética. Este trabalho teve como objetivo avaliar o potencial de embriogênese somática e organogênese em nove cultivares de soja e uma linhagem, do banco de germoplasma da Coodetec. Cotilédones imaturos de soja com 4 a 6 mm de comprimento foram utilizados para produção de embriões somáticos, induzidos com 2,4-D, e sementes maduras de soja foram usadas para avaliação do potencial organogênico. As cultivares CD 201, CD 216 e CD 220 mostram-se promissoras para produção de embriões somáticos, enquanto que para o sistema organogênico a cultivar CD 217 apresentou a maior quantidade de explantes diferenciados e manteve a maior eficiência de regeneração. Para obtenção de uma planta via embriogênese somática foram necessários 10 meses, em virtude do período necessário para obtenção dos explantes. O sistema organogênico se mostrou mais rápido, e plantas completas de soja foram obtidas em quatro meses de cultivo in vitro.

Background The history of the world citrus industry is marked by a series of diseases caused by different etiologic agents. Several of them are characterized as biotrophic pathogens like Xanthomonas citri subsp. citri ,t hreeCandidatus Liberibacter spp and Citrus leprosis virus C (CiLV-C). In plant-pathogen interactions, the role of salicylic acid (SA) in activating defense related genes is well recognized [1]. The SABP2 (Salicylic acid-binding protein 2) is required to convert methyl salicylate to SA as part of the signal transduction pathway that activates systemic acquired resistance, induces PR expression, and enhances disease resistance. Due to the reduced or absence of genetic resistance to these pathogens in commercial sweet orange cultivars, the genetic transformation to over-express a gene involved in the defense response of plants is a possible alternative to produce tolerant or resistant plants to biotrophic pathogens. Methods The aim of this study was to produce ‘Hamlin’ sweet orange (Citrus sinensis L. Osb.) transgenic plants, via Agrobacterium tumefaciens, over-expressing the SABP2 gene from sweet orange driven by the constitutive promoter ubiquitin (Ubq10). The SABP2 gene was cloned into pCambia 2301 and inserted into A. tumefaciens EHA 105. The genetic transformation was performed using epicotyl segments from seedlings [2]. Results and conclusions A total of 620 explants in three independent experiments were introduced and approximately 336 shoots were regenerated. The GUS histochemical test was performed and confirmed the transformation of 30 positive shoots. These shoots are being grafted onto Carrizo citrange [Citrus sinensis x Poncirus trifoliata (L.) Raf.] seedlings grown in test tubes containing MS culture medium. The presence of the transgene will be evaluated by PCR using specifics primers that amplify part of the ubiquitin promoter and part of the gene. SABP2 expression levels in transgenic plants will be assessed through qPCR. After bud multiplication, transgenic plants will be evaluated for their response to citrus canker, HLB and leprosis.

Tolerância à seca em plantas não é uma característica simples, mas sim um complexo de mecanismos que trabalham em conjunto ou isoladamente para evitar ou tolerar períodos de déficit hídrico. Genótipos que diferem em tolerância ao déficit hídrico devem apresentar diferenças qualitativas e quantitativas na expressão gênica quando submetidos a períodos de seca. Três cultivares de soja (BR-4, BR-16 e MG/BR-46 Conquista), com respostas contrastantes ao déficit hídrico, foram estudadas com o uso da técnica "Differential Display" (DD) para identificar e isolar genes que podem apresentar diferenças de expressão durante períodos de seca entre os genótipos estudados. Um total de 84 fragmentos de DNAc diferencialmente expressos foram detectados. Trinta e cinco fragmentos foram clonados em vetores pGEM-T, onde vinte e oito puderam ser seqüenciados. Comparação das seqüências obtidas com seqüências em bancos de genes mostraram identidade de seqüência com genes conhecidos. Um dos clones, por exemplo, mostrou homologia com um ativador de transcrição encontrado em Arabidopsis thaliana, ao passo que outro clone mostrou-se homólogo a uma subunidade de NADH Desidrogenase de Spinacia oleracea. O ativador de transcrição apresentou-se diferencialmente expresso somente em raízes, e o homólogo à subunidade de NADH Desidrogenase foi expresso somente no genótipo considerado tolerante à seca e somente durante a condição de estresse hídrico. Pelos resultados deste trabalho, foi possível identificar alguns fragmentos potencialmente envolvidos em respostas à condição de seca em soja. Com essas informações, o estudo desses genes pode ser aprofundado, visando à confirmação desses resultados e a auxiliar no desenvolvimento de novos genótipos mais adaptados às condições de seca em soja e possivelmente em outras culturas.

The citrus greening (or huanglongbing) disease has caused serious problems in citrus crops around the world. An early diagnostic method to detect this malady is needed due to the rapid dissemination of Candidatus Liberibacter asiaticus (CLas) in the field. This analytical study investigated the fluorescence responses of leaves from healthy citrus plants and those inoculated with CLas by images from a stereomicroscope and also evaluated their potential for the early diagnosis of the infection caused by this bacterium. The plants were measured monthly, and the evolution of the bacteria on inoculated plants was monitored by real-time quantitative polymerase chain reaction (RT-qPCR) amplification of CLas sequences. A statistical method was used to analyse the data. The selection of variables from histograms of colours (colourgrams) of the images was optimized using a paired Student's t-test. The intensity of counts for green colours from images of fluorescence had clearly minor variations for healthy plants than diseased ones. The darker green colours were the indicators of healthy plants and the light colours for the diseased. The method of fluorescence images is novel for fingerprinting healthy and diseased plants and provides an alternative to the current method represented by PCR and visual inspection. A new, non-subjective pattern of analysis and a non-destructive method has been introduced that can minimize the time and costs of analyses.

A simple criterion for the approximate validity of the single projection Peierls-Yoccoz method is discussed. A procedure for chosing the generator coordinate is proposed.