Search

Calo embriogênico tem sido o tecido-alvo mais utilizado para transformação genética de cereais. O objetivo deste trabalho foi investigar o estabelecimento de calos embriogênicos e a regenera ção de plantas in vitro a partir de embriões maduros de genótipos de aveia (Avena sativa L.). Embriões maduros foram retirados das sementes e colocados em meio MS (Murashige & Skoog), contendo 30,0 g L-1 de sacarose e 2,0 mg L-1 de ácido 2,4-diclorofenoxiacético (2,4-D). Após o período de indução de calos, agregados embriogênicos foram isolados e subcultivados a cada 21 dias para meio fresco. Os calos embriogênicos foram então transferidos para meio de indução de parte aérea, e, na seqüência, as partes aéreas foram transferidas para meio de indução de raízes. Houve diferenças entre genótipos quanto à capacidade de embriogênese somática e regeneração de plantas in vitro a partir de embrião maduro. Este explante permitiu a indução de calos embriogênicos, que se multiplicaram, e que regeneraram in vitro um grande número de plantas de genótipos como UFRGS 7 e UFRGS 19, o que o faz passível de ser utilizado na transformação genética da aveia. Embryogenic callus has been the most used target tissue for cereal genetic transformation. Therefore, the objective of this study was to investigate the establishment of embryogenic calli and the in vitro plant regeneration from mature embryos of oat genotypes (Avena sativa L.). Mature embryos were taken out of the seeds and placed on a culture medium MS (Murashige & Skoog), containing 30,0 mg L-1 of sucrose and 2,0 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D). From the induction period, embryogenic aggregates were isolated and subcultivated each 21 days into a fresh medium. After this period, embryogenic calli were transferred to a medium for shoot regeneration. Subsequently, the shoot was transferred to a medium for root induction. There was variability among genotypes for somatic embryogenesis and in vitro plant regeneration from mature embryos. This explant allowed the induction of calli with ability to multiply and regenerate high number of plants from genotypes as UFRGS 7 and UFRGS 19, what makes it capable for oat genetic transformation.

Apple (Malus × domestica Borkh.) is the most important deciduous tree fruit crop grown around the world. Comparisons of gene expression profiles from different tissues, conditions or cultivars are valuable scientific tools to better understand the gene expression changes behind important silvicultural and nutritional traits. However, the accuracy of techniques employed to access gene expression is dependent on the evaluation of stable reference genes for data normalization to avoid statistical significance undue or incorrect conclusions. The objective of this work was to select the best genes to be used as references for gene expression studies in apple trees by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Vegetative and reproductive tissues of the apple “Gala” cultivar were evaluated during their seasonal cycle of growth and dormancy. The expression of 23 traditional housekeeping genes or genes suggested as constitutive by microarray data was investigated. Tested combinations of primers allowed the specific amplification and the generation of suitable efficiency curves for gene expression studies by RT-qPCR. Gene stability was determined by two different statistical descriptors, geNorm and NormFinder. The known variable PAL gene expression was used to validate selected normalizers. Results obtained allowed us to conclude that MDH, SAND, THFS, TMp1 and WD40 are the best reference genes to accurately normalize the relative transcript abundances using RT-qPCR in various tissues of apple.

A regeneração de plantas in vitro é uma limitação encontrada na maioria dos cereais. Uma forma de minimizar essa dificuldade tem sido a busca de fontes alternativas de tecidos com habilidade para esse fim. O objetivo deste trabalho foi investigar o potencial e o período necessário para regeneração de plantas in vitro de genótipos brasileiros de aveia (Avena sativa L.) a partir de segmentos da base da folha. Segmentos de 1-2mm retirados do coleóptilo de genótipos de aveia foram cultivados por 21 dias em meio MS (MURASHIGE & SKOOG, 1962) suplementado com 2,0mg L-1 de 2,4-D (ácido 2,4- diclorofenoxiacético) e, posteriormente, transferidos para meio de regeneração de plantas. Não foi encontrada variabilidade entre os genótipos quanto à capacidade de induzir calos embriogênicos e regenerar plantas a partir de segmentos da base da folha, tendo sido possível regenerar genótipos de aveia antes considerados sem habilidade para o cultivo in vitro. O uso desse explante possibilitou reduzir o tempo de regeneração de plantas de aveia para cinco meses e a não correlação entre a percentagem de embriogênese somática e regeneração de plantas indica que a embriogênese não é o único caminho de regeneração a partir desse explante. The ability for in vitro plant regeneration is limited in most of the cereal crops. One way to minimize this problem has been to search for alternative sources of tissues with the ability for in vitro cultivation. This report aims at the investigation of the potential and the amount of time necessary for in vitro plant regeneration of Brazilian oat (Avena sativa L.) genotypes from leaf base segments. One to two millimeter coleoptile segments of oat genotypes were cultivated for 21 days on MS medium (MURASHIGE & SKOOG, 1962) plus 2,0mg L-1 de 2,4-D (2,4- dichlorophenoxyacetic acid), and transferred to plant regeneration media. Variability among genotypes for somatic embryogenesis and plant regeneration from leaf base segments was not found, but it was possible to regenerate oat genotypes previously considered recalcitrants for in vitro regeneration. Besides that, it was possible to reduce to 5 months the plant regeneration period with this explant. The non-significant correlation between the percentage of somatic embryogenesis and plant regeneration indicates that the embryogenesis is not the only way for plant regeneration from this explant

The bacterium Bacillus thuringiensis (Bt) is characterized by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the patogenicity of twelve Bt isolates was tested against Anticarsia gemmatalis, one of the most important insect pests of soybeans. Spore-crystal complex was applied to the surface of artificial diets and the mortality of A. gemmatalis larvae was assessed seven days after each treatment. When compared to a control Bt isolate known by its high toxicity to A. gemmatalis larvae, four novel Bt isolates exhibited even higher toxic activities against the insect, resulting in more than 90% mortality. PCR was used to amplify DNA fragments related to known cry1 genes. Bt strains with high toxicity produced expected PCR products of around 280 bp, whereas non-toxic or low toxic strains did not produce any PCR product or showed amplified fragments of different sizes. Toxic Bt isolates also exhibited an expected protein profile when total protein extracts were evaluated by SDS-PAGE.

of the panel presented at the SBBq annual meeting (see attachament).

Allium cyrilli was long misinterpreted by Italian botanists and often confused with other taxa. In order to fix the application of this name, a lectotype is selected. The taxonomy of this species is clarified and discussed, especially in relation to the similar A. nigrum . Moreover, the distribution data of A. cyrilli in Italy are significantly updated, since the species is newly recorded for northern Italy (Piedmont, Emilia-Romagna and Veneto). The most useful qualitative and quantitative morphological characters, aimed to distinguish this species, are highlighted.

A convenient vector system was developed to evaluate transcriptional promoter activities in plants. Two primary vectors, optionally containing the cauliflower mosaic virus (CaMV) 35S −47 or −90 minimal promoters, offer multiple sites for cloning the sequence of interest upstream from the β-glucuronidase gene (gusA). The piomoter-gusA cassette can be transferred to a binary vector containing the selectable neomycin phosphotransferase II-encoding gene (nptII) next to the left border. In addition, the transferred DNA (T-DNA) contains the chloramphenicol acetyltransferase gene (cat) driven by the CaMV 35S promoter. Activity of cat can serve as a reference for gusA expression to correct for effects of chromosomal position or T-DNA copy number.

Iron-sulfur [Fe-S] clusters are prosthetic groups required to maintain life processes including respiration, photosynthesis, metabolic reactions, sensing, signaling,and gene regulation. In plants the biogenesis of Fe-S proteins is compartmentalized and adapted to specific needs of the eukaryotic and photosynthetic cell. Although critical to so many fundamental metabolic pathways and drastically affecting plant adaptability and productivity, Fe-S proteins were never investigated in woody species. Eucalyptus grandis is an important economical tree widely cultivated in subtropical regions which suffers under low temperature stress. Here we describe a transcriptional analysis of the E. grandis NFS1, ISU1 and ISA1, three genes involved in the biogenesis of [Fe-S] clusters. Microarray analyses were carried out for the comparison of global gene expression in leaves and vascular tissues (xylem) of E. grandis and vascular tissues of E. globulus. In general, leaves from E. grandis demonstrated higher expression of these genes than xylem. EgrISU1 had a constitutive expression in E. grandis, but its expression pattern was higher in this species than in E. globulus xylem. Differences observed in the relative gene expression profile between xylem tissues from the two Eucalyptus species suggest that these genes may be implicated in the contrasting phenotypic characteristics of their wood. The response of these genes to a series of hormonal and stress signals over E. grandis seedlings was also evaluated by RT-qPCR. After the chilling treatment of seedlings, EgrNFS1 and EgrISU1 showed 6 to 8-fold and 0.6 to 1.7-fold increase respectively; andEgrISA1 exhibited a drastic 69 to 114-fold increase. These data are the same observed in Arabidopsis microarrays available in GeneVestigator database. These results suggest that (i) NFS1 and ISA1 may be related to the cellular response to stress caused by chilling, and (ii) the increase in the expression is probably due to sulfur metabolism. A time-course chilling experiment was also carried out. The ISU1 gene expression was higher in the first two hours of treatment and decreased right after that period. The ISA1 gene, which showed the highest expression in the previous experiment, didn’t show significant differences in the expression pattern during the 16 hours of chilling, as well as the NFS1 gene. Our data indicated that Fe-S proteins are possibly involved in the recovery of plants after chilling stress.

Motivated by the Brazilian Ministry of Agriculture, Farming and Supply (MAPA), we created in 2009 the “Collaborating Center in Agriculture Defense Relative to the Biosafety of Genetically Modified Eucalypts” (Project “CDA Eucalyptus”) in order to collect information and conduct research to assess the biosafety of GM eucalypts in the Brazilian context. The Normative Resolution Nr. 5 of the National Biosafety Technical Commission (CTNBio) is the official document presenting all information needed to propose the commercial release of GMOs in Brazil. Based on this document and along with the personnel of the Suzano Paper & Cellulose Co., we conducted a series of experiments with GM and non-GM eucalypts planted in a test field in the state of Sao Paulo to start collecting the necessary information. Two independent groups of transgenic plants, harboring two different transgene constructions along with non-GM control plants are being assayed. The genetic traits, the identity or names of the transgenes as well as the identity of each tree individual will not be revealed due to intellectual property request still pending. Each group of plants was represented by four independent events in triplicates (2 groups x 4 events x 3 clonal trees + 3 non-GM clonal trees), therefore totaling 27 individuals under analysis. Samples were identified by random numbers and all assays were conducted in a simple-blind or a double-blind fashion. Tests concluded until now included (i) the detection of transgene regulatory sequences in purified DNA samples by conventional PCR and RT-qPCR, confirming the expected sampling conducted; (ii) extraction, chemical characterization and analysis of the antifungal effects of essential (volatile) oils extracted from leaves; (iii) pollen germination in vitro; (iv) flower morphology; (v) seed production; (vi) initial seedling development; (vii) leaf allelopathy; (viii) measurements of total phenolic compounds in leaves and roots; and (ix) effects of leaf extracts on the viability of human colon cells. All results obtained from experiments (ii) to (ix) revealed no statistical differences between GM- and non-GM-derived samples. A second round of experiments will be conducted to confirm these results. Proteomic and transcriptomic profiling of GM and non-GM trees are under analysis, as well as a series of experiments that include the chemical, nutritional and biological analysis of honey samples derived from bee hives located in fields of GM versus non-GM plants; and bee (Apis mellifera) population dynamics.

Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO 2 . These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli . The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H + and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10 −6 –10 −7 M.

The bacterium Bacillus thuringiensis (Bt) is characterized by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the patogenicity of twelve Bt isolates was tested against Anticarsia gemmatalis, one of the most important insect pests of soybeans. Spore-crystal complex was applied to the surface of artificial diets and the mortality of A. gemmatalis larvae was assessed seven days after each treatment. When compared to a control Bt isolate known by its high toxicity to A. gemmatalis larvae, four novel Bt isolates exhibited even higher toxic activities against the insect, resulting in more than 90% mortality. PCR was used to amplify DNA fragments related to known cry1 genes. Bt strains with high toxicity produced expected PCR products of around 280 bp, whereas non-toxic or low toxic strains did not produce any PCR product or showed amplified fragments of different sizes. Toxic Bt isolates also exhibited an expected protein profile when total protein extracts were evaluated by SDS-PAGE.

Background Micro RNAs (miRNAs) are a class of small (~21 nucleotide) non-coding RNAs that recently gained much attention due to their perceived role as master regulators of gene expression in Eukaryotes, responsible for fine tuning gene expression regulation and, in plants, has been shown to be involved in a diverse range of biological processes such as plant development and architecture, flowering, cell differentiation and response to biotic and abiotic stresses [1]. The repertoire of expressed miRNAs differs among cell types, tissues, development, environmental condition, etc [2]. Notwithstanding, the exact function of thousands miRNAs sequences present in miRBase [http://www.mirbase.org/] is not elucidated. At this point, discovery and profiling of new and conserved miRNAs are critical in the attempt to understand their function and mechanism. Deep sequencing through next generation sequencing is the methodology of choice for this purpose as its ultra high throughput permits a comprehensive interrogation of the small RNA transcriptome, permitting de novo identification and relative quantification of different small RNA species [3]. Due to its economic importance the Eucalyptus grandis genome has been sequenced by JGI and the annotation of miRNAs is pivotal. In order to provide the first large scale experimental characterization of Eucalyptus miRNAs we performed an Illumina deep sequencing run that allowed us to discover and quantify the miRNA levels in two different tissues – xylem and leaves. Additionally, to get insights of the observed phenotypic differences in wood quality among Eucalyptus species, we characterized the xylem small RNA transcriptome of two different E. globulus individuals and

Dof proteins are a family of transcription factors specific to the plant kingdom that contain a particular class of zinc finger DNA binding domain. Members of this family are involved in the regulation of genes related to a plethora of metabolic processes including stress or hormone response, seed and endosperm development, flowering, carbohydrate metabolism, and cell or tissue specificity. Dof proteins and encoding genes were characterized in several plant species. Nevertheless poplar is the only woody species whose Dof genes were better characterized. The recent availability of the Eucalyptus grandis genome and transcriptome, along with transcription factor databases of several plant species allowed us to identify and run a valuable comparative analysis of the Dof protein family in this tree. These species included Arabidopsis thaliana, Arabidopsis lyrata, Carica papaya, Populus trichocarpa, Vitis vinifera, Sorghum bicolor, Chlamydomonas reinhardtii, Oryza sativa indica and Zea mays.The phylogenetic relationships among Dof proteins from E. grandis and Arabidopsis thaliana - a popular model for studying the genomics of many plants - is a fundamental step to unravel functionality of new Dof genes not yet characterized. Twenty-three distinct DNA sequences were predicted to belong to the Dof gene family after the analysis of the complete available genome of E. grandis. The deduced protein sequences of 22 members do contain a conserved Dof domain. One sequence seemed to have lost the conserved Dof domain, suggesting it to be a pseudogene or to present an activity not directly linked to the Dof family. Gene structures, including exon/intron positions, and amino acid sequences were predicted for each gene based on the available E. grandis transcriptome. In order to determine the relationship and function of the genes putatively encoding Dof proteins, we carried out a phylogenetic analysis with 43 Dof protein sequences from A. thaliana. Our analysis allowed us to classify the E. grandis Dof sequences into five groups of orthologous genes. Gene expression analysis via real time, quantitative PCR was also conducted with ten of the E. grandis Dof genes, using samples obtained from flowers, leaves and vascular tissue. Generally, Dof steady-state mRNA levels were higher in E. grandis vascular tissues, with more reduced levels in flowers. Dof genes showed an increase in steady-state mRNA levels after hormone signaling, and reduced levels following abiotic stress. This is the first study that aimed the identification of Dof genes in E. grandis which are possibly involved in numerous plant metabolic processes. The phylogenetic relationship to A. thaliana counterparts and the patterns of mRNA accumulation in E. grandis allowed us to speculate on possible roles for some of the Dof-encoding genes. Financial support: Brazilian Ministry of Agriculture, Farming & Supply (MAPA), and The National Council for the Development of Science & Technology (CNPq), Brazilian Ministry of Science & Technology (MCT).

Key Message Cytokinin together with MdoBRR1, MdoBRR8 and MdoBRR10 genes participate in the downregulation of MdoDAM1, contributing to the transition from endo- to ecodormancy in apple buds. Abstract The fnal step of cytokinin (CK) signaling pathway culminates in the activation of type-B response regulators (BRRs), important transcriptional factors in the modulation of CK-responsive genes. In this study, we performed a genomewide analysis aiming to identify apple BRR family members and understand their involvement in bud dormancy control. The investigation identifed ten MdoBRR protein-coding genes. A higher expression of three MdoBRR (MdoBRR1, MdoBRR9 and MdoBRR10) was observed in dormant buds in comparison to other developmental stages. Interestingly, in ecodormant buds these three MdoBRR genes were upregulated in a CK-dependent manner. Transcription profles, determined during dormancy cycle under feld and artifcially controlled conditions, revealed that MdoBRR1 and MdoBRR8 played important roles in the transition from endo- to ecodormancy, probably mediated by endogenous CK stimuli. The expression of MdoBRR7, MdoBRR9, and MdoBRR10 was induced in ecodormant buds exposed to warm temperatures, indicating a putative role in growth resumption after chilling requirement fulfllment. Contrasting expression patternsin vivo between MdoBRRs and MdoDAM1, an essential dormancy establishment regulator, were observed during dormancy cycle and in CK-treated buds. Thereafter, in vivo transactivation assays showed that CK stimuli combined with transient overexpression of MdoBRR1, MdoBRR8, and MdoBRR10 resulted in downregulation of the reporter gene gusA driven by the MdoDAM1 promoter. These pieces of evidences point to the integration of CK-triggered responses through MdoBRRs that are able to downregulate MdoDAM1, contributing to dormancy release in apple. Keywords MdoBRR · Cytokinin · Bud dormancy · MdoDAM1 · Apple Made available in DSpace on 2020-12-10T09:03:34Z (GMT). No. of bitstreams: 1 10.1007-s00299-020-02595-z.pdf: 3550323 bytes, checksum: 05420c80b17b97786fe271e78e5df069 (MD5) Previous issue date: 2020

Seedlessness in grapes is a desirable trait, especially for in natura consumption. Previously, we showed that VviAGL11 is the main responsible gene for seed morphogenesis in grapevine. Here we tested the function of this gene in grapevine with the use of plant plasmids. VviAGL11 was cloned into silencing and overexpression versions of p28iIR plasmid. Reproductive grapevine bunches from di ff erent seeded and seedless cultivars were separately treated with VviAGL11 -harboring plasmids, along with controls. Plasmids were detected in leaves after a month of treatment, and berries, leaves, stems and seeds were analyzed for ectopic gene expression by RT- qPCR after 90 days of plasmid injection. Fruits from the seedless ? Linda ? treated with the VviAGL11-over- expression plasmid showed high expression levels of VviAGL11 and exhibited small seeds that were not found in the untreated control samples. Mature grapes from seeded ? Italia ? and ? Ruby ? bunches treated with the VviAGL11-silencing plasmid showed decreased VviAGL11 expression, reduced number of seeds and increased number of seed traces. The present study con fi rms that VviAGL11 is a key master regulator of seed morpho- genesis in grapevine and corroborates with the applicability of plant plasmids as promising biotechnological tools to functionally test genes in perennial plants in a rapid and confident way. Keywords: Plant plasmid Grapevine Seed Silencing Overexpression VviAGL11 Made available in DSpace on 2019-01-07T23:54:59Z (GMT). No. of bitstreams: 1 MalabarbaPlantScience.pdf: 1102884 bytes, checksum: fe400c77873521d4a93caed7d657c8b5 (MD5) Previous issue date: 2019-01-07

Despite the wide appreciation of seedless grapes, little is known about the molecular mechanisms that drive the stenospermocarpic seedless-type phenotype in grapevine. In order to address the molecular mechanisms that control seedlessness in grapevine, our study aimed to characterize VviAGL11, a class D MADS-box transcription factor gene that has been proposed as the major candidate gene involved in Vitis vinifera seed morphogenesis. VviAGL11 allelic variations in seeded and seedless grapevine cultivars were determined, and its correlations with allele-specific steady-state mRNA levels were investigated. VviAGL11 relative expression was significantly higher in seeds at 2, 4, and 6 weeks after fruit set, whereas in the seedless grape its transcript levels were extremely low in all stages analyzed. In situ hybridization revealed transcript accumulation specifically in the dual endotesta layer of the seeds, which is responsible for elongation and an increase of cell number, a necessary step to determine the lignification and the final seed size. No hybridization signals were visible in the seedless grapevine tissues, and a morphoanatomical analysis showed an apparent loss of identity of the endotesta layer of the seed traces. Ectopic expression of VviAGL11 in the Arabidopsis SEEDSTICK mutant background restored the wild-type phenotype and confirmed the direct role of VviAGL11 in seed morphogenesis, suggesting that depletion of its expression is responsible for the erroneous development of a highly essential seed layer, therefore culminating in the typical apirenic phenotype. Key words: Apireny, grapevine, in situ hybridization, seedlessness, Sultanine, VviAGL11. Made available in DSpace on 2018-01-04T23:25:30Z (GMT). No. of bitstreams: 1 MalabarbaVviAGL111.pdf: 1779650 bytes, checksum: 9f378cfbfebcbf171e6b738925749389 (MD5) Previous issue date: 2018-01-04

Made available in DSpace on 2016-02-23T06:46:58Z (GMT). No. of bitstreams: 1 FALAVIGNAPlantMolBiolRp2013.pdf: 1004721 bytes, checksum: db1027744078f244a46a39df28872ee5 (MD5) Previous issue date: 2014-01-13