Andréa Almeida Carneiro

Otimização dos parâmetros de bombardeamento de partículas para a transformação genética de linhagens brasileiras de milho

The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditions ranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics.

A Phosphate Transporter Promoter from Arabidopsis thaliana AtPHT1;4 Gene Drives Preferential Gene Expression in Transgenic Maize Roots Under Phosphorus Starvation

Phosphorus (P) stress responsive genes have been identified and characterized, including the high-affinity phosphate transporter AtPHT1;4 from Arabidopsis thaliana. This gene encodes a membrane protein that is primarily expressed in roots under phosphorus deficiency. A 2.3-kb promoter region from AtPHT1;4 has been fused with the β-glucuronidase (GUS) encoding gene and introduced into maize via biolistic bombardment to evaluate its spatiotemporal activity in a heterologous system. AtPHT1;4::GUS expression is detected preferentially in transgenic maize roots under P deficiency. Further analysis of transgenic plants has also revealed that GUS activity is higher in roots than in leaves by about sixfold. These results demonstrate the ability of AtPHT1;4 promoter to direct expression of the reporter gene in a monocot root system under P stress. This property of AtPHT1;4 promoter makes it useful to engineer maize plants to modify the soil’s rhizosphere and increase efficiency of P acquisition under P stress conditions.

Molecular characterization and pathogenicity of isolates of Beauveria spp. to fall armyworm

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs.

Resposta de eixos embrionários de cupuaçu (Theobroma grandiflorum Schum.) à concentração de sais, doses de sacarose e renovação do meio de cultivo

The effects of concentration of salts and sucrose and culture medium renewal frequency on cupuassu (Theobroma grandiflorum Schum.) embryonic axes development were studied. Two salts concentrations of MS (100 and 50%), two sucrose levels (1.5 and 3%) and three explants subcultives (without medium renewal, renewal at 30 and 60 days) were tested, with NAA (0.5 mg/L) and BAP (0.3 mg/L) in the MS medium. The use of MS at full concentration, with addition of 3% of sucrose, and renewals of the medium at 30 day intervals promoted better cupuassu embryonic axes development.

Indução de calos embriogênicos em explantes de cupuaçuzeiro

It was studied the induction of embryogenics calli in cupuassu, in function of kind of explant and culture medium. Cotyledons segments and embryonic axes were tested and divided in three parts: region of plumule, radicule and hypocotile. The explants were cultivated in two different culture medium: 1) MS supplemented with 2,4-D (1 mg L-1) and Kinetin (0,25 mg L-1); 2) MS supplemented with NAA (5 mg L-1) and Kinetin (0,25 mg L-1). The hypocotile region demonstrated to be more responsive segment of the embryonic axe, forming callus with white and friable aspect. No somatic embryogenesis was evidenced in callus of cupuassu with auxines testeds in the medium.

DESENVOLVIMENTO DE CALOS EM EXPLANTES DE CUPUAÇUZEIRO (Theobroma grandiflorum Schum.) EM FUNÇÃO DA CONCENTRAÇÃO DE AUXINAS E DO MEIO LÍQUIDO

Objetivou-se estudar o efeito da concentracao de auxina e do meio liquido sobre o desenvolvimento de calos de cupuacuzeiro. Segmentos de eixos embrionarios e cotiledones, obtidos de frutos de cupuacu dos tipos Mamorana e Redondo, foram cultivados em 4 meios de cultura diferentes: 1) meio MS (50%), suplementado com 2,4-D (1; 2; 4; 8 mg/L); 2) sais N6 (SIGMA) (4 g/L), acrescidos de 2,4-D (0; 2; 4 mg/L) e ANA (0; 3; 5 mg/L); 3) igual ao anterior, suplementado apenas com ANA (3 mg/L); e 4) meio MS, acrescido com ANA (1 mM). Calos com aspecto branco e brilhante foram observados em segmentos de eixos embrionarios e cotiledones, cultivados nas menores concentracoes de meio 1 (1 e 2 mg/L), enquanto nas maiores concentracoes (4 e 8 mg/L) se observou a formacao de calos e massa calosa branco-opaca, em eixos embrionarios e em segmentos cotiledonares, estas estruturas tornaram-se escuras dentro de oito semanas. Usando o meio 2, um grande numero de raizes foram formadas, enquanto o mesmo meio suplementado apenas com ANA (3; 5 mg/L) originou uma massa calosa. A combinacao de ANA e 2,4-D, 3 e 2 mg/L, respectivamente, promoveu a formacao de calos brancos e raizes. A transferencia das culturas para meio liquido, sem regulador de crescimento, promoveu aumento de tamanho dos explantes e escurecimento dos mesmos. O cultivo desses explantes no meio 3 resultou no aparecimento de calos amarelos, com aspecto friavel, que permaneceram com a mesma aparencia no meio 4.

FluxTransgenics: a flexible LIMS-based tool for management of plant transformation experimental data

BackgroundThe production and commercial release of Genetically Modified Organisms (GMOs) are currently the focus of important discussions. In order to guarantee the quality and reliability of their trials, companies and institutions working on this subject must adopt new approaches on management, organization and recording of laboratory conditions where field studies are performed. Computational systems for management and storage of laboratory data known as Laboratory Information Management Systems (LIMS) are essential tools to achieve this.ResultsIn this work, we have used the SIGLa system – a workflow based LIMS as a framework to develop the FluxTransgenics system for a GMOs laboratory of Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Maize and Sorghum (Sete Lagoas, MG - Brazil). A workflow representing all stages of the transgenic maize plants generation has been developed and uploaded in FluxTransgenics. This workflow models the activities involved in maize and sorghum transformation using the Agrobacterium tumefaciens method. By uploading this workflow in the SIGLa system we have created Fluxtransgenics, a complete LIMS for managing plant transformation data.ConclusionsFluxTransgenics presents a solution for the management of the data produced by a laboratory of genetically modified plants that is efficient and supports different kinds of information. Its adoption will contribute to guarantee the quality of activities and products in the process of transgenic production and enforce the use of Good Laboratory Practices (GLP).The adoption of the transformation protocol associated to the use of FluxTransgenics has made it possible to increase productivity by at least 300%, increasing the efficiency of the experiments from between 0.5 and 1 percent to about 3%. This has been achieved by an increase in the number of experiments performed and a more accurate choice of parameters, all of which have been made possible because it became easier to identify which were the most promising next steps of the experiments. The FluxTransgenics system is available for use by other laboratories, and the workflows that have been developed can be adapted to other contexts.

Genetic Transformation of Immature Sorghum Inflorescence via Microprojectile Bombardment

Sorghum bicolor is one of the most important cereals in the world after rice, maize, wheat and barley. In 2010, more than 60 million tons were produced from approximately 50 million ha around the world. It is an important crop in the arid and semi-arid regions of the world and it is primarily used in Brazil as a supply for an increasing livestock market. However, the Brazilian sorghum productivity is low (1,500 to 2,500 kg/ha) and extremely variable along the years, typical of a culture sowed in marginal climate conditions and mainly without the use of high input technologies. Conventional breeding programs have already done a great deal of research to increase the sorghum productivity, though in some fields, the gains obtained by these programs are reaching stationary levels due to the lack of genetic variability (Nwanze et al., 1995). Alternatively, recombinant DNA technology and the generation of transgenic plants can increment conventional breeding programs through the amplification of the gene pool that can be used to improve sorghum environmental fitness and nutritional qualities.

Maize Transformation to Obtain Plants Tolerant to Viruses by RNAi Technology

Plants represent the principal source of human foods and livestock feeds and efforts to improve them in many agronomic aspects have focused on plant breeding. The biotechnology revolution in the past decade made possible for plant breeders access new sources of genetic variability for the development of superior cultivars. It has been possible to define additional strategies for crop improvement through the introduction and stable integration of heterologous genes in plant cells with the knowledge of the regulation of the important agronomic characteristics. The genetic manipulation of plants allows their adaptation to different environmental stresses, whether biotic or abiotic. Currently, the production of genetically modified plants occupies a prominent place in both, basic and applied plant research. Genetically modified crops are generated through a process known as genetic engineering, in which genes of interest are transferred to plants without the need of natural crossing. The most widely used methods for introducing transgenes into the genome of plants are Agrobacterium mediated transformation and microprojectile bombardment. In the first case, scientists took advantage of the natural ability of Agrobacterium to transfer some of its wild genes to plant cells causing the diseases known as crown gall or hairy roots, and replace them by other genes expressing traits of agronomic interest. However, Agrobacterium is not able to infect all plants in a very efficient way, as a consequence, new systems for direct transfer of genes to plants emerged. The microprojectile bombardment system is a direct transfer of genes that involves an equipment known as gene gun. The DNA to be introduced into plant cells is physically attached to metal microparticles that are then propelled against the plant cells, using the gene gun. DNA that penetrates the plant cell can be integrated into the plant genome. Maize is one of the most cultivated cereals in the world. The main maize producer’s countries are the United States, China, and Brazil, followed by Mexico, France, Argentina and India. Among the big losses faced by agriculture are the attacks of pests and diseases. For maize, these problems have worsened since 1990 because of the increase of the cultivated areas in both the normal growing season and the off season, mainly due to intensive cultivation of maize in the irrigated areas, and lack of adoption of crop rotation in certain fields. In recent years, diseases that were not a problem, increased in importance such as the viruses. Among the strains of the virus complexes, potyviruses cause significant losses in grain and forage of maize susceptible genotypes. Plants have different mechanisms for protection against invasion by pathogens, and different genes directly related to

Introdução de genes em segmentos foliares de cupuaçu (Theobroma grandiflorum schumm.) usando biobalística

The biolistic technique was used with the objective of adapting a transformation protocol to cupuassu plants. Foliar paths of cupuassu were bombed with a plasmid, containing the antocianine reporter gene with helium pressures of 650, 1000 and 1100 psi. The bombarded explants were transferred to half MS medium for 24 hours for incubation and red points were detected using a stereomicroscope Stemi SV11 Zeiss (Germany). The expression of the antocianine gene (red points) was observed with pressures of 650 and 1000 psi and necroses were found when 1100 psi were used. Genes C1 and R ‘ of the antocianina synthesis, under the control of the promoter 35S, can be used as reporters to monitor transformation events in cupuassu.