Development of transgenic imazapyr-tolerant cowpea (Vigna unguiculata).

KEY MESSAGE: Here we present the development of cowpea lines tolerant to a herbicide from imidazoline class (imazapyr). Plants presented tolerance to fourfold the commercial recommended dose for weed control. Cowpea is one of the most important and widely cultivated legumes in many parts of the world. Its cultivation is drastically affected by weeds, causing damages during growth and development of plants, competing for light, nutrients and water. Consequently, weed control is critical, especially using no-tillage farming systems. In tropical regions, no-till farming is much easier with the use of herbicides to control weeds. This study was conducted to evaluate the possibility of obtaining transgenic cowpea plants resistant to imidazolinone, which would facilitate weed control during the summer season. The biolistic process was used to insert a mutated acetohydroxyacid synthase coding gene (Atahas) which confers tolerance to imazapyr. The transgene integration was confirmed by Southern blot analysis. Out of ten lines tested for tolerance to 100 g ha(-1) imazapyr, eight presented some tolerance. One line (named 59) revealed high herbicide tolerance and developmental growth comparable to non-transgenic plants. This line was further tested for tolerance to higher herbicide concentrations and presented tolerance to 400 g ha(-1) imazapyr (fourfold the commercial recommended dose) with no visible symptoms. Line 59 will be the foundation for generating imidazolinone-tolerant cowpea varieties, which will facilitate cultivation of this crop in large areas.

Diversity among isolates of cowpea severe mosaic virus infecting cowpeas in northeastern Brazil.

Eleven isolates of cowpea severe mosaic virus (CPSMV), a member of the genus Comovirus, were selected from 50 samples collected of nine cowpea fields in Northeastern Brazil (Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe, Bahia, and Distrito Federal) and partially sequenced. The RNA1 partial sequence, corresponding to the helicase, viral genome-linked protein, picornain 3C-like protease, and the RNA-directed RNA polymerase genes from CPSMV, had high identity among isolates, varying from 98 to 100%. No evidence was found for intermolecular or intramolecular recombination. Phylogenetic analysis confirmed that the Brazilian CPSMV isolates are substantially different from the CPSMV strain USA. Despite the low variability found among Brazilian CPSMV isolates, there were notable differences in the symptomatology of infected cowpea plants, ranging from mild to moderate. Previous reports have demonstrated an association between CPSMV symptom determinants and helicase. However, we found no correlation between the helicase mutations and symptoms caused by CPSMV. Nevertheless, all isolates with mutation R to K in the protease provoked severe symptoms. This type of information can provide a foundation for the development of strategies to produce durable resistant cowpea lines. It is crucial for strategies based on DNA sequence-dependent technologies, such as inhibition with RNAi.

A minimal DNA cassette as a vector for genetic transformation of soybean (Glycine max).

Currently, the market demands products committed to protecting human health and the environment, known as clean products. We developed a protocol using DNA fragments containing only the gene sequence of interest, to replace the circular vectors containing genes for antibiotic resistance and other undesirable sequences, for obtaining transgenic soybeans for microparticle bombardment. Vector pAC321 was digested with the restriction enzyme PvuII to produce the 6159 bp ahas fragment, which contains the mutated ahas gene from Arabidopsis thaliana (Brassicaceae), under the control of its own promoter and terminator. This gene confers resistance against imazapyr, a herbicidal molecule of the imidazolinone class, capable of systemically translocating and concentrating in the apical meristematic region of the plant, the same region used for the introduction of the transgenes. This fragment was used to generate 10 putative transgenic soybean lines.

Isolation and characterization of the promoter sequence of a cassava gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in storage roots.

Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.

Suppression of SERK gene expression affects fungus tolerance and somatic embryogenesis in transgenic lettuce.

The Somatic embryogenesis receptor-like kinase (SERK) gene plays an important role in plant somatic and zygotic embryogenesis induction. The gene encodes an LRR-containing receptor-like kinase protein. Studies have been carried out focusing on different aspects of its function, but definitive conclusions on its role are far from being reached. SERK expression is generally detected in cells in which somatic or zygotic embryogenesis has been triggered. Transgenic lettuce lines were produced to silence the endogenous SERK gene using antisense RNA. The average number of seeds per flower in the R(1) and R(2) generations was similar for both transgenic and non-transgenic lines. However, a reduction in the number of viable grained seeds was observed in four studied transgenic lines. Endogenous SERK expression analysis revealed the absence of detectable LsSERK gene transcripts in three transgenic lines, which presented a reduction in their ability to form in vitro somatic embryonic structures. In addition, transgenic lines showed enhanced susceptibility to the pathogenic fungus Sclerotinia sclerotiorum, when compared to control plants. The results support the idea that SERK genes might not only be involved in plant growth and development, but probably also in a general mechanism of biotic and abiotic stress perception.

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil.

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.

Transgene elimination in genetically modified dry bean and soybean lines

Transgene elimination is a poorly studied phenomenon in plants. We made genetic and molecular studies of a transgenic dry bean line immune to bean golden mosaic geminivirus and a soybean line. In both lines, the transgenes were stable during the vegetative phase but were eliminated during meiosis. Due to its potential biotechnological value, this transgenic line was micropropagated by grafting and the vegetative copies were studied for more than two years. More than 300 plants of progeny were obtained during this period, demonstrating that the phenomenon of elimination was consistently repeated and offering an opportunity for detailed study of transgene elimination, including the characterization of the integration sites. Cloning and sequencing of the transgenic loci, reciprocal crosses to untransformed plants, genomic DNA blots, and GUS assays were performed in the transgenic lines. Based on the molecular and genetic characterization, possible mechanisms involved in transgene elimination include intrachromosomal recombination, genetic instability resulting from the tissue culture manipulations, and co-elimination of transgenes, triggered by a process of genome defense.

Effectiveness of liposomes to transfect livestock fibroblasts

The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation

Inheritance of a recessive transgene-associated character controlling albinism in transgenic bean (Phaseolus vulgaris L.).

We identified a transgenic line exhibiting albinism during our work to introduce genes through genetic engineering in dry bean (Phaseolus vulgaris). The transgenic mother plant (R0) presented a normal phenotype and generated albino and normal green plants in the first generation (R1). The segregation ratio of the albino character in the R1 and R2 generations fitted the expected ratio for a character controlled by a single recessive gene linked to a foreign gus gene, suggesting that albinism could be a consequence of insertional mutation caused by introduction of the exogenous gene. Analysis by electron microscope revealed that the albino cells possessed no chloroplasts and a greater number of mitochondria when compared to normal green plants. This transgenic bean line may be used in understanding the genetic control of chloroplast genesis, for acquiring additional knowledge of genomic structure or in physiological studies. This is the first described transgene-associated mutant bean plant.

Expression of human bone morphogenetic protein (BMP-2 and BMP-4) genes in transgenic bovine fibroblasts

cDNAs dos genes bone morphogenetic protein-2 (BMP-2) e bone morphogenetic protein-4 (BMP-4) foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma) e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV). Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

Biolistic co-transformation of Metarhizium anisopliae var. acridum strain CG423 with green fluorescent protein and resistance to glufosinate ammonium.

Metarhizium anisopliae var. acridum (syn. M. flavoviride) is recognized as a highly specific and virulent mycopathogen of locusts and grasshoppers and is currently being developed as a biological control agent for this group of insects in Brazil. Intact conidia of M. anisopliae var. acridum strain CG423 were transformed using microparticle bombardment. Plasmids used were: (1) pBARKS1 carrying the bar gene of Streptomyces hygroscopicus fused to the Aspergillus nidulans trpC promoter, encoding resistance to glufosinate ammonium (or phosphinothricin) and modified by addition of the telomeric repeat (TTAGGG)(18) of Fusarium oxysporum and 2.pEGFP/gpd/tel carrying a red-shifted variant gene for Aequorea victoria green fluorescent protein (EGFP) which we have fused to the A. nidulans gpd promoter and trpC terminator. Highly fluorescent co-transformants were selected on solid minimal medium containing 100 microg ml(-1) glufosinate ammonium using an inverted microscope with 450-490 nm excitation/510 nm emission filter set. Southern blot analysis of co-transformants revealed varying multiple chromosomal integrations of both bar and egfp genes at both telomeric and non-telomeric loci. Transformants retained pathogenicity in bioassays against Rhammatocerus schistocercoides and showed unaltered lack of pathogenicity against larvae of the non-target insect Anticarsia gemmatalis. One co-transformant from four tested, however, showed a significant, but non-dose-dependent, elevation in virulence against Tenebrio molitor.

[An evaluation of municipal actions in the control of dengue vectors in the region of São José do Rio Prêto, São Paulo, 1989 to 1995]. / Avaliação de ações municipais de combate a vetores da dengue na região de São José do Rio Prêto, São Paulo, 1989 a 1995.

The aim of this study was to determine the coverage of municipal activities in terms of the control of Aedes aegypti and/or Aedes albopictus by routine house-to-house visits and by emergency activity, carried out between 1989 and 1995 in the area of São José do Rio Prêto, São Paulo State, and to evaluate the cross-correlation between them and the Breteau index (BI). For towns with up to 50,000 real estate properties, the joint coverage by routine and emergency activities was mostly appropriate and the routine activities showed a negative cross-correlation with the BI. For the county seat (more than 50,000 real estate properties), the coverage provided by the above activities was not correlated with the BI. In general, the coverage was inversely proportional to town size. Emergency activities did not show a correlation with the BI in any town size range, proving to be ineffective.

High frequency gene transfer by microprojectile bombardment of intact conidia from the entomopathogenic fungus Paecilomyces fumosoroseus.

Two different methods, (i) PEG and (ii) biolistic, were employed to transform protoplasts and conidia of Paecilomyces fumosoroseus using hygromycin resistance as selectable marker. Transformation frequencies varied from 1.9 to 2.5 transformants microgram-1 of DNA by the PEG method, and from 33 to 153 transformants microgram-1 of DNA by the biolistic procedure.

Transient expression of GUS and the 2S albumin gene from Brazil nut in peanut (Arachis hypogaea L.) seed explants using particle bombardment.

The effect of parameters involved in the transformation efficiency of peanut (Arachis hypogaea L.) seed tissues by direct gene transfer using a helium inflow particle bombardment device was evaluated. Transient gene expression was affected by both particle and DNA amounts, and was positively correlated with gene copy number, as determined byß-glucuronidase (GUS) activity assays. No influence of plasmid size on GUS gene expression was observed. Transcriptional control of GUS by either the CaMV 35S or the 2S promoter from Brazil nut 2S albumin gene varied with the developmental stage of the seed and was approximately tenfold greater under the influence of the 35S promoter than under the 2S promoter. The gene products of both the Brazil nut methionine-rich 2S albumin and GUS genes under the transcriptional control of the 35S promoter were detected by ELISA assays.

Biolistic-mediated gene expression in guinea pigs and cattle tissue in vivo.

Foreign genes were introduced and expressed in vivo in guinea pigs and cattle utilizing a new hand-held device based on high-pressure helium gas to accelerate DNA-coated microparticles. Guinea pigs were used to evaluate the physical parameters to introduce and express the exogenous DNA. The best conditions were applied to conduct bombardments in cattle. The results showed a high frequency of gene expression in all the bombarded cattle. This procedure could be used to study the immune responses in cattle and in a wide variety of animals through genetic immunization.

Biolistic-mediated gene expression in guinea pigs and cattle tissues in vivo

Foreign genes were introduced and expressed in vivo in guinea pigs and cattle utilizing a new hand-held device based on high-pressure helium gas to accelerate DNA-coated microparticles. Guinea pigs were used to evaluate the physical parameters to introduce and express the exogenous DNA. The best conditions were applied to conduct bombardments in cattle. The results showed a high frequency of gene expression in all the bombarded cattle. This procedure could be used to study the immune responses in cattle and in a wide variety of animals through genetic immunization.

High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae.

Three different methods, (i) PEG, (ii) electroporation and (iii) biolistic, were employed to transform Metarhizium anisopliae using benomyl resistance as a selectable marker. Transformation frequencies and mitotic stability varied for each method, from 0.8 to 6.9 transformants micrograms-1 of DNA and 46%, respectively, by the PEG method; 1.3 to 1.8 transformants micrograms-1 of DNA and 67% by electroporation; and 32 to 201 transformants micrograms-1 of DNA and 90% by biolistic. We demonstrate by PCR that 60% of the transformants were generated by gene conversion.

Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment.

Exploiting the biolistic process we have generated stable transgenic bean (Phaseolus vulgaris L.) plants with unlinked and linked foreign genes. Co-transformation was conducted using plasmid constructions containing a fusion of the gus and neo genes, which were co-introduced with the methionine-rich 2S albumin gene isolated from the Brazil nut and the antisense sequence of AC1, AC2, AC3 and BC1 genes from the bean golden mosaic geminivirus. The results revealed a co-transformation frequency ranging from 40% to 50% when using unlinked genes and 100% for linked genes. The introduced foreign genes were inherited in a Mendelian fashion in most of the transgenic bean lines. PCR and Southern blot hybridization confirmed the integration of the foreign genes in the plant genome.