Elibio L. Rech

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.

Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean [Glycine max (L.) Merril] plants at a high frequency

Abstract Imazapyr is a herbicidal molecule that concentrates in the apical meristematic region of the plant. Its mechanism of action is the inhibition of the enzymatic activity of acetohydroxyacid synthase, which catalyses the initial step in the biosynthesis of isoleucine, leucine and valine. The selectable marker gene, ahas, was previously isolated from Arabidopsis thaliana and contains a mutation at position 653 bp. Combining the use of imazapyr, the ahas gene and a multiple shooting induction protocol has allowed us to develop a novel system to select transgenic meristematic cells after the physical introduction of foreign genes. In this study, we describe a protocol to obtain a high frequency of fertile transgenic soybean plants that is variety-independent.

Rnai-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content

Inositol plays a role in membrane trafficking and signaling in addition to regulating cellular metabolism and controlling growth. In plants, the myo-inositol-1-phosphate is synthesized from glucose 6-phosphate in a reaction catalyzed by the enzyme myo-inositol-1-phosphate synthase (EC 5.5.1.4). Inositol can be converted into phytic acid (phytate), the most abundant form of phosphate in seeds. The path to phytate has been suggested to proceed via the sequential phosphorylation of inositol phosphates, and/or in part via phosphatidylinositol phosphate. Soybean [Glycine max (L.) Merrill] lines were produced using interfering RNA (RNAi) construct in order to silence the myo-inositol-1-phosphate (GmMIPS1) gene. We have observed an absence of seed development in lines in which the presence of GmMIPS1 transcripts was not detected. In addition, a drastic reduction of phytate (InsP6) content was achieved in transgenic lines (up to 94.5%). Our results demonstrated an important correlation between GmMIPS1 gene expression and seed development.

A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.

High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7–10 months.

Electro-enhancement of division of plant protoplast-derived cells

SummaryElectric field pulses, ranging from 250 to 2000 V and of 10 to 50 μsec duration, were assessed for their effect on the growth in culture of isolated protoplasts ofGlycine canescens, Prunus avium × pseudocerasus, Pyrus communis, Solanum dulcamara andSolanum viarum. Three successive voltage pulses between 250 and 1000 V caused a small decrease in protoplast viability, but promoted cell division and enhanced significantly the plating efficiency. A higher percentage of electro-pulsed protoplasts showed sustained growth in culture to the microcallus stage compared to untreated protoplasts. The rate of cell division was also stimulated in electro-treated protoplasts. These observations are discussed in relation to present knowledge of the effects of electrical treatments on plant and animal cells.

Transgenic beans (Phaseolus vulgaris L.) engineered to express viral antisense RNAs show delayed and attenuated symptoms to bean golden mosaic geminivirus

The genes Rep-TrAP-REn and BC1 from the Brazilian isolate bean golden mosaic geminivirus (BGMV-BR) were cloned in antisense orientation under the transcriptional control of the CaMV 35S promoter. This construct was used to transform common bean (Phaseolus vulgaris L.) using the biolistic method. Transgenic plants from the R3 and R4 generations were challenged by inoculation with a BGMV-BR viruliferous whitefly population. Of the four transgenic lines tested, two had both delayed and attenuated viral symptoms. Un-transformed plants or plants transformed with a construct containing only the gus-neo gene developed typical BGMV-BR symptoms 10–15 days after inoculation.

Recombinant DNA production of spider silk proteins

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein‐based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large‐scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.

Particle bombardment-mediated transient expression of a Brazil nut methionine-rich albumin in bean (Phaseolus vulgaris L.)

Bean (Phaseolus vulgaris L.) mature embryos were transformed using biolistic methods with a plasmid containing 2S albumin and β-glucuronidase structural sequences, both under the control of the 35S CaMV promoter. We have shown that chimaeric tissues could be obtained and that both structural sequences were expressed to similar levels.

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

Cellular immune responses are a critical part of the hosts defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer.