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Improving nitrogen use efficiency (NUE) is one of the main ways of increasing plant productivity through genetic engineering. The modification of nitrogen (N) metabolism can affect the hormonal content, but in transgenic plants, this aspect has not been sufficiently studied. Transgenic birch (Betula pubescens) plants with the pine glutamine synthetase gene GS1 were evaluated for hormone levels during rooting in vitro and budburst under outdoor conditions. In the shoots of the transgenic lines, the content of indoleacetic acid (IAA) was 1.5–3 times higher than in the wild type. The addition of phosphinothricin (PPT), a glutamine synthetase (GS) inhibitor, to the medium reduced the IAA content in transgenic plants, but it did not change in the control. In the roots of birch plants, PPT had the opposite effect. PPT decreased the content of free amino acids in the leaves of nontransgenic birch, but their content increased in GS-overexpressing plants. A three-year pot experiment with different N availability showed that the productivity of the transgenic birch line was significantly higher than in the control under N deficiency, but not excess, conditions. Nitrogen availability did not affect budburst in the spring of the fourth year; however, bud breaking in transgenic plants was delayed compared to the control. The IAA and abscisic acid (ABA) contents in the buds of birch plants at dormancy and budburst depended both on N availability and the transgenic status. These results enable a better understanding of the interaction between phytohormones and nutrients in woody plants. [ABSTRACT FROM AUTHOR]

The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin—a well-known nanomolar inhibitor of glutamine synthetase—after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen. [ABSTRACT FROM AUTHOR]

ABSTRACT: With the possibility of using ammonium glufosinate in corn, studies on its association with other herbicides are essential. The aim of this study was to assess weed control and herbicide selectivity in association with ammonium glufosinate in corn containing the pat and cp4-epsps genes. The experiment was carried out under field conditions in two sites in a randomized block design with four replications. Treatments consisted of ammonium glufosinate, glyphosate, ammonium glufosinate + glyphosate, ammonium glufosinate + nicosulfuron, ammonium glufosinate + atrazine, ammonium glufosinate + tembotrione, ammonium glufosinate + mesotrione, ammonium glufosinate + carfentrazone ethyl, ammonium glufosinate + bentazon, ammonium glufosinate + 2,4-D, control without weeding, and hand-weeded control. Visual assessments were performed regarding damage, electron transport rate in photosystem II (ETR), ammonia quantification, height, and grain yield of corn plants, as well as weed control at both sites. The application of ammonium glufosinate and its associations did not provide a reduction in productivity when compared to the hand-weeded control. Some treatments presented higher levels of visual damage and ammonia accumulation, but without a yield reduction. Therefore, herbicide mixture with ammonium glufosinate may be an alternative to weed control without damaging corn crop, which presents in its genetic constitution the cp4-epsps and pat genes.

Sugarcane being the major contributor of sugar and potential source of biofuel around the globe, occupies significant commercial importance. Red rot is the most devastating disease of sugarcane, severely affecting its quality as well as yield. Here we report the overexpression of SUGARWIN1 and SUGARWIN2 genes in any field crop for the first time. For this purpose, SUGAWIN1 and SUGARWIN2 were cloned downstream of maize ubiquitin (Ubi-1) promoter to construct two independent expression cassettes. The bar gene conferring resistance against phosphinothricin was used as selectable marker. Embryogenic calli of sugarcane were bombarded with both expression cassettes and selected on regeneration medium supplemented with phosphinothricin. The phosphinothricin-resistant shoots were rooted and then, analyzed using molecular tools at the genomic as well as transcriptomic levels. The transcriptomic analysis, using real time qPCR, showed that expression of SUGARWIN1 (SWO) and SUGARWIN2 (SWT) was higher in transgenic plants as compared to untransformed plants. Our results further demonstrated that over expression of these genes under maize ubiquitin (Ubi-1) promoter causes significant restriction in proliferation of red rot causal agent, Colletotrichum falcatum in sugarcane transgenic plants, under in vitro conditions. This report may open up exciting possibilities to extend this technology to other monocots for the development of crops with better ability to withstand fungal pathogens.

Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetase (GS) activity in plants, and its phytotoxicity is ascribed to ammonium accumulation and reactive oxygen species bursts, which drives rapid lipid peroxidation of cell membranes. In agricultural fields, PPT is extensively sprayed on plant foliage; however, a portion of the herbicide reaches the soil. According to the present study, PPT absorbed via roots can be phytotoxic to Arabidopsis, inducing more adverse effects in roots than in shoots. Alterations in plant physiology caused by 10 days exposure to herbicide via roots are reflected through growth suppression, reduced chlorophyll content, perturbations in the sugar and organic acid metabolism, modifications in the activities and abundances of GS, catalase, peroxidase, and superoxide dismutase. Antagonistic interaction of Nepeta rtanjensis essential oil (NrEO) and PPT, emphasizes the existence of complex control mechanisms at the transcriptional and posttranslational level, which result in the mitigation of PPT-induced ammonium toxicity and in providing more efficient antioxidant defense of plants. Simultaneous application of the two agents in the field cannot be recommended; however, NrEO might be considered as the PPT post-treatment for reducing harmful effects of herbicide residues in the soil on non-target plants.

l-Phosphinothricin (glufosinate or 2-amino-4-((hydroxy(methyl) phosphinyl) butyric acid ammonium salt (AHPB)), which is a structural analog of glutamate, is a recognized herbicide that acts on weeds through inhibition of glutamine synthetase. Due to the structural similarity between phosphinothricin and some bisphosphonates (BPs), this study focuses on investigating the possibility of repurposing phosphinothricin as a bisphosphonate analogue, particularly in two medicine-related activities: image probing and as an anti-cancer drug. As BP is a competitive inhibitor of human farnesyl pyrophosphate synthase (HFPPS), in silico molecular docking and dynamic simulations studies were established to evaluate the binding and stability of phosphinothricin with HFPPS, while the results showed good binding and stability in the active site of the enzyme in relation to alendronate. For the purpose of inspecting bone-tissue accumulation of phosphinothricin, a technetium (99mTc)–phosphinothricin complex was developed and its stability and tissue distribution were scrutinized. The radioactive complex showed rapid, high and sustained uptake into bone tissues. Finally, the cytotoxic activity of phosphinothricin was tested against breast and lung cancer cells, with the results indicating cytotoxic activity in relation to alendronate. All the above results provide support for the use of phosphinothricin as a potential anti-cancer drug and of its technetium complex as an imaging probe. [ABSTRACT FROM AUTHOR]

O objetivo deste trabalho foi avaliar a sensibilidade de plantas transgênicas de citrumelo Swingle com elevada produção de prolina, ao herbicida glufosinato de amônio. As plantas utilizadas apresentavam a inserção do gene mutante da enzima delta1-pirrolina-5-carboxilato sintetase (P5CS), responsável pela biossíntese de prolina. A expressão do gene p5cs em plantas transgênicas causou aumento nas quantidades de prolina em tecidos foliares, em até cinco vezes, quando comparadas às plantas-controle tratadas com 200 µM de glufosinato de amônio. As plantas transgênicas acumularam maior quantidade de NH4+ nas folhas, em relação às plantas não-transgênicas. Os danos causados pelo herbicida foram avaliados in vitro, utilizando-se discos foliares cultivados em meio MS com diferentes concentrações de glufosinato de amônio. Observou-se maior clorose em discos foliares das plantas transgênicas, o que comprova a maior suscetibilidade de plantas de citrumelo Swingle com alta produção de prolina ao herbicida.The objective of this work was to evaluate the susceptibility to glufosinate ammonium of transgenic plants of Swingle citrumelo with high proline production. The mutant gene of the enzyme delta1-pyrroline-5-carboxylate synthetase (P5CS), the rate-limiting enzyme in proline biosynthesis, was inserted into Swingle citrumelo plants. The expression of the gene p5cs caused up to 5-fold increase on the proline content in leaf tissues of transgenic plants treated with 200 µM glufosinate ammonium, when compared with control plants. Leaves of transgenic plants accumulated higher amounts of NH4+ than the nontransgenic control. The herbicide toxicity was evaluated using leaf disks cultivated in MS medium, containing different concentrations of glufosinate ammonium. The severity of the chlorosis, observed in leaf disks of transgenic plants, confirmed the higher susceptibility of Swingle citrumelo plants, with high proline production, to this herbicide.

Objectives: To develop a host-vector system for heterologous protein co-expression and purification in the Trichoderma reesei (teleomorph Hypocrea jecorina) industrial strain RUT-C30. Results: The co-expression and purification system is based on: (i) an efficient and reliable selectable marker using bar (phosphinothricin acetyltransferase gene); (ii) a compact hygromycin B resistance marker; and (iii) a versatile integration plasmid for gene expression and knockout. Fluorescent protein genes were introduced into T. reesei and the corresponding proteins were purified from fermentation broth. The host-vector system was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. Conclusions: This protocol can be used at an industrial scale to produce large amounts of proteins in T. reesei. [ABSTRACT FROM AUTHOR]

The establishment of a genetic transformation program requires a selection in vitro system of transformed tissues. In the Glycine genus exists few references that employ the Glufosinate-ammoniun that selective agent in transformed somatic embryos. For this reason this work had as objective to determine the minimum lethalconcentration of the herbicide Glufosinate-ammoniun in somatic embryos of Cuban soybean cultivar INCASoy-27, to use as selective agent in genetic transformation programs. The soybean somatic embryos were placed on multiplication medium that contained 2,4-dichlorophenoxyacetic acid 20 mg L-1 with different concentrations of Glufosinate-ammonium (1,0; 2,0; 3,0; 4,0; 5,0; 6,0 mg L-1). All concentrations of selective agent caused necrosis in not transformed soybean somatic embryos. The increments of selective agent concentration and culture time increased the necrosis of somatic embryos. Finally was determined the minimum lethal concentration of 6,0 mg L-1 Glufosinate-ammoniun in semisolid medium, that selective agent in the genetic transformation of somatic embryos of soybean cultivar INCASoy-27. [ABSTRACT FROM AUTHOR]

We have developed marker-free transgenic wheat using a transcription factor, AtDREB1A cloned from Arabidopsis. Southern hybridization confirmed a transgenic event with a single copy insertion. PCR analysis of the T plants showed four were positive only for AtDREB1A. A T1 plant (HRCB3#17-37) was marker-free and had good expression of drought tolerance in comparison with untransformed plants. The leaf relative water content of this T transgenic plant was 12-15 % higher than that of the wild type during stress with an 8 % higher yield under water deficit conditions compared with wild type plants. [ABSTRACT FROM AUTHOR]

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤50 μM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoenzymes is proposed. The mechanism is in concurrence with all experimental and literature data. [ABSTRACT FROM AUTHOR]

l-Methionine sulfoximine (MSO) and dl-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators. [ABSTRACT FROM AUTHOR]

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II ( nptII) and phosphinothricin resistance genes ( bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, ) medium supplemented with 1 mg L−1 benzyladenine (BA), 20 mg L−1 l-glutamine and 2 mg L−1 phosphinothricin (PPT) or 100 mg L−1 kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L−1) containing MS media with 0.5 mg L−1 gibberellic acid (GA3) and 0.6 mg L−1 indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T0 and the progenies. T1 segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants. [ABSTRACT FROM AUTHOR]

The article presents a s study which identifies a novel bacterial Phosphinothricin (PPT) N-acetyltransferase, methione sulfone N-acetylransferase (MAT) through the isolation of the PPT-gene from a soil bacterium Nocardia sp. It was found that MAT has the ability to detoxify the herbicide PPT. In addition, MAT's chloroplast expression can lead to a higher herbicide resistance than expression in the cytosol. It also shows that MAT can be used a selectable marker in plants' transformation.

Genetic modification of nitrogen metabolism via bacterial NADPH- dependent glutamate dehydrogenase (GDH; E.C.4.1.2.1) favorably alters growth and metabolism of C3 plants. The aim of this study was to examine the effect of expression of GDH in the cytoplasmic compartment of Zea mays cells. The gdhA gene from Escherichia coli , that encoded a NADPH-GDH, was ligated to the ubiquitin promoter that incorporated the first intron enhancer and used to transform Z. mays cv. ‘H99’ embryo cultures by biolistics. R0–R3 generations included selfed inbreds, back-crossed inbreds, and hybrids with B73 derivatives. The lines with the highest GDH specific activity produced infertile R0 plants. The highest specific activity of GDH from the fertile Z. mays plants was sufficient to alter phenotypes. Plant damage caused by the phosphinothricin in gluphosinate-type herbicides, glutamine synthetase (GS; EC 6.1.3.2) inhibitors, was less pronounced in Z. mays plants with gdhA pat than in gusA pat plants. Germination and grain biomass production were increased in gdhA transgenic plants in the field during seasons with significant water deficits but not over all locations. Water deficit tolerance under controlled conditions was increased. Crops modified with gdhA may have value in semi-arid locations. [ABSTRACT FROM AUTHOR]

This work describes the development of a fast and efficient methodology for Agrobacterium tumefaciens-mediated genetic transformation of internodal stem segments from potato (Solanum tuberosum L.), cultivar Désirée, using phosphinothricin (PPT, glufosinate ammonium) as a selection agent. This methodology has a transformation efficiency of 68%, inducing shoots after 4 to 5 weeks in the presence of 2 mg/L of phosphinothricin. The expression of the bar gene in leaf segments from resistant, transformed plants was demonstrated by the chlorophenol red assay, as was its presence in the genome of explants by Polymerase Chain Reaction. An additional observation was the presence of high levels of resistance to phosphinothricin (up to 500 mg/L) in the transgenic clones, with an otherwise normal phenotype. These transgenic plants were transplanted to greenhouses and after aspersion with the herbicide Finale® (Bayer CropScience) at 2.5 L/ha remained healthy and green, in contrast with the non-transgenic controls which died after 7 days. [ABSTRACT FROM AUTHOR]

A fast and efficient methodology for transforming internodal segments in stems of potato (Solanum tuberosum L.) Désirée cultivar, with Agrobacterium tumefaciens and by using phosphinothricin (PPT, gluphosinate ammonium) as selection agent, was developed. Shoots obtained with 68% transformation efficiency were induced 4 to 5 weeks later in the presence of phosphinothricin at 2 mg/L. The chlorophenol red assay was successfully employed in segments of leaves from transformed plants, demonstrating the expression of the bar gene, and its presence was confirmed in the genome of explants by polymerase chain reaction. Moreover, increased resistance of transgenic clones at phosphinothricin concentrations up to 500 mg/L was observed, also showing a normal phenotypic development. These transgenic plants were transplanted into greenhouses where they also showed to be resistant to the Finale® herbicide (Bayer CropScience) at a concentration 2.5 L/ha, growing green and vigorously as did not the non-transgenic control plants that died 7 days after treatment. [ABSTRACT FROM AUTHOR]

Methionine sulfoximine, like phosphinothricin (PPT), the active agent in the herbicide BASTA®, is a glutamate analogue that inhibits growth of wildtype Arabidopsis plants through its action on glutamine synthetase. The bar gene, which confers resistance to PPT, also confers resistance to methionine sulfoximine. In this study we show that methionine sulfoximine is an effective and economical alternative to PPT as a selective agent in agar medium. [Copyright &y& Elsevier]

The toxicity of three antibiotics commonly used to eliminate Agrobacterium tumefaciens from plant tissue during transformation were tested to determine their effect on Pinus pinea L. morphogenesis. Cefotaxime and vancomycin at a dose of 250 µg ml−1, as well as ticarcillin at 300 µg ml−1, were essentially nontoxic to the culture and significantly enhanced regeneration and shoot development. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. On the contrary, phosphinothricin and its ammonium salt glufosinate in the commercial formulation Finale® proved to be the best selection agents because they were less toxic to stone pine cotyledons. Thus, they could be applied immediately after transformation to avoid escapes. Other schemes for selection and recovery of transgenic stone pine trees are discussed. [ABSTRACT FROM AUTHOR]

In tomato ( Lycopersicon esculentum Mill.) leaves, the predominant glutamine synthetase (GS; EC 6.3.1.2) is chloroplastic (GS2; 45 kDa) whereas the cytosolic isoform (GS1; 39 kDa) is represented as a minor enzyme. Following either infection by Pseudomonas syringae pv. tomato (Pst) or treatment with phosphinothricin (PPT), a GS inhibitor, GS1 accumulated in the leaves. In contrast to healthy control leaves, where GS1 was restricted to the veins, in infected and PPT-treated leaves the GS1 polypeptide was also detected in the leaf blade; moreover, it was more abundant than GS2. Different immunological approaches were therefore used to investigate whether or not the GS1 polypeptide expressed in Pst-infected and PPT-treated tomato leaves was distributed among different tissues and subcellular compartments in the same way as the constitutive GS1 expressed in healthy leaves. By tissue-printing analysis, a similar GS immunostaining was observed in epidermis, mesophyll and phloem of leaflet midrib cross-sections of control, infected and PPT-treated leaves. Immunocytochemical localization revealed that GS protein was present in the chloroplast of mesophyll cells and the cytoplasm of phloem cells in healthy leaves; however, in Pst-infected or PPT-treated leaves, a strong labelling was observed in the cytoplasm of mesophyll cells. Two-dimensional analysis of GS polypeptides showed that, in addition to the constitutive GS1, a GS1 polypeptide different in charge was present in tomato leaflets after microbial infection or herbicide treatment. All these results indicate that a novel cytosolic GS is induced in mesophyll cells of Pst-infected or PPT-treated leaves. A possible role for this new cytosolic GS in the remobilization of leaf nitrogen during infection is proposed. [ABSTRACT FROM AUTHOR]

The co-action of light and the N-source in the regulation of the expression of the single-copy gene encoding plastidic glutamine synthetase (GS-2) and of the multigene family encoding cytosolic glutamine synthetase (GS-1) was investigated in the cotyledons of tomato ( Lycopersicon esculentum L.). Light, acting at red/far red or at blue regions of the spectrum increased the abundance of the GS-2 gene product and induced a modification of GS-2 subunits, resulting in the appearance of two GS-2 proteins exhibiting different molecular weights. The magnitude of the light stimulation of GS-2 gene expression was independent of the nitrogen source. However, following red- or far-red-light treatment of etiolated tomato cotyledons, two GS-2 proteins were found when nitrate was the N-source, while only one GS-2 protein was present with ammonium as the sole nitrogen source. Thus, light of specific wavelengths and N-substrates seem to act in concert to regulate GS-2 subunit composition. Tomato GS-1 gene expression was unaffected by light. Ammonium provided externally increased the level of the tomato GS-1 protein. Irrespective of the N-source or the light quality, the GS-1 subunits were represented by polypeptides of similar molecular weight in tomato cotyledons. However, phosphinothricin-induced inhibition of GS activity resulted in the appearance of at least one additional GS-1 polypeptide in etiolated or in green tomato cotyledons. In addition, impairment of GS activity in green tomato cotyledons by phosphinothricin was correlated with an increased level of the GS-1 transcript. Taken together, our data suggest a metabolic control of GS-1 gene expression in green tomato cotyledons. [ABSTRACT FROM AUTHOR]

In a study to optimize the use of the bar gene as a selectable marker gene in plant transformation, the selection mechanism of phosphinothricin in vitro was studied. It was found that tissues with a high anabolic activity were more sensitive to the toxicity of ammonium than tissues with a low anabolic activity. However, tissues with a low anabolic and high catabolic activity were more sensitive to glutamine deprivation. The metabolic (anabolic and catabolic) activity of callusing hypocotyl explants of Brassica napus L. was influenced by the carbon source of the medium and the light condition. The expression of the bar gene regulated by the light-inducible promoter derived from the gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase was also influenced by both light condition and carbon source. Finally, a bioassay to screen for, and to quantify the resistance of expiants to, phosphinothricin is presented. [ABSTRACT FROM AUTHOR]

Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks with Agrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration. [ABSTRACT FROM AUTHOR]

Callus was initiated from in vitro-grown plants of Gladiolus cultivars ‘Jenny Lee’ and ‘Florida Flame.’ The age of callus used for regeneration of plants was either 9 mo. old or 8 yr old from ‘Jenny Lee,’ and 4 mo. old from ‘Florida Flame.’ Regeneration medium consisted of Murashige and Skoog’s basal salts medium supplemented with 2.0 mg/l (9.3 µ M) kinetin. This medium was supplemented with various concentrations of either bialaphos (Meiji Seika, Tokyo, Japan) or phosphinothricin (Hoechst-Roussell, Frankfurt, Germany). Bialaphos was more effective than phosphinothricin at stimulating plant regeneration. Plants regenerated from 8-yr-old callus of ‘Jenny Lee’ only when the regeneration medium was supplemented with 0.10 mg/l bialaphos. A bialaphos concentration of 0.01 mg/l stimulated regeneration from 9-mo.-old callus of cultivar ‘Jenny Lee’ and 4-mo.-old callus of ‘Florida Flame.’ [ABSTRACT FROM AUTHOR]

Kinetics for the inhibition of glutamine synthetase (EC 6.3.1.2) in situ by the herbicidal glutamate analogue, phosphinothricin, have been generated, and produce an inhibitor dissociation constant (K) of 6.5 μM. This has been achieved through the development of a rapid technique for the isolation of mesophyll cells from the cladophylls of young asparagus ( Asparagus sprengeri) plants to provide starting material for the direct measurement of enzyme activities in situ. A modification of the technique developed by Rhodes and Stewart (Planta 118, 133-144 (1974) for the direct determination of enzyme activities in higher-plant tissues has been applied to these asparagus cells. Treatment of the cells by a single freezing in liquid nitrogen for a very short period (10 s), followed by thawing, alters the permeability of cell and organelle membranes allowing enzymes to become accessible to many small molecules, and yet remain concentrated and active within the cell. The activities of enzymes known to be located specifically in the organelles as well as the cytoplasm can be measured in asparagus cells treated in this way. Comparisons have been made between the activity and inhibition of glutamine synthetase in situ, and the enzyme isolated and partially purified from asparagus cells by fast protein liquid chromatography. Similarities in K and K values obtained between these two emphasize the efficacy of the freeze-thaw technique. There is only a single glutamine-synthetase isoenzyme in asparagus mesophyll cells, which copurifies with the one normally associated with the chloroplast (GS2). [ABSTRACT FROM AUTHOR]

Sugarcane being the major contributor of sugar and potential source of biofuel around the globe, occupies significant commercial importance. Red rot is the most devastating disease of sugarcane, severely affecting its quality as well as yield. Here we report the overexpression of SUGARWIN1 and SUGARWIN2 genes in any field crop for the first time. For this purpose, SUGAWIN1 and SUGARWIN2 were cloned downstream of maize ubiquitin (Ubi-1) promoter to construct two independent expression cassettes. The bar gene conferring resistance against phosphinothricin was used as selectable marker. Embryogenic calli of sugarcane were bombarded with both expression cassettes and selected on regeneration medium supplemented with phosphinothricin. The phosphinothricin-resistant shoots were rooted and then, analyzed using molecular tools at the genomic as well as transcriptomic levels. The transcriptomic analysis, using real time qPCR, showed that expression of SUGARWIN1 (SWO) and SUGARWIN2 (SWT) was higher in transgenic plants as compared to untransformed plants. Our results further demonstrated that over expression of these genes under maize ubiquitin (Ubi-1) promoter causes significant restriction in proliferation of red rot causal agent, Colletotrichum falcatum in sugarcane transgenic plants, under in vitro conditions. This report may open up exciting possibilities to extend this technology to other monocots for the development of crops with better ability to withstand fungal pathogens. [ABSTRACT FROM AUTHOR]

A rapid, efficient assay that is nondestructive and semiquantitative for identifying transgenic plants and progeny from Biolistic and protoplast transformations is described. Leaf sections of maize and wheat plants are placed on an indicator medium containing chlorophenol red and the selection agent. Changes in the color of the medium from red to yellow resulting from altered pH indicate transformed plants within 2-5 d. The method is particularly suited to use with phosphinothricin and could be used with other suitable selectable markers. [ABSTRACT FROM AUTHOR]

In the work, metagenomic sequencing was conducted to investigate the microbial gene catalogue in two samples of phosphinothricin (PPT)-utilized soils from South China. The gene sets contained an overwhelming majority of prevalent microbial genes, and were largely shared between these two samples. Several genus with high abundance were shared, such as norank_d__Bacteria, Nitrososphaera, Candidatus_Nitrosotalea, Candidatus_Nitrosocosmicus, and Rhodanobacter. Bacitracin resistance genes (61.4%) were the most dominant antibiotic resistance genes in two samples, followed by multidrug resistance efflux pump (12.5%). A lot of common virulence factors with high abundance were found in two samples, such as Alginate, Capsule I, ClpC, FbpABC, and HitABC, many of which were used for the iron uptake system. Total 57 putative PPT acetyltransferase were annotated, and two of them were found to be novel putative acetyltransferases for acetylation and detoxification of PPT. In conclusion, the work revealed microbial gene catalogue of PPT-utilized soils and found two novel putative PPT acetyltransferases using metagenomics. The work facilitates the understanding of impact of PPT on complex microbial community structure and physiology resides in PPT-utilized soils. Moreover, two annotated PPT acetyltransferases show important potential for the development of transgenic herbicide-resistant crops., (Copyright © 2019 Elsevier B.V. All rights reserved.)