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Developing simple and efficient multi-gene expression systems is crucial for multi-trait improvement or bioproduction in transgenic plants. In previous research, an IGG6-based bicistronic system from the nonpathogenic fungus Glarea lozoyensis efficiently expressed multiple enzyme proteins in yeast and maize, and the heterologous enzymes successfully performed their catalytic activity to reconstruct the biosynthetic pathway in the host organism. Unlike enzyme proteins, some heterologous functional proteins (such as insecticidal proteins) are dose-dependent and they need to express sufficient levels to perform their biological functions. It remains unclear whether the IGG6-based bicistronic system can achieve high expression of the functional proteins for practical applications in crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, vip3Aa and cry1Ab, were linked via IGG6 to form a bicistron, while two glyphosate resistance genes, gr79epsps and gat, served as monocistronic selectable marker genes. Regenerated maize plants were produced through genetic transformation. RNA and immunoblot analyses revealed that the vip3Aa-IGG6-cry1Ab bicistron was transcribed as a single transcript, which was then translated into two separate proteins. Notably, the transcription and translation of cry1Ab were significantly positively correlated with those of vip3Aa. Through ELISA and leaf bioassay, we identified two transgenic maize lines, VICGG-15 and VICGG-20, that exhibited high insecticidal activity against fall armyworm (FAW; Spodoptera frugiperda) and Asian corn borer (ACB; Ostrinia furnacalis), both of which had high expression of Vip3Aa and Cry1Ab proteins. Subsequent evaluations, including silk, ear, and field bioassays, as well as glyphosate tolerance assessments, indicated that the VICGG-15 plants displayed high resistance to FAW and ACB, and could tolerate up to 3600 g acid equivalent (a.e.) glyphosate per hectare without adversely affecting phenotype or yield. Our finding established that the IGG6-based bicistronic system can achieve high expression of functional proteins in maize, and it is a potential candidate for multi-gene assembly and expression in plants. [ABSTRACT FROM AUTHOR]

BRS 700FL B3RF is an extra-long staple (ELS) upland cotton cultivar. It features exceptional fiber quality, with fiber length exceeding 33.0 mm and fiber strength above 32.5 gf tex-1. This cultivar includes transgenic events Bollgard® III and Roundup Ready Flex™, providing enhanced resistance to lepidopteran pests and tolerance to glyphosate. [ABSTRACT FROM AUTHOR]

Clary sage (Salvia sclarea L.) is well known as a highly aromatic herbaceous plant and is commonly used in aromatic products and medicine. The biggest problem in the cultivation of clary sage is weeds, which not only entails a great cost of labor for weeding, but also leads to a great fluctuation in the yield and quality of its products. Therefore, there is a need to breed herbicide-resistant clary sage using genetic engineering. However, the lack of suitable regeneration and genetic transformation systems for clary sage has limited the process of molecular breeding. In this study, we firstly reported the regeneration system of clary sage, where explants from hypocotyls of sage induced adventitious shoots at a rate of 22.22 % when cultured on MS medium supplemented with 2.5 mg·L−1 6-BA, 0.15 mg·L−1 IAA, and 0.25 mg·L−1 VC. Rooting of adventitious shoots was achieved at a rate of 94.44 % on 1/2 MS medium containing 0.125 mg·L−1 NAA, 0.5 mg·L−1 IAA, and 0.1 mg·L−1 IBA. Secondly, based on the regeneration system, an efficient and stable Agrobacterium -mediated genetic transformation system was established in this paper, and the rate of genetic transformation was determined to be in the range of 3 %-5 % by transforming the hypocotyls of clary sage with pCAMBIA1301 vector and pCAMBIA1301- GR79 - GAT vector. Finally, glyphosate tolerance was obtained from GR79 and GAT co-expressed clary sage plants in this study. This study not only lays the foundation of genetic transformation for the functional gene research and in-depth investigation of various biological problems of clary sage, but also provides a feasible genetic engineering solution for its industry to solve the weed problem, as well as a promotional role for precise molecular breeding of clary sage. • The regeneration system for clary sage was established for the first time. • The genetic transformation system for clary sage was established for the first time. • The EPSPS GR79 and GAT herbicide resistance genes were introduced into clary sage for the first time. • T1 generation clary sage plants with some level of glyphosate tolerance were obtained. [ABSTRACT FROM AUTHOR]

Glyphosate is a widely used nonselective herbicide. Probing the glyphosate tolerance mechanism is necessary for the screening and development of resistant cultivars. In this study, a hyperspectral image was used to develop a more robust leaf chlorophyll content (LCC) prediction model based on different datasets to finally analyze the response of LCC to glyphosate-stress. Chlorophyll a fluorescence (ChlF) was used to dynamically monitor the photosynthetic physiological response of transgenic glyphosate-resistant and wild glyphosate-sensitive maize seedlings and applying chemometrics methods to extract time-series features to screen resistant cultivars. Six days after glyphosate treatment, glyphosate-sensitive seedlings exhibited significant changes in leaf reflection and photosynthetic activity. By updating source domain and transfer component analysis, LCC prediction model performance was improved effectively (the coefficient of determination value increased from 0.65 to 0.84). Based on the predicted LCC and ChlF data, glyphosate-sensitive plants are too fragile to protect themselves from glyphosate stress, while glyphosate-resistant plants were able to maintain normal photosynthetic physiological activity. JIP-test parameters, φE0, VJ, ψE0, and M0, were used to indicate the degree of plant damage caused by glyphosate. This study constructed a transferable model for LCC monitoring to finally evaluate glyphosate tolerance in a time-series manner and verified the feasibility of ChlF in screening glyphosate-resistant cultivars. [ABSTRACT FROM AUTHOR]

Background: Glyphosate is a broad-spectrum, non-selective systemic herbicide. Introduction of glyphosate tolerance genes such as EPSPS or detoxification genes such as GAT can confer glyphosate tolerance on plants. Our previous study revealed that co-expression of EPSPS and GAT genes conferred higher glyphosate tolerance without "yellow flashing". However, the plant response to glyphosate at the transcriptional level was not investigated. Methods and results: To investigate the glyphosate tolerance mechanism, RNA-seq was conducted using four soybean genotypes, including two non-transgenic (NT) soybeans, ZH10 and MD12, and two GM soybeans, HJ698 and ZH10-6. Differentially expressed genes (DEGs) were identified in these soybeans before and after glyphosate treatment. Similar response to glyphosate in the two NT soybeans and the different effects of glyphosate on the two GM soybeans were identified. As treatment time was prolonged, the expression level of some DEGs involved in shikimate biosynthetic pathway and herbicide targeted cross-pathways was increased or declined continuously in NT soybeans, and altered slightly in HJ698. However, the expression level of some DEGs was altered in ZH10-6 at 12 hpt, while similar expression level of some DEGs involved in shikimate biosynthetic pathway and herbicide targeted cross-pathways was observed in ZH10-6 at 0 hpt and 72 hpt. These observations likely explain the higher glyphosate tolerance in ZH10-6 than in HJ698 and NT soybeans. Conclusions: These results suggested that GAT and EPSPS genes together play a crucial role in response to glyphosate, the GAT gene may work at the early stage of glyphosate exposure, whereas the EPSPS gene may be activated after the uptake of glyphosate by plants. These findings will provide valuable insight for the molecular basis underlying glyphosate tolerance or glyphosate detoxication. [ABSTRACT FROM AUTHOR]

Modern agriculture has gained significant economic benefits worldwide with the use of genetically modified (GM) technologies. While GM crops provide convenience to humans, their biosafety has attracted increasing concern. In this study, the Illumina MiSeq was used to perform a high-throughput sequencing of the V3-V4 hypervariable regions of 16S rRNA gene (16S rDNA) amplicons to compare the rhizosphere bacterial communities of the EPSPS/GAT dual transgenic glyphosate-tolerant soybean line Z106, its recipient variety ZH10, and Z106 with glyphosate application (Z106G) during flowering, seed filling, and maturing stages under field settings. At each of the three stages, the alpha and beta diversity of rhizosphere bacterial communities revealed no significant differences between ZH10, Z106, and Z106G. However, some bacterial taxa demonstrated a greater proportional contribution, particularly the nitrogen-fixing rhizobium Ensifer fredii, in the rhizospheric soil of Z106 at the seed filling and maturing stages, when compared to ZH10 and Z106G. The present study therefore suggests that the EPSPS/GAT dual transgenic line Z106 and exogenous glyphosate application have a minimal effect on the composition of the soybean rhizosphere bacterial community but have no impact on the structure of the rhizosphere microbial community during a single planting season. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L. we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS , resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape. [ABSTRACT FROM AUTHOR]

The large-scale use of the herbicide glyphosate leads to growing ecotoxicological and human health concerns. Microbe-assisted phytoremediation arises as a good option to remove, contain, or degrade glyphosate from soils and waterbodies, and thus avoid further spreading to non-target areas. To achieve this, availability of plant-colonizing, glyphosate-tolerant and -degrading strains is required and at the same time, it must be linked to plant-microorganism interaction studies focusing on a substantive ability to colonize the roots and degrade or transform the herbicide. In this work, we isolated bacteria from a chronically glyphosate-exposed site in Argentina, evaluated their glyphosate tolerance using the minimum inhibitory concentration assay, their in vitro degradation potential, their plant growth-promotion traits, and performed whole genome sequencing to gain insight into the application of a phytoremediation strategy to remediate glyphosate contaminated agronomic soils. Twenty-four soil and root-associated bacterial strains were isolated. Sixteen could grow using glyphosate as the sole source of phosphorous. As shown in MIC assay, some strains tolerated up to 10000 mg kg–1 of glyphosate. Most of them also demonstrated a diverse spectrum of in vitro plant growth-promotion traits, confirmed in their genome sequences. Two representative isolates were studied for their root colonization. An isolate of Ochrobactrum haematophilum exhibited different colonization patterns in the rhizoplane compared to an isolate of Rhizobium sp. Both strains were able to metabolize almost 50% of the original glyphosate concentration of 50 mg l–1 in 9 days. In a microcosms experiment with Lotus corniculatus L, O. haematophilum performed better than Rhizobium, with 97% of glyphosate transformed after 20 days. The results suggest that L. corniculatus in combination with to O. haematophilum can be adopted for phytoremediation of glyphosate on agricultural soils. An effective strategy is presented of linking the experimental data from the isolation of tolerant bacteria with performing plant-bacteria interaction tests to demonstrate positive effects on the removal of glyphosate from soils.

The large-scale use of the herbicide glyphosate leads to growing ecotoxicological and human health concerns. Microbe-assisted phytoremediation arises as a good option to remove, contain, or degrade glyphosate from soils and waterbodies, and thus avoid further spreading to non-target areas. To achieve this, availability of plant-colonizing, glyphosate-tolerant and -degrading strains is required and at the same time, it must be linked to plant-microorganism interaction studies focusing on a substantive ability to colonize the roots and degrade or transform the herbicide. In this work, we isolated bacteria from a chronically glyphosate-exposed site in Argentina, evaluated their glyphosate tolerance using the minimum inhibitory concentration assay, their in vitro degradation potential, their plant growth-promotion traits, and performed whole genome sequencing to gain insight into the application of a phytoremediation strategy to remediate glyphosate contaminated agronomic soils. Twenty-four soil and root-associated bacterial strains were isolated. Sixteen could grow using glyphosate as the sole source of phosphorous. As shown in MIC assay, some strains tolerated up to 10000 mg kg–1 of glyphosate. Most of them also demonstrated a diverse spectrum of in vitro plant growth-promotion traits, confirmed in their genome sequences. Two representative isolates were studied for their root colonization. An isolate of Ochrobactrum haematophilum exhibited different colonization patterns in the rhizoplane compared to an isolate of Rhizobium sp. Both strains were able to metabolize almost 50% of the original glyphosate concentration of 50 mg l–1 in 9 days. In a microcosms experiment with Lotus corniculatus L, O. haematophilum performed better than Rhizobium , with 97% of glyphosate transformed after 20 days. The results suggest that L. corniculatus in combination with to O. haematophilum can be adopted for phytoremediation of glyphosate on agricultural soils. An effective strategy is presented of linking the experimental data from the isolation of tolerant bacteria with performing plant-bacteria interaction tests to demonstrate positive effects on the removal of glyphosate from soils. [ABSTRACT FROM AUTHOR]

Cultivar BRS 433FL B2RF is a high-quality fiber upland cotton with high lint yield potential and resistance to several diseases. The introgressed transgenic events Bollgard II® and Roundup Ready Flex® confer enhanced resistance to lepidopteran pests and tolerance to the herbicide glyphosate, respectively. Fiber length exceeds 32.5 mm and fiber strength 33 gf tex-1. [ABSTRACT FROM AUTHOR]

The bad cide in adopting cide-tolerant alfalfa cultivar is the shift in weed communities with rise proportion of tolerant species. The main objective of the recent study was to trace competition indicator of alfalfa populations as affected by recurrent selection to glyphosate tolerance. Two cycles of recurrent selection for Glyphosate tolerance were imposed on each base population. Competition indicators following selection included plant characters and weed bioassay. In most studied populations, plant height decreased with the first cycle of selection to glyphosate tolerance by -8.991, -3.225, -1.240 and -0.426% in Siwa, Hasawi, C.U.F101 and Baladi1 populations, respectively. Except for, Sirivar population an increase by +6.740 % was accompanied with selection to glyphosate tolerance. On the other hand, by cycle two of selection, populations had different response relative to C1, where Baladi1, C.U.F101 and Sirivar populations had a decrease in plant height with values reached -1.761, -0.720 and 0.617 cm, respectively. While, Hasawi and Siwa populations showed an increased plant height by +3.816 and +3.197cm, respectively. Glyphosate treated plots (+ and -) were evaluated by weed bioassay in terms of green and dry weight during a course of three successive cuttings. Exposed genetic materials to glyphosate treatments were selection cycles (C0, C1 and C2) of the five studied populations.The first cycle of selection to glyphosate tolerance expressed a significant increase of 9.59 and 10.93% relative to the base population as green and dry weed weights, when evaluated under glyphosate treating condition. The second cycle of selection was associated with a reduction of 29.63 and 28.88% in green and dry weight of weed. This might indicate a proliferation of alfalfa plants that were selected for glyphosate tolerance, the matter that reduced the mass of weeds, whether, as green or dry weight. [ABSTRACT FROM AUTHOR]

Hybrid rice breeding offers an important strategy to improve rice production, and therefore is crucial for ensuring worldwide food security. Purity of hybrids is a key factor in keeping the high production of hybrid rice. While another important thing is mechanical weeding of hybrid rice, which could greatly release labor from laborious work and cut the production cost. Both of these two problems could be partly resolved by developing glyphosate-tolerant restorer lines. In this study, a novel glyphosate-tolerant gene I.variabilis-EPSPS* was transferred into elite indica restorer line Minghui 86 (MH86) by Agrobacterium-mediated transformation. By Southern blot and inverse PCR analysis, 3 transgenic restorer lines (MY28, MY50, and MY51) with a single copy of I.variabilis-EPSPS* integrated into the intergenic regions were selected from 72 regenerated transgenic plants. The inheritance of I.variabilis-EPSPS* in the 3 transgenic restorer lines was proved to follow Mendelian inheritance by germinating test. In the following generations, according to Southern blot, Northern blot, 3'RACE, and Western blot, I.variabilis-EPSPS* were proved to stably inherit and express in the candidate transgenic restorer lines. At the same time, glyphosate tolerance assay was performed in the field, in which the candidate transgenic restorer lines kept normal agronomic traits under glyphosate treatments with a dosage as high as 8400 g/ha. Therefore, the candidate transgenic restorer lines were highly tolerant to glyphosate. The 3 candidate transgenic restorer lines were respectively hybridized with the male sterile line II-32A, and their hybrids were respectively named as MY28H, MY50H, and MY51H. According to Southern blot and Western blot analysis, I.variabilis-EPSPS* could stably inherit to F1 hybrids and express in these hybrids, which indicated that the true hybrids could be distinguished by I.variabilis-EPSPS*. In the field test, the transgenic hybrids with I.variabilis-EPSPS* could suffer 3360 g/ha glyphosate without the damage of agronomic traits, which indicated the application potential of these hybrids in direct seeding. Collectively, the transgenic glyphosate-tolerant restore lines developed in this study were highly tolerant to glyphosate, and they had potential to be used in commercial production of hybrid rice. [ABSTRACT FROM AUTHOR]

The recent study was an attempt to measure the influence of selection for glyphosate tolerance in alfalfa germplasm on green and dry forage yields. Two cycles of recurrent selection for Glyphosate tolerance were imposed on each of five base population. Evaluation of selected cycles (C1 and C2) along with base populations (C0) was carried out for each population as a split plot design with Glyphosate treatment (+ and -) as main plots and populations (C0, C1 and C2) as a sub –plot. The recorded dry forage yield from the studied population was similar irrespective of glyphosate treatment. Meanwhile, green, and dry forages of studied population, significantly varied (p≥0.01) in ranke or magnitude depending on selection cycle (significant population × selection cycle interaction). In addition, green forage yields of glyphosate treatments significantly varied (p≥0.05) among populations and selection cycles (significant glyphosate treatment × population × selection cycle).The least green forage yield resulted from C.U.F. 101population when treated with glyphosate (110.32 ton.ha-1). That was not significantly different from green forage yields produced by any of Baladi 1and Siwa populations under glyphosate treatment. The highest significant green forage yields were obtained from any of Siriver or Baladi 1 population without glyphosate treatment (194.12 and 142.77 tons. ha-1 for the former and the latter, respectively). On another words, the most sensitive population to glyphosate treatment in terms of green forage yield was Baladi 1, since, it produced the least green forage yield with glyphosate application (110.91 tons.ha-1) versus significantly the highest green forage yield without glyphosate application (192.77 tons.ha-1).The least green forage yield resulted from C.U.F. 101population when treated with glyphosate (110.32 ton.ha-1).That was not significantly different from green forage yields produced by any of Baladi 1and Siwa populations under glyphosate treatment.Response of dry forage yield to population × selection cycles interaction took similar pattern to what noticed in green forage yield. C.U.F. 101 showed a significant reduction of 10.26% in dry forage yield of cycle two relative to cycle one. While, Hasawi population, recorded a significant increase in dry forage yield of 15.52% with the second cycle of selection relative to cycle one. Siwa population, showed lowering in dry forage yield with one cycle of selection for glyphosate tolerance (-7.373%) and an increase of 6.998% at second cycle of selection relative to cycle one. [ABSTRACT FROM AUTHOR]

BRS 369RF and BRS 370RF were developed by the EMBRAPA as a part of efforts to create high-yielding germplasm with combinations of transgenic traits. BRS 369RF and BRS 370RF are midseason cultivars and have yield stability, adaptation to the central Brazilian savanna, good fiber quality and tolerance to glyphosate herbicide.

It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops (Cao et al. 2012). In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301UbiSpAM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase (rbcS) small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize (Zea mays) immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis (Bt) cry gene and epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1Ah gene with the 2mG2-epsps gene and combined the wide-used manA gene as a selective marker to construct one coordinated expression vector called p2EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

Summary: Glyphosate has been associated with control failures for Spermacoce verticillata in some Brazilian States. The objective of this work was to evaluate and determine the possible causes of this and propose alternative herbicides to use. Glyphosate was applied at three plant stages of development (2–4 leaves, 4–6 leaves and full bloom) where foliar absorption and translocation of glyphosate to various plants parts and time were analysed using the 14C technique. Data were submitted to nonlinear regressions and analysis of variance, where means were compared by a Tukey test at 5% probability. Plant control by the application of different herbicides (19) in the same three stages of development of weed was evaluated. Twenty‐one days after herbicide application, control was visually evaluated and data analysed and means were compared. Due to absorption and/or translocation problems, S. verticillata plants were not controlled by glyphosate. Plants with 4–6 leaves showed lower absorption and translocation of the herbicide to the leaf/root regions compared with plants at the beginning of their development. Plants at full bloom showed lower translocation of the herbicide to the root. In addition to the application of glyphosate at early stages of development, the application of paraquat, flumioxazin and mixtures of glyphosate with flumioxazin or cloransulam is recommended. Late applications could result in poor control, principally if glyphosate is used. Therefore, early applications are strongly recommended for control of this species. [ABSTRACT FROM AUTHOR]

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line. [ABSTRACT FROM AUTHOR]

Main conclusion: The glyphosate-resistant gene, GR79Ms, was successfully introduced into the genome of alfalfa. The transgenic events may serve as novel germplasm resources in alfalfa breeding.Weed competition can reduce the alfalfa yield, generating new alfalfa germplasm with herbicide resistance is essential. To obtain transgenic alfalfa lines with glyphosate resistance, a new synthetic glyphosate-resistant gene GR79Ms encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was introduced into alfalfa germplasm by Agrobacterium tumefaciens-mediated transformation. In total, 67 transformants were obtained. PCR and Southern blot analyses confirmed that GR79Ms was successfully inserted into the genome of alfalfa. Reverse transcription-PCR and western blot analyses further demonstrated the expression of GR79Ms and its product, GR79Ms EPSPS. Moreover, two homozygous transgenic lines were developed in the T2 generation by means of molecular-assisted selection. Herbicide tolerance spray tests showed that the transgenic plants T0-GR1, T0-GR2, T0-GR3 and two homozygous lines were able to tolerate fourfold higher commercial usage of glyphosate than non-transgenic plants. [ABSTRACT FROM AUTHOR]

Transgenic cotton cultivars BRS 430 B2RF and BRS 432 B2RF, medium and late season respectively, were developed by EMBRAPA in partnership with Bahia Foundation, aiming to create a high-yielding germplasm. The Bt toxins Cry1Ac and Cry2Ab target lepidopteran pests and the CP4-EPSPS gene confers tolerance to the herbicide glyphosate. [ABSTRACT FROM AUTHOR]

The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate acetyltransferase (GAT) effectively detoxifies glyphosate by N-acetylation. With the aim of identifying a new strategy for development of glyphosate-tolerant crops, the plant expression vector pG2-GAT harboring gat and G2-aroA (encoding EPSPS) has been transformed into tobacco (Nicotiana tabacum) to develop novel plants with higher tolerance to glyphosate. Results from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein level. Glyphosate tolerance was compared among transgenic tobacco plants containing gat, G2-aroA, or both genes. Plants containing both gat and G2-aroA genes were the most glyphosate-tolerant. This study has shown that a combination of different strategies may result in higher tolerance in transgenic crops, providing a new approach for development of glyphosate-tolerant crops.

Cotton cultivar BRS 368RF was developed by EMBRAPA in a partnership with the Bahia Foundation, aiming to create high-yielding germplasm by adding transgenic traits addressed to Brazilian Savanna (cerrado) of the Northeast and North regions. BRS 368RF is a midseason cultivar and has yield stability and transgenic tolerance to glyphosate. [ABSTRACT FROM AUTHOR]

Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at four-fold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.

In recent years, glyphosate-tolerant legumes have been used as cover crops for weed management in tropical areas of Mexico. Mexican cologania ( Cologania broussonetii (Balb.) DC.) is an innate glyphosate-tolerant legume with a potential as a cover crop in temperate areas of the country. In this work, glyphosate tolerance was characterized in two Mexican cologania (a treated (T) and an untreated (UT)) populations as being representatives of the species, compared in turn to a glyphosate-susceptible hairy fleabane (S) ( Conyza bonariensis (L.) Cronq.) population. Experiments revealed that T and UT Mexican cologania populations had a higher tolerance index (TI), and a lower shikimic acid accumulation and foliar retention than the hairy fleabane S population. Absorption and translocation, leaf morphology and metabolism studies were only carried out in the Mexican cologania T population and the hairy fleabane S population. The latter absorbed 37% more 14 C-glyphosate compared to the Mexican cologania T at 96 h after treatment (HAT). Mexican cologania T translocated less herbicide from the treated leaf to the remainder of the plant than hairy fleabane S. The Mexican cologania T presented a greater epicuticular wax coverage percentage than the hairy fleabane S. This morphological characteristic contributed to the low glyphosate absorption observed in the Mexican cologania. In addition, the Mexican cologania T metabolized glyphosate mainly into AMPA, formaldehyde and sarcosine. These results indicate that the high glyphosate tolerance observed in Mexican cologania is mainly due to the poor penetration and translocation of glyphosate into the active site, and the high glyphosate degradation into non-toxic substances. [ABSTRACT FROM AUTHOR]

To date, only AroA variant derived from Agrobacterium tumefaciens CP4 has been used to generate the commercial glyphosate-resistant crops currently available in the market. This single source of the EPSPS gene may have caused the decrease in herbicide tolerance, which has become a major concern of those involved in field management programs. Therefore, it is of interest to increase aroA/EPSPS gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In the current study, EPSPS gene from Vitis vinifera (VvEPSPS) was cloned using reverse transcription polymerase chain reaction. However, wild type VvEPSPS cannot be directly used for developing transgenic crops because of its extreme glyphosate sensitivity. Recent studies have demonstrated that DNA shuffling is an effective strategy in producing multi-mutated EPSPS resourced from plants (EPSPSplant) with improved glyphosate resistance in bacteria and plants. After performing DNA shuffling on VvEPSPS gene, one highly glyphosate-resistant mutant with seven amino acid variations was isolated after five rounds of shuffling and screening. The mutant showed seven amino acid changes in the EPSPS gene, namely, Q93R, T113A, P117L, G126A, C160Y, N239H, and V343A. The assay of glyphosate resistance further confirmed the potential of the VvEPSPS mutant in developing glyphosate-resistant crops. [ABSTRACT FROM AUTHOR]

As part of an ecological risk assessment, Roundup Ready 2 Yield® soybean (MON 89788) was compared to a conventional control soybean variety, A3244, for disease and arthropod damage, plant response to abiotic stress and cold, effects on succeeding plant growth (allelopathic effects), plant response to a bacterial symbiont, and effects on the ability of seed to survive and volunteer in a subsequent growing season. Statistically significant differences between MON 89788 and A3244 were considered in the context of the genetic variation known to occur in soybean and were assessed for their potential impact on plant pest (weed) potential and adverse environmental impact. The results of these studies revealed no effects of the genetic modification that would result in increased pest potential or adverse environmental impact of MON 89788 compared with A3244. This paper illustrates how such characterization studies conducted in a range of environments where the crop is grown are used in an ecological risk assessment of the genetically modified (GM) crop. Furthermore, risk assessors and decision makers use this information when deciding whether to approve a GM crop for cultivation in—or grain import into—their country. [ABSTRACT FROM AUTHOR]

During the development of a genetically modified (GM) crop product, extensive phenotypic and agronomic data are collected to characterize the plant in comparison to a conventional control with a similar genetic background. The data are evaluated for potential differences resulting from the genetic modification process or the GM trait, and the differences-if any-are subsequently considered in the context of contributing to the pest potential of the GM crop. Ultimately, these study results and those of other studies are used in an ecological risk assessment of the GM crop. In the studies reported here, seed germination, vegetative and reproductive growth, and pollen morphology of Roundup Ready 2 Yield soybean, MON 89788, were compared to those of A3244, a conventional control soybean variety with the same genetic background. Any statistically significant differences were considered in the context of the genetic variation known to occur in soybean and were evaluated as indicators of an effect of the genetic modification process and assessed for impact on plant pest (weed) characteristics and adverse ecological impact (ecological risk). The results of these studies revealed no effects attributable to the genetic modification process or to the GM trait in the plant that would result in increased pest potential or adverse ecological impact of MON 89788 compared with A3244. These results and the associated risk assessments obtained from diverse geographic and environmental conditions in the United States and Argentina can be used by regulators in other countries to inform various assessments of ecological risk. [ABSTRACT FROM AUTHOR]

The 5-enolpyruvylshikimate-3-phosphate synthases (EPSPSs) encoded by aroA genes are traditionally divided into two classes, and only the class II EPSPS is naturally insensitive to herbicide glyphosate. Here, a novel EPSPS from Isoptericola variabilis , designated as I. variabilis EPSPS, was characterized. Its aroA gene contained 1374 bp with 77% of GC contents, encoding a peptide of 458 amino acids. The EPSPS carried a highly conserved region involved in substrate bindings in EPSPSs and displayed a high sequence and structure similarity to class I EPSPS. However, this EPSPS had high naturally glyphosate-tolerant capability and enzymatic kinetics constants similar to that of class II EPSPSs. Escherichia coli AB2829 harboring the aroA gene grew well in medium containing 150 mM glyphosate. The purified EPSPS had the highest enzymatic activity at pH 8.0 and 30 °C, and retained its activity at pH 6–10 and 20–60 °C. This enzyme had a K m value of 28 μM for substrate phosphoenolpyruvate and 111 μM for shikimate-3-phosphate, respectively, with a rather high K i value for glyphosate. Cations increased substrate affinity but slightly reduced glyphosate tolerance. The I. variabilis EPSPS may be used for generating transgenic glyphosate-tolerant plants and dissecting mechanisms underlying the glyphosate tolerance. [ABSTRACT FROM AUTHOR]

Herbicide resistance is the most widely used transgenic crop trait for broad-spectrum control of weeds. Here we report a novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene ( Gr5) isolated from glyphosate-contaminated soil. The full Gr5 gene was 1,819 bp and contained a 1,341-bp open reading frame encoding a 47-kDa protein. Phylogenetic analysis showed that Gr5 is a class I EPSPS even though most such enzymes are naturally sensitive to glyphosate. Interestingly, Gr5 protein contained highly conserved PEP and S3P binding residues (Glu-351) and several motifs insensitive to glyphosate. Transgenic Gr5 plants ( T) grew normally and produced seeds which we treated with a high-glyphosate solution (4× recommended spray). Analysis of the T progenies showed that Gr5 was inherited at a Mendelian 3:1 segregation ratios and that glyphosate tolerance in T plants was unchanged. Our results show the Gr5 gene to be a promising candidate for the development of commercial transgenic crops with high glyphosate tolerance. [ABSTRACT FROM AUTHOR]

Glyphosate is a widely used herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase ( EPSPS) activity. Most plants and microbes are sensitive to glyphosate. However, transgenic-resistant crops that contain a modified epsps obtained from the resistant microbes have been commercially successful and therefore, new resistance genes and their adaptive regulatory mechanisms are of great interest. In this study, a soil-borne, glyphosate-resistant bacterium was selected and identified as Enterobacter. The EPSPS in this strain was found to have been altered to a resistant one. A total of 42 differentially expressed genes ( DEGs) in the glyphosate were screened using microarray techniques. Under treatment, argF, sdhA, ivbL, rrfA-H were downregulated, whereas the transcripts of speA, osmY, pflB, ahpC, fusA, deoA, uxaC, rpoD and a few ribosomal protein genes were upregulated. Data were verified by quantitative real-time PCR on selected genes. All transcriptional changes appeared to protect the bacteria from glyphosate and associated osmotic, acidic and oxidative stresses. Many DEGs may have the potential to confer resistance to glyphosate alone, and some may be closely related to the shikimate pathway, reflecting the complex gene interaction network for glyphosate resistance. [ABSTRACT FROM AUTHOR]

Changes in leaf anatomy were examined in two S. African populations of Conyza canadensis of which one was presumed to be resistant (CCPR) and the other susceptible (CCS) to glyphosate. Glyphosate was applied to plants, which were grown from seed collected from these populations, at rates of 1, 2 and 4 kg a.e. ha-1 of TOUCHDOWN® [active substance: glyphosate trimesium salt, 500 g L-1] that are equivalent to 2, 4 and 8 L ha -1 of the herbicide Touchdown. Leaf samples for the light microscope (LM) analysis were collected 3, 7 and 24 h after treatment (HAT). Changes in chlorophyll and shikimate content of leaf material were also examined. Changes in the palisade and pith tissue of leaves were not detected in the investigated populations at 3 and 7 HAT. However, at 24 HAT the different herbicide doses caused changes in leaf anatomy. These changes (injuries) were detected in the CCS at all tested doses, but in the CCPR population of C. canadensis the injuries were observed at only the two highest rates, 2 and 4 kg a.e. ha-1. Chlorophyll and shikimate contents indicated significant differences between the treated and untreated plants of susceptible population only. Difference in glyphosate resistance between the CCS and CCPR populations was confirmed with an index of resistance of 1.58. This value of the index of resistance indicates that CCS population is 1.58 times more susceptible to glyphosate compared to CCPR population. [ABSTRACT FROM AUTHOR]

A new 5-enolpyruvylshikimate-3-phosphate synthase ( EPSPS) gene from Malus domestica ( MdEPSPS) was cloned and characterized by rapid amplification of c DNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids ( N63 D, N86 S, T101 A, A187 T, D230 G, H317 R, Y399 R and C413 A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes ( T101 A and A187 T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187 T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops. [ABSTRACT FROM AUTHOR]

The advantages of gene 'stacking' or 'pyramiding' are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG -epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG, were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector. [ABSTRACT FROM AUTHOR]

With 2 figures and 4 tables Stepwise selection approach was adopted to obtain glyphosate-tolerant upland cotton mutant (R1098) from the embryogenic calli of ‘Coker 312’ ( Gossypium hirsutum L.). The calli were transferred to the selection medium and multi-step selection pressure process was carried out until the calli could proliferate in the presence of 20 mm glyphosate. The regenerated plants from the glyphosate-tolerant calli were analysed for glyphosate response, tolerance of progenies and shikimate accumulation on whole-plant basis. ‘R1098’ could tolerate 1.48 kg acid equivalent/ha glyphosate. Progeny tests demonstrated that glyphosate tolerance trait was controlled by one dominant gene. Shikimate accumulation assay showed that significantly different amounts of shikimate were present between the mutant (R1098) and its wild parent (Coker 312) after glyphosate treatment. This indicates that ‘R1098’ has sufficient 5-enolpyruvylshikimate-3-phosphate synthase activity in the presence of glyphosate, thereby maintaining normal carbon flow through the shikimate pathway. Moreover, ‘R1098’ offers a greater potential to improve the weed control system in cotton production. [ABSTRACT FROM AUTHOR]

Abstract: Glyphosate tolerance can be conferred by decreasing the herbicide’s ability to inhibit the enzyme 5-enol pyruvylshikimate-3-phosphate synthase, which is essential for the biosynthesis of aromatic amino acids in all plants, fungi, and bacteria. Glyphosate tolerance is based upon the expression of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) protein. The 2mEPSPS protein, with a lower binding affinity for glyphosate, is highly resistant to the inhibition by glyphosate and thus allows sufficient enzyme activity for the plants to grow in the presence of herbicides that contain glyphosate. Based on both a review of published literature and experimental studies, the potential safety concerns related to the transgenic 2mEPSPS protein were assessed. The safety evaluation supports that the expressed protein is innocuous. The 2mEPSPS enzyme does not possess any of the properties associated with known toxins or allergens, including a lack of amino acid sequence similarity to known toxins and allergens, a rapid degradation in simulated gastric and intestinal fluids, and no adverse effects in mice after intravenous or oral administration (at 10 or 2000mg/kg body weight, respectively). In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the 2mEPSPS protein in human food or in animal feed. [Copyright &y& Elsevier]

Glyphosate, a powerful nonselective herbicide, acts as an inhibitor of the activity of the enzyme 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by the aroA gene involved in aromatic amino acid biosynthesis. An Escherichia coli mutant AKM4188 was constructed by insertion a kanamycin cassette within the aroA coding sequence. The mutant strain is an aromatic amino acids auxotroph and fails to grow on M9 minimal media due to the inactive aroA. A DNA metagenomic library was constructed with samples from a glyphosate-polluted area and was screened by using the mutant AKM4188 as recipient. Three plasmid clones, which restored growth to the aroA mutant in M9 minimal media supplemented with chloramphenicol, kanamycin, and 50 m M glyphosate, were obtained from the DNA metagenomic library. One of them, which conferred glyphosate tolerance up to 150 m M, was further characterized. The cloned fragment encoded a polypeptide, designated RD, sharing high similarity with other Class II EPSPS proteins. A His-tagged RD fusion protein was produced into E. coli to characterize the enzymatic properties of the RD EPSP protein. [ABSTRACT FROM AUTHOR]

A novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene of 1.35 kb was cloned from a cosmid library of Halomonas variabilis HTG7, inserted into vector pET-28a (+) and transformed in Escherichia coli BL21 (DE3). EPSPS was over-expressed in soluble form after induction with IPTG at 30°C and it showed a single band in SDS-PAGE, which corresponds to a molecular weight of 51 kD. Deduced amino acid sequence analysis showed that there is little homology with the aroA genes which encode glyphosate-tolerant EPSPS in known sources, such as E. coli K12 and Agrobacterium sp. CP4. The over-expressed EPSPS was purified on nickel-nitrilotriacetic acid resin and detected by Western blotting analysis. Enzyme activity measurements demonstrated that there were 4.27 units/mg in cell extract, compared with 0.049 units/mg of the control. There is an 87-fold increase in specific activity for EPSPS. [ABSTRACT FROM AUTHOR]

Abstract: 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, and is the target of the herbicide glyphosate. Glyphosate tolerance activity of the enzyme could be obtained by natural occurrence or by site-directed mutagenesis. A functional Pseudomonas putida AroA was obtained by co-expression of two protein fragments AroA P. putida -N210 and AroA P. putida -C212 in Escherichia coli aroA mutant strain AB2829. From sequence analysis, the equivalent split site on E. coli AroA was chosen for further study. The result indicated that functional E. coli AroA could also be reconstituted from two protein fragments AroA E. coli -N218 and AroA E. coli -C219, under both in vivo and in vitro conditions. This result suggested that the fragment complementation property of this family of enzyme may be general. Additional experiments indicated that the glyphosate tolerance property of AroA could also be reconstituted in parallel with its enzyme activity. The implication of this finding is discussed. [Copyright &y& Elsevier]

N-Acetylation is a modification of glyphosate that could potentially be used in transgenic crops, given a suitable acetyltransferase. Weak enzymatic activity (kcat = 5 min-1, KM = 1 mM) for N-acetylation of glyphosate was discovered in several strains of Bacillus licheniformis (Weigmann) Chester by screening a microbial collection with a mass spectrometric assay. The parental enzyme conferred no tolerance to glyphosate in any host when expressed as a transgene. Eleven iterations of DNA shuffling resulted in a 7000-fold improvement in catalytic efficiency (kcat/KM), sufficient for conferring robust tolerance to field rates of glyphosate in transgenic tobacco and maize. In terms of kcat/KM, the native enzyme exhibited weak activity (4-450% of that with glyphosate) with seven of the common amino acids. Evolution of the enzyme towards an improved kcat/KM for glyphosate resulted in increased activity toward aspartate (40-fold improved kcat), but activity with serine and phosphoserine almost completely vanished. No activity was observed among a broad sampling of nucleotides and antibiotics. Improved catalysis with glyphosate coincided with increased thermal stability. [ABSTRACT FROM AUTHOR]

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338-16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain. [ABSTRACT FROM AUTHOR]