Glyphosate Tolerance Research Articles

Injury Indicator of Glyphosate to Alfalfa as affected by Recurrent Selection

In Egypt, alfalfa represts a suitable choice for forage cultivation expansion, since, the available land and water are oflower quality. The early seeding stage of alfalfa is the most val near able to weed competition. Research results regardingimprovement of alfalfa tolerance to glyphosate in Egypt is relatively scare. The recent study was an attempt to tracevariability in glyphosate tolerance of alfalfa germplasm. The improvement in tolerance due to recurrent selection was alsoconsidered. Alfalfa plant materials (Medicago sativa,L. ) used in that recent study were five base populations .Two cyclesof recurrent selection for Glyphosate tolerance were imposed on each base population. Evaluation of selected cycles (C1and C2) along with base populations (C0) was carried-out for each population as a split plot design with Glyphosatetreatment (+ and -) as main plots and populations (C0, C1 and C2) as a sub –plots. Glyphosate treated plots were evaluatedfor injury characters, i.e.; total chlorophyll, injury level, percent of death and shikimic acid content.Significant (p≥0.01) variations were detected among the studied alfalfa population. Also, the recorded values for allinjury indicator characters significantly varied among selection cycles. In the meantime, the interactions between thestudied population and selection cycles were significant (p ≥0.05). Chlorophyll content expressed as units was notchanged after one cycle of selection (30.22 and 29.15 spade for base population (C0) and first cycle of selection (C1) as anaverage over the studied population, respectively). While, the second cycle of selection resulted in significantly higherlevel of total chlorophyll content (34.51 spade). This might indicates that tolerance to glyphosate was associated to higherchlorophyll content. The value of injury level decreased in both of Siwa and Hasawi populations after the first cycle ofselection to glyphosate tolerance(-19.51 and -13.22%, respectively). Meanwhile, the progress of selection for glyphosatetolerance in alfalfa populations, gave a substantial decrease in value of injury level in all studied populations, except for,Siwa population that recorded an increase in value of injury level (-47.05, -44.44, -40.91 and -4.35 percent of therespective value recorded for injury level at the first cycle of selection in C.U.F101, Baladi 1, Sirivar and Hasawipopulations, respectively).Change in shikimic acid content represented by a decrease reached -35.05, -22.99, -5.49 and -4.26% in Siwa, Baldi 1, C.U.F 101 and Sirivar populations, respectively, after the first cycle of selection for glyphosatetolerance.Only Hasawi population recorded an increase in shikimic acid content reached +251.8% relative to base population.While, after the second cycle of selection an increase in shikimic acid content reached +63.60 and +4.688% for C.U.F101and Baldi1 populations relative to (C1). Sharing genes among tolerant individuals, increase the frequency of genesresponsible for tolerance, consequently expressing higher levels of cide- tolerance. The recent results assume thepossibility of obtaining glyphosate tolerant alfalfa population depending on frequent cycles of recurrent selection.

Effect on metabolome of the grains of transgenic rice containing insecticidal cry and glyphosate tolerance epsps genes

Genetically modified organisms (GMOs) need to be evaluated for safety before their release. Metabolome techniques provide effective methods to evaluate unintended effects. In this study, the grain metabolome of six transgenic rice lines containing secticidal cry and glyphosate tolerance epsps genes were tested by using an non-targeted metabolite profiling, and 161 and 138 metabolites were identified in grains at grain-filling stage and mature stage, respectively. The metabolic profiles of genetically modified (GM) lines and non-GM lines were all significantly different from each other at grain-filling stage. Although the levels of many metabolites were significantly changed in each transgenic line, only seven of them were simultaneously changed in all the six lines compared with those in non-GM lines at grain-filling stage. The number of significantly changed metabolites was much less at mature stage than that at grain-filling stage. Besides, none of these metabolites was simultaneously changed in all the six lines, suggesting that the metabolites of GMOs are different at different stages of GMO development. This study provides useful information about the metabolic variation between GMO and non-GMO in two development stages of rice grains. These findings suggest that non-targeted metabolite profiling can strengthen the assessment of risk based on metabolites.

Ochrobactrum cytisi IPA7.2 promotes growth of potato microplants and is resistant to abiotic stress.

Bacteria in natural associations with agricultural crops are promising for use in the improvement of clonal micropropagation of plants. We clarified the taxonomic position of Ochrobactrum cytisi strain IPA7.2 and investigated its tolerance for salinity, high temperature, and glyphosate pollution. We also tested the strain's potential to promote the growth of potato (Solanum tuberosum L.) microplants. Using the IPA7.2 draft genome (no. NZ_MOEC00000000), we searched for housekeeping genes and also for the target genes encoding glyphosate tolerance and plant-growth-promoting ability. A multilocus sequence analysis of the gap, rpoB, dnaK, trpE, aroC, and recA housekeeping genes led us to identify isolate IPA7.2 as O. cytisi. The strain tolerated temperatures up to 50°C and NaCl concentrations up to 3-4%, and it produced 8µgml-1 of indole-3-acetic acid. It also tolerated 6mM glyphosate owing to the presence of type II 5-enolpyruvylshikimate-3-phosphate synthase. Finally, it was able to colonize the roots and tissues of potato microplants, an ability preserved by several generations after subculturing. We identified the development phase of potato microplants that was optimal for inoculation with O. cytisi IPA7.2. Inoculation of in vitro-grown 15-day-old microplants increased the mitotic index of root meristem cells (by 50%), the length of shoots (by 34%), the number of leaves (by 7%), and the number of roots (by 16%). Under ex vitro conditions, the inoculated plants had a greater leaf area (by 77%) and greater shoot and root dry weight (by 84 and 61%, respectively) than did the control plants. We recommend O. cytisi IPA 7.2 for use in the growing of potato microplants to improve the production of elite seed material.

Investigation of alternate herbicides for effective weed management in glyphosate-tolerant cotton

ABSTRACTGlyphosate-resistant (GR) weeds are the biggest concern for all cotton stakeholders worldwide. Currently, 43 weeds species are resistant to glyphosate and the number is increasing at an alarming rate. Soil residual/pre-emergence (PRE) herbicides like Pendimethalin and S-metolachlor can be effectively used for the control of GR weeds; however, their use is very limited at farmer’s side due to the adoption of herbicide-tolerant technology with complete reliance on glyphosate. The present study was conducted to evaluate the performance of PRE and post-emergence (POST) herbicides in glyphosate-tolerant (GT) cotton. The herbicide treatments were pendimethalin and S-metolachlor as PRE-residual, and glyphosate was applied as POST at 20 days after sowing (DAS) either alone or in combination with other herbicides like S-metolachlor, pendimethalin, and haloxyfop. A second application of glyphosate was made at 35 DAS. Results revealed that pendimethalin and S-metolachlor treatments gave 100% suppression of all dominant weeds and increased lint yield by 310–350% as compared to weedy control. In contrast, glyphosate applied once and twice, gave weed biomass reduction of only 10–86%, and increased lint yield by 136–185% over weedy control. This research established that PRE application of pendimethalin and S-metolachlor can be included in the weed management program of GT cotton.

Growth-stage and temperature influence glyphosate resistance in Conyza bonariensis (L.) Cronquist

Glyphosate, currently the world's most extensively used herbicide was on the market for more than 20 years since its introduction in 1975 without reported evolution of resistant weed cases. Glyphosate is the only reported herbicide to inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and endogenous accumulation of shikimic acid has been used as a biomarker for the herbicide's activity in plants after spraying. Increased levels of shikimic acid indicate sensitivity or injury while a lack or limited accumulation of shikimic acid shows resistance to glyphosate. Conyza bonariensis seed germinates in flushes in South Africa and its emergence is staggered throughout the year under conducive temperature conditions. This variability in growth stages in the same field poses a challenge in chemical control. Glyphosate herbicide efficacy is affected by environmental conditions, particularly temperature. A glyphosate resistance screening study was carried out based on reports from farmers that C. bonariensis has become increasingly difficult to control especially in conservation tillage systems. This study has confirmed the occurrence of glyphosate-resistant C. bonariensis (hairy fleabane) cases in the western and southern Cape regions of South Africa. A glasshouse screening experiment of 24 biotypes indicates resistance levels ranging from 0.6 to 26.9-fold, with GR50 values of up to 3908.42 g.ae.ha−1 being reported in a population collected from a vineyard in the Piketberg district of the western Cape. The influence of growth stage and temperature on glyphosate resistance in hairy fleabane was evaluated by using a shikimate assay. Results from this study have shown that response of hairy fleabane to glyphosate is influenced by the phenological stage, with sensitivity or injury decreasing with growth stage. The significant statistical tests verified the dependence of the shikimate pathway on temperature, with more shikimic acid accumulating at 15 °C compared to 27 °C across all biotypes, regardless of origin and glyphosate tolerance status. More tolerance to the herbicide was demonstrated under warm temperature conditions. Possible outcomes of applying glyphosate at the bolting stage and under warm temperature conditions are escapes and no control of susceptible and resistant plants, respectively.

Alternative Herbicides for Control of Glyphosate-Resistant Giant Ragweed in Nebraska

Giant ragweed is an early emerging and one of the most competitive summer annual species found in many fields throughout North America. Extensive use of glyphosate in glyphosate-tolerant (GT) crops has evolved giant ragweed populations with glyphosate resistance. Field dose-response studies were conducted to determine the influence of growth stage on the level of glyphosate resistance in a suspected giant ragweed population. In addition, efficacy of alternative pre-plant, pre-emergence (PRE) and post-emergence (POST) herbicides were evaluated in corn and soybeans for glyphosate-resistant (GR) giant ragweed control. The field glyphosate dose-response studies confirmed that the suspected giant ragweed population were resistant ranging from 14- to 32-fold resistance depending on the growth stage of glyphosate application. The 10, 20 and 30 cm tall giant ragweed had 14, 17 and 32X resistance level, respectively. The dose-response studies indicated that the 10, 20, and 30 cm tall GR giant ragweed was controlled 90% with 214, 402 and 482 g ae ha-1 of dicamba, respectively, when tank-mixed with glyphosate (1060 g ae ha-1) 21 days after treatment (DAT). All evaluated pre-plant herbicides for corn provided ≥ 90% control of the GR giant ragweed at 30 DAT; among which the best control (100%) was achieved with pre-plant application of atrazine (2240 g ai ha-1), isoxaflutole (90 g ai ha-1), and premix of flumioxazin/pyroxasulfone (315 g ai ha-1). Herbicide combinations of different site of action provided greater than 90% control of the GR giant ragweed population in a PRE followed by POST herbicide program in corn and soybean, suggesting that alternative herbicide for giant ragweed control are available.

Edaphic Entomofauna Variation Depending on Glyphosate Application in Roundup Ready Soybean Crops

ABSTRACT: Currently, biomonitoring is a methodology used to verify the environmental impact of new technologies in the agricultural environment, highlighting edaphic entomofauna that is traditionally used as a bioindicator in this sort of research. Therefore, the objective of this study was to investigate the edaphic entomofauna variation depending on glyphosate application in Roundup Ready soybeans. The experiment was carried out in Coimbra, MG during the 2007/2008 and 2008/2009 cropping seasons. The experimental design was a randomized block design with five replications. The treatments were: non-transgenic soybean with mechanical weeding of weeds; transgenic soybean with mechanical weeding of weeds; transgenic soybean with one glyphosate application and transgenic soybean with three glyphosate applications. The populations of the edaphic entomofauna were sampled during two crops. The insertion of the glyphosate tolerance gene did not affect the richness and the abundance of arthropods in the soil. The arthropod richness was reduced in treatments where glyphosate was applied one and three times. The glyphosate application in transgenic soybean reduced the density of the predatory mite Galumnidae (Acari); predator ants Neivamyrmex sp. (Hymenoptera: Formicidae) and Solenopsis sp. (Hymenoptera: Formicidae); and springtails Entomobryidae (Collembola), Hypogastrura sp. (Collembola: Hypogastruridae) and Onychiuridae (Collembola). Therefore, it is essential to follow the use recommendations of the herbicide glyphosate and adopt good agricultural practices that promote pesticide biodegradation, thereby contributing to the reduction of the toxicological potential of glyphosate on the edaphic entomofauna.

Complex gene response of herbicide-resistant Enterobacter strain NRS-1 under different glyphosate stresses.

Knowledge of biological evolution and genetic mechanisms is gained by studying the adaptation of bacteria to survive in adverse environmental conditions. In this regard, transcriptomic profiling of a glyphosate-tolerant Enterobacter strain NRS-1 was studied under four different treatments to investigate the gene-regulatory system for glyphosate tolerance. A total of 83, 83, 60 and 74 genes were up-regulated and 108, 87, 178 and 117 genes down-regulated under 60-NPG, 110-NPG, NaCl (355mM) and HCl (pH 4.46) stress treatments, respectively. Complex gene network was identified to be involved in regulating tolerance to glyphosate. This study revealed that NRS-1 has gained glyphosate tolerance at the cost of osmotic and acidic resistance. The 25 differentially expressed genes are reported to may have partly changed the function for providing resistance to glyphosate directly, among them genes metK, mtbK, fdnG and wzb that might detoxify/degrade the glyphosate. However, under 110-NPG condition, NRS-1 might have utilized economical and efficient ways by depressing its metabolism and activity to pass through this stress. Hence, the present study provides insights into the genes involved in glyphosate tolerance, which can be effectively utilized to engineer herbicide-resistant crop varieties after their proper validation to manage weed growth.

Comparação entre métodos químicos de controle de buva com início de dessecação em período seco ou úmido

Diversas são as ocasiões onde o clima interfere diretamente na eficácia dos herbicidas aplicados para o controle de plantas daninhas. Dessa forma, objetivou-se com este trabalho avaliar o melhor momento climático para iniciar o controle químico da buva (Conyza spp.). Os experimentos foram conduzidos no município de Indianópolis/ MG. O experimento 1 foi realizado em esquema fatorial (2x5), tendo como primeiro fator as condições climáticas, período seco ou úmido, no momento da primeira aplicação do herbicida glifosato + 2,4-D (1.200 + 670 g e.a. ha-1), e o segundo fator a aplicação sequencial representada pelos tratamentos herbicidas (g e.a ou i.a. ha-1): glifosato + 2,4-D (1.200 + 670) ou paraquat (500), isolados ou associados ao diclosulam (29,4), fluroxipir (200) isolado, além da testemunha absoluta sem aplicação de herbicida. O experimento 2 visou comprovar o nível de dificuldade de controle da buva ao herbicida glifosato na área de ambos experimentos, em aplicações sequenciais, espaçadas 35 dias entre as mesmas, de doses (g e.a ha-1) crescentes de glifosato (0, 480, 960, 1.920 e 3.840), 2,4-D (670) e a associação de glifosato + 2,4-D (960 + 670). De forma geral, para se obter controle efetivo, iniciando a aplicação em período seco ou úmido, foi necessário realizar aplicação sequencial usando na primeira o glifosato + 2,4-D seguida de uma segunda aplicação, independente dos herbicidas testados. A população de buva presente na área demonstrou não ser controlada somente com aplicações do herbicida glifosato.

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3-Phosphate Synthase.

Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site-directed mutagenesis (SDM). The mutant significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile and Pro 102 to Ser. These mutation sites in E.coli have been studied as significant active sites of glyphosate resistance. However, in our research they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate-resistant plants.

Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance.

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.

Expression of GNA and biting site-restricted cry1Ac in cotton; an efficient attribution to insect pest management strategies

Insect-resistant transgenic cotton has been commercialized for two decades. Most of the introduced cultivars express Bt gene(s) constitutively under the control of 35S promoter in whole-plant tissues. However, there have been other promoters considered by researchers to confine the toxin expression to targeted organ and tissues. We developed a triple-gene construct including GNA, cry1Ac and cp4 epsps genes. We attempted to confine cry1Ac expression to insect biting sites by cloning it to downstream of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1). Moreover, to broaden the range of resistance, GNA was driven by the 35S promoter to target the sap-sucking insects like aphids which impose large losses in cotton production. To select the transformants in selection medium and for glyphosate tolerance, GNA and cry1Ac genes were accompanied with cp4 epsps gene. Two binary vectors harboring desired genes were constructed and utilized in the study (pGTGNAoC1AC and pGTGN35C1AC). Transformation of cultivar GSN-12 was carried out by employing Agrobacterium tumefaciens strain EHA105. Plantlets were primarily screened under glyphosate (N-phosphonomethyl glycine) selection pressure and subsequently subjected to molecular and biotoxicity assays. Introduction of cry1Ac and GNA to cotton plant conferred resistance to Spodoptera littoralis and Aphis gossypii Glover. Restriction of cry1Ac toxin protein to insect biting sites along with a plant lectin attributes significantly to insect pest management strategies.

Response of Glyphosate-resistant and Conventional Soybean Grafted Plants to Glyphosate

AbstractGrafting is a common technique used to impart desirable traits to a plant’s scion. Herbicide resistant rootstocks have the potential to confer non–genetically modified (non-GM) scions with herbicide tolerance while mitigating some societal concerns regarding GM crops and food. We examined the impacts of soybean cultivar and growth stage and environmental temperature on transference of glyphosate tolerance to conventional (CN) soybean scions when grafted to glyphosate-resistant (RR) rootstocks. Small CN/RR (scion/rootstock) plants (3-leaf stage) and medium-sized plants (6-leaf stage) were injured more than large plants (10-leaf stage) 34 d after treatment (DAT) with 0.84 and 1.68 kg ae ha−1 glyphosate. All CN/CN combinations died, and RR/RR were uninjured. The cultivar of the scion had a greater effect on glyphosate tolerance than the cultivar of the rootstock. CN scions 352 and 5418 were more tolerant than CN scion 5388 across all RR rootstocks 35 DAT when treated with 0.84 kg ha−1 glyphosate. CN/RR construct 5388/9392 was more sensitive to temperature compared with 352/9392. Less leaf regrowth of 5388/9392 was observed under the warmer temperature. Our experiments demonstrated that grafting imparted robust glyphosate tolerance across different plant sizes, environmental temperatures, and scion/rootstock cultivars.

Sugar Beet Improvement using Agrobacterium-mediated Transformation technology

Since discovering Agrobacterium tumefaciens distinctive capacity to incorporate a specified part of their transfer-DNA (T-DNA) into eukaryotic cells, the bacteria were commonly used for crop transformation originally of dicotyledonous crops and subsequently of nearly all organisms. To achieve this, the tumor-inducing (Ti) plasmid was changed to extract phytohormone and opine biosynthetic proteins (cytokinin and auxin) so as not to interfere with ordinary morphological growth. Overall, the conversion mediated by Agrobacterium was easier, more effective and less costly relative to other technologies. It also results in insertions with small copy count. Tumor development in crops has also proved the susceptibility of explants from field-grown sugar beet crops to Agrobacterium tumefaciens. Early efforts by Agrobacterium tumefaciens to transform sugar beet were unsuccessful, primarily owing to inability to regenerate crops from stably modified callus or suspended cells. A genotype-independent method was defined under which cotyledonary explants of various sugar beet genotypes are inoculated with Agrobacterium tumefaciens comprising whether kanamycin tolerance and GUS activity or kanamycin resistance, GUS activity and glyphosate tolerance. GUS expression, NPT dot blot as well as EPSPS assays verified the presence of transgenes; progeny showed Mendelian genetically modified inheritance and glyphosate tolerance at deadly concentrations to control plants. Unfortunately, there was no publication of technical information of the technique. Here we reviewed the concept Agrobacterium-mediated transformation and how to be applicable

Management of Field-Evolved Resistance to Bt Maize in Argentina: A Multi-Institutional Approach

Evolution of resistance to control measures in insect populations is a natural process, and management practices are intended to delay or mitigate resistance when it occurs. During the 2012/13 season the first reports of unexpected damage by Diatraea saccharalis on some Bt maize hybrids occurred in the northeast of San Luis province, Argentina. The affected Bt technologies were Herculex I® (HX-TC1507) and VT3PRO® (MON 89034 × MON 88017*). Event TC1507 expresses Cry1F and event MON 89034 expresses Cry1A.105 and Cry2Ab2, whichr are all Bt proteins with activity against the lepidopterans D. saccharalis and Spodoptera frugiperda (MON 88017 expresses the protein Cry3Bb1 for control of coleopteran insects and the enzyme CP4EPSPS for glyphosate tolerance). The affected area is an isolated region surrounded by sierra systems to the northeast and west, with a hot semi-arid climate, long frost-free period, warm winters, hot dry summers, and woody shrubs as native flora. To manage and mitigate the development of resistance, joint actions were taken by the industry, growers and Governmental Agencies. Hybrids expressing Vip3A protein (event MIR162) and/or Cry1Ab protein (events MON 810 and Bt11) as single or stacked events are used in early plantings to control the first generations of D. saccharalis, and in later plantings date's technologies with good control of S. frugiperda. A commitment was made to plant the refuge, and pest damage is monitored. As a result, maize production in the area is sustainable and profitable with yields above the average.

Identification of Major Rhizobacterial Taxa Affected by a Glyphosate-Tolerant Soybean Line via Shotgun Metagenomic Approach.

The worldwide commercial cultivation of transgenic crops, including glyphosate-tolerant (GT) soybeans, has increased widely during the past 20 years. However, it is accompanied with a growing concern about potential effects of transgenic crops on the soil microbial communities, especially on rhizosphere bacterial communities. Our previous study found that the GT soybean line NZL06-698 (N698) significantly affected rhizosphere bacteria, including some unidentified taxa, through 16S rRNA gene (16S rDNA) V4 region amplicon deep sequencing via Illumina MiSeq. In this study, we performed 16S rDNA V5–V7 region amplicon deep sequencing via Illumina MiSeq and shotgun metagenomic approaches to identify those major taxa. Results of these processes revealed that the species richness and evenness increased in the rhizosphere bacterial communities of N698, the beta diversity of the rhizosphere bacterial communities of N698 was affected, and that certain dominant bacterial phyla and genera were related to N698 compared with its control cultivar Mengdou12. Consistent with our previous findings, this study showed that N698 affects the rhizosphere bacterial communities. In specific, N698 negatively affects Rahnella, Janthinobacterium, Stenotrophomonas, Sphingomonas and Luteibacter while positively affecting Arthrobacter, Bradyrhizobium, Ramlibacter and Nitrospira.

Non-destructive Determination of Shikimic Acid Concentration in Transgenic Maize Exhibiting Glyphosate Tolerance Using Chlorophyll Fluorescence and Hyperspectral Imaging.

The development of transgenic glyphosate-tolerant crops has revolutionized weed control in crops in many regions of the world. The early, non-destructive identification of superior plant phenotypes is an important stage in plant breeding programs. Here, glyphosate-tolerant transgenic maize and its parental wild-type control were studied at 2, 4, 6, and 8 days after glyphosate treatment. Visible and near-infrared hyperspectral imaging and chlorophyll fluorescence imaging techniques were applied to monitor the performance of plants. In our research, transgenic maize, which was highly tolerant to glyphosate, was phenotyped using these high-throughput non-destructive methods to validate low levels of shikimic acid accumulation and high photochemical efficiency of photosystem II as reflected by maximum quantum yield and non-photochemical quenching in response to glyphosate. For hyperspectral imaging analysis, the combination of spectroscopy and chemometric methods was used to predict shikimic acid concentration. Our results indicated that a partial least-squares regression model, built on optimal wavelengths, effectively predicted shikimic acid concentrations, with a coefficient of determination value of 0.79 for the calibration set, and 0.82 for the prediction set. Moreover, shikimic acid concentration estimates from hyperspectral images were visualized on the prediction maps by spectral features, which could help in developing a simple multispectral imaging instrument for non-destructive phenotyping. Specific physiological effects of glyphosate affected the photochemical processes of maize, which induced substantial changes in chlorophyll fluorescence characteristics. A new data-driven method, combining mean fluorescence parameters and featuring a screening approach, provided a satisfactory relationship between fluorescence parameters and shikimic acid content. The glyphosate-tolerant transgenic plants can be identified with the developed discrimination model established on important wavelengths or sensitive fluorescence parameters 6 days after glyphosate treatment. The overall results indicated that both hyperspectral imaging and chlorophyll fluorescence imaging techniques could provide useful tools for stress phenotyping in maize breeding programs and could enable the detection and evaluation of superior genotypes, such as glyphosate tolerance, with a non-destructive high-throughput technique.

Glyphosate application increased catabolic activity of gram-negative bacteria but impaired soil fungal community.

Glyphosate is a non-selective organophosphate herbicide that is widely used in agriculture, but its effects on soil microbial communities are highly variable and often contradictory, especially for high dose applications. We applied glyphosate at two rates: the recommended rate of 50mg active ingredientkg-1 soil and 10-fold this rate to simulate multiple glyphosate applications during a growing season. After 6months, we investigated the effects on the composition of soil microbial community, the catabolic activity and the genetic diversity of the bacterial community using phospholipid fatty acids (PLFAs), community level catabolic profiles (CLCPs), and 16S rRNA denaturing gradient gel electrophoresis (DGGE). Microbial biomass carbon (Cmic) was reduced by 45%, and the numbers of the cultivable bacteria and fungi were decreased by 84 and 63%, respectively, under the higher glyphosate application rate. According to the PLFA analysis, the fungal biomass was reduced by 29% under both application rates. However, the CLCPs showed that the catabolic activity of the gram-negative (G-) bacterial community was significantly increased under the high glyphosate application rate. Furthermore, the DGGE analysis indicated that the bacterial community in the soil that had received the high glyphosate application rate was dominated by G- bacteria. Real-time PCR results suggested that copies of the glyphosate tolerance gene (EPSPS) increased significantly in the treatment with the high glyphosate application rate. Our results indicated that fungi were impaired through glyphosate while G- bacteria played an important role in the tolerance of microbiota to glyphosate applications.

Overexpressing Exogenous 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Genes Increases Fecundity and Auxin Content of Transgenic Arabidopsis Plants

Transgenic glyphosate-tolerant plants overproducing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) may exhibit enhanced fitness in glyphosate-free environments. If so, introgression of transgenes overexpressing EPSPS into wild relative species may lead to increased competitiveness of crop-wild hybrids, resulting in unpredicted environmental impact. Assessing fitness effects of transgenes overexpressing EPSPS in a model plant species can help address this question, while elucidating how overproducing EPSPS affects the fitness-related traits of plants. We produced segregating T2 and T3 Arabidopsis thaliana lineages with or without a transgene overexpressing EPSPS isolated from rice or Agrobacterium (CP4). For each of the three transgenes, we compared glyphosate tolerance, some fitness-related traits, and auxin (indole-3-acetic acid) content in transgene-present, transgene-absent, empty vector (EV), and parental lineages in a common-garden experiment. We detected substantially increased glyphosate tolerance in T2 plants of transgene-present lineages that overproduced EPSPS. We also documented significant increases in fecundity, which was associated with increased auxin content in T3 transgene-present lineages containing rice EPSPS genes, compared with their segregating transgene-absent lineages, EV, and parental controls. Our results from Arabidopsis with nine transgenic events provide a strong support to the hypothesis that transgenic plants overproducing EPSPS can benefit from a fecundity advantage in glyphosate-free environments. Stimulated biosynthesis of auxin, an important plant growth hormone, by overproducing EPSPS may play a role in enhanced fecundity of the transgenic Arabidopsis plants. The obtained knowledge is useful for assessing environmental impact caused by introgression of transgenes overproducing EPSPS from any GE crop into populations of its wild relatives.

Effect on transcriptome and metabolome of stacked transgenic maize containing insecticidal cry and glyphosate tolerance epsps genes.

Gene stacking is a developing trend in agricultural biotechnology. Unintended effects in stacked transgenic plants are safety issues considered by the public and researchers. Omics techniques provide useful tools to assess unintended effects. In this paper, stacked transgenic maize 12-5×IE034 that contained insecticidal cry and glyphosate tolerance G10-epsps genes was obtained by crossing of transgenic maize varieties 12-5 and IE034. Transcriptome and metabolome analyses were performed for different maize varieties, including 12-5×IE034, 12-5, IE034, and conventional varieties collected from different provinces in China. The transcriptome results were as follows. The nine maize varieties had obvious differences in gene expression. There were 3561-5538 differentially expressed genes between 12-5×IE034 and its parents and transgenic receptor, which were far fewer than the number of differentially expressed genes in different traditional maize varieties. Cluster analysis indicated that there were close relationships between 12-5×IE034 and its parents. The metabolome results were as follows. For the nine detected maize varieties, the number of different metabolites ranged from 0 to 240. Compared with its parents, 12-5 and IE034, the hybrid variety 12-5×IE034 had 15 and 112 different metabolites, respectively. Hierarchical cluster analysis with Pearson's correlation analysis showed that the differences between 12-5×IE034 and its parents were fewer than those between other maize varieties. Shikimate pathway-related genes and metabolites analysis results showed that the effects of hybrid stacking are less than those from transformation and differing genotypes. Thus, the differences due to breeding stack were fewer than those due to natural variation among maize varieties. This paper provides scientific data for assessing unintended effects in stacked transgenic plants.