Study on Antioxidant Enzyme Activity and Physiology and Biochemistry of Solanum nigrum L. under Glyphosate Stress

[Objectives] This study was conducted to investigate the scientific prevention and control of Solanum nigrum L. [Methods] Through experiments on S. nigrum from different sources, it was found that glyphosate stress had significant effects on antioxidant enzyme activity and oxidative damage of sensitive S. nigrum plants. [Results] Sensitive S. nigrum showed oxidative damage under glyphosate stress, while resistant S. nigrum responded to adversity damage by improving its antioxidant enzyme activity. The experimental results showed that the antioxidant enzymes and reduced glutathione of S. nigrum had certain metabolic detoxification effects under glyphosate stress. [Conclusions] This study provides a theoretical basis for scientific prevention and control of S. nigrum , and has a certain reference value for revealing the glyphosate resistance mechanism of S. nigrum .

Size-controlled preparation of hollow silica spheres and glyphosate release

Different-sized hollow SiO2 spheres of 249–1348 nm in diameter were successfully prepared by using Na2SiO3 as the precursor and using polystyrene and polystyrene-methyl acrylic acid latexes as the templates. The diameter and shell thickness of the hollow SiO2 spheres increase with increasing the latex template diameter at a given mass ratio of SiO2 to latex template. The diameter and shell thickness of the hollow SiO2 spheres also increase with increasing the mass ratios of SiO2 to latex template. The presence of carboxylic acid groups on the surfaces of polystyrene-methyl acrylic acid latex templates favors the formation of dense and uniform SiO2 shells. The hollow SiO2 sphere is constructed by mesoporous shell with large specific surface area. When glyphosate is used as a release model chemical, glyphosate release rate is tuned by varying the shell thickness.

Screening for Glyphosate Resistant Wild Soybean(Glycine soja) and Study on Its Physiological Mechanisms of Resistance

[Objective] This study aimed to preliminarily determine the physiological mechanism of glyphosate resistance produced in wild soybean（Glycine soja） and further provide a basis for the breeding of glyphosate-resistant soybean. [Method] First,a screening for glyphosate resistant varieties among sixty-seven wild soybean materials was done in a field trial; subsequently, physiological indexes of the screened resistant variety ZYD0685 and the sensitive variety ZYD0790 were studied. [Result]At the glyphosate dose of 1.23 kg a.i/hm2, glyphoaste resistance varied greatly among different wild soybean materials, with the highest survival rate of 87% and83% occurring in ZYD0685 and ZYD2405, respectively, and that of another seven accessions ranged from 2.7% to 38%, and all the remaining fifty-eight soybean materials died. After treatment with glyphoaste at different doses, there were no significant differences in chlorophyll content and shikimate content in the resistant ZYD0685, but there was an evident increase in the activity of gultathione-S-transferases（GSTs）; while in the sensitive ZYD0790, the content of shikimic acid increased significantly, and chlorophyll content decreased significantly, and GSTs activity revealed a slight change. [Conclusion] Therefore, lowering the amount of accumulated shikimic acid is the major physiological response to glyphosate in wild soybean.

Establishment of PCR-ELISA for Detecting Glyphosate Resistant Transgenic Soybean

A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows： the concentration of digoxin tag probe was 0.5 μmol · L^-1, the time of hybridization reaction was 15 min and the chromogenic reaction should last for 30 min. The sensitivity and the repeatability of our PCR-ELISA method were evaluated, and the results showed that it could be detected when the concentration of DNA template from transgenic soybean samples was 0.01% or higher, and the coefficient of variation of this method was less than 5% in our research condition. These results suggested that PCR-ELISA method establishment in this study had good repeatability and high precision for detecting the transgenic soybean samples.

Selection and Characterization of a Novel Glyphosate Tolerant Upland Cotton(Gossypium hirsutum L.) Mutant(R1098)

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 selection medium and multi-step selection pressure process was carried out until the

Weed and insect control affected by mixing insecticides with glyphosate in cotton

Field studies were conducted in 2012 and 2013 to evaluate weed and insect control efficacy with glyphosate at 1 230 g ai（active ingredient） ha^（-1） and the insecticides acephate（728 g ai ha^（-1））,carbosulfan（135 g ai ha^（-1））,endosulfan（683 g ai ha^（-1））,imidacloprid（32 g ai ha^（-1））,or lambda-cyhalothrin（23 g ai ha^（-1））,as well as glyphosate tank-mixed with these insecticides.Four of the most common weeds in cotton,common purslane,false daisy,goosegrass,and lambsquarters,were manually sown in the cotton field and treated with glyphosate alone or in combination with insecticides.Glyphosate efficacy,based on visual estimates of control and weed fresh weight at 21 d after treatment（DAT）,was unaffected by the addition of insecticides.Four weeds were controlled by 93-97%and 86-100%（visual rating） and reduced weed fresh biomass by98-99%and 96-100%with glyphosate alone and its combination with insecticides,respectively.Addition of glyphosate to acephate improved cotton aphid control compared with acephate alone.However,addition of glyphosate to carbosulfan,endosulfan,imidacloprid,or lambda-cyhalothrin did not affect the aphid control when compared with the insecticide alone treatments.These results indicate that cotton producers could potentially integrate weed and insect management strategies by choosing suitable insecticide mixing partners with glyphosate,thereby reducing the application costs without sacrificing the efficacy of the glyphosate or the insecticides.

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha–1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.

Occurrence of Glyphosate-Resistant Horseweed (Conyza canadensis) Population in China

Horseweed （Conyza canadensis）, an invasive alien weed, is one of the main weeds in orchards in China. Although glyphosate has been used for control of horseweed and many other weeds in orchards for more than 25 years in China, a case of glyphosate-resistant horseweed has not been identified in orchard in China so far despite glyphosate-resistant horseweed cases have been reported in some other countries. Seeds of 25 horseweed populations were collected from different orchards with different glyphosate application history. Potted seedlings with 11-13-leaf growth stage were treated with glyphosate at 0.035, 0.07, 0.14, 0.28, 0.56, 1.12, 2.24, 4.48, and 8.96 kg a.i. ha-1. The dosage dependence response curve of each population was constructed with Log-logistic dose response regression equations. The ED50 value of each population was calculated and compared with the susceptible population from China. Different populations had different relative glyphosate-resistant levels which increased with the number of years of glyphosate application. Two populations with the highest resistance levels, 8.28 and 7.95 times, were found in Ningbo, Zhejiang Province, China, where glyphosate was used for weed control in orchards twice each year for 15 yr. The two resistant populations accumulated approximately two to four times less shikimic acid than the two susceptible populations 48 h after glyphosate application.

Cadmium adsorption in montmorillonite as affected by glyphosate

Behaviors of soil heavy metals are often affected by coexisting herbicides due to their physical and chemical interaction. Effect of glyphosate, an herbicide containing -PO 2- 3 and -COOH groups, on cadmium adsorption in montmorillonite was studied in detail. The results showed that the cadmium adsorption quantity in montmorillonite increased with increasing soil solution pH and cadmium concentration as usual, but decreased with glyphosate, which is due to the formation of a low affinity complex of Cd and glyphosate and decreasing solution pH induced by glyphosate addition. When the equilibrium solution pH was below 6.7, glyphosate has little effect on cadmium adsorption, but when the equilibrium solution pH was above 6.7, glyphosate significantly decreased cadmium adsorption quantity in montmorillonite. In addition, the adding order of Cd and glyphosate also influenced Cd adsorption quantity in montmorillonite.

Effects of phosphate on the adsorption of glyphosate on three different types of Chinese soils

Glyphosate （GPS） is a non-selective, post-mergence herbicide that is widely used throughout the world. Due to the similar molecular structures of glyphosate and phosphate, adsorption of glyphosate on soil is easily affected by coexisting phosphate, especially when phosphate is applied at a significant rate in farmland. This paper studied the effects of phosphate on the adsorption of glyphosate on three different types of Chinese soils including two variable charge soils and one permanent charge soil. The results indicated that Freundlich equations used to simulate glyphosate adsorption isotherms gave high correlation coefficients（0.990-0.998） with K values of 2751, 2451 and 166 for the zhuanhong soil（ZH soil, Laterite）, red soil（ RS, Udic Ferrisol） and Wushan paddy soil（WS soil, Anthrosol）, respectively. The more the soil iron and aluminum oxides and clay contained, the more glyphosate adsorbed. The presence of phosphate significantly decreased the adsorption of glyphosate to the soils by competing with glyphosate for adsorption sites of soils. Meanwhile, the effects of phosphate on adsorption of glyphosate on the two variable charge soils were more significant than that on the permanent charge soil. When phosphate and glyphosate were added in the soils in different orders, the adsorption quantities of glyphosate on the soils were different, which followed GPS-soil 〉 GPS-P-soil = GPS-Soil-P 〉 P-soil-GPS, meaning a complex interaction occurred among glyphosate, phosphate and the soils.

Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes

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.

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Use of chlorophyll fluorescence and P700 absorbance to rapidly detect glyphosate resistance in goosegrass(Eleusine indica)

The rapid detection of glyphosate resistance in goosegrass（Eleusine indica） will enhance our ability to respond to new resistant populations of this major weed. Chlorophyll fluorescence（Fluo） and P700（reaction center chlorophyll of photosystem I） absorbance were analyzed in one biotype of goosegrass that is resistant to glyphosate and in another that remains sensitive to the herbicide. Both biotypes were treated with a foliar spray of glyphosate. Differences in photosystem II maximum quantum yield（Fv/Fm）, effective photochemical quantum yield（Y（II））, and non-photochemical quenching（NPQ） between the biotypes increased over time. Values for Fv/Fm and Y（II） differed between the two biotypes 24 h after treatment（HAT）. Differentiated activities and energy dissipation processes of photosystem II（PSII） and energy dissipation processes of photosystem I（PSI） were manifested in the two biotypes 24 HAT with 20 mmol L–1 glyphosate. Differentiated energy dissipation processes of PSI were still apparent 24 HAT with 200 mmol L–1 glyphosate. These results indicate that the Fluo parameters related to PSII activity and energy dissipation and the P700 parameters related to energy dissipation are suitable indicators that enable rapid detection of glyphosate resistance in goosegrass.

Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate

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 pM3301 Ubi Sp AM79, 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.

Development of glyphosate-tolerant transgenic cotton plants harboring the G2-aroA gene

Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China,the development of glyphosate-resistant cotton holds great promise.Using an Agrobacterium-mediated transformation method,a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS）was transformed into cotton cultivar K312.The transgenic cotton plants were regenerated from a callus tissue culture via kanamycin selection.Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit.The results from polymerase chain reaction（PCR）and Southern and Western blot analyses indicated that the target gene was integrated into the cotton chromosome and was expressed effectively at the protein level.The glyphosate tolerance analysis showed that the transgenic cotton had a high resistance to glyphosate.Further,even cotton treated with 45.0 mmol L^–1 of glyphosate was able to slowly grow,bloom and seed.The transgenic cotton may be used for cotton breeding research of glyphosate-tolerant cotton.

Control of Glyphosate-Resistant Waterhemp with Two-Pass Weed Control Strategies in Glyphosate/Dicamba-Resistant Soybean

Waterhemp is a small-seeded, dioecious, broadleaf weed that emerges throughout the growing season. If left uncontrolled, waterhemp interference can reduce soybean yield up to 73%. Glyphosate-resistant (GR) waterhemp was first discovered in one county in Ontario in 2014;as of 2017, it has been found in two other counties. Glyphosate/dicamba-resistant soybean can be sprayed with glyphosate and/or dicamba preplant (PP), preemergence (PRE) and/or postemergence (POST). The objective of this study was to determine the control of GR waterhemp in glyphosate/dicamba-resistant soybean with PRE residual herbicides, glyphosate/dicamba applied POST or a two-pass program of a PRE residual herbicide followed by glyphosate/dicamba applied POST. At 8 weeks after application (WAA), pyroxasulfone (150 g ai ha-1), S-metolachlor/metribuzin (1943 g ai ha-1), pyroxasulfone/sulfentrazone (300 g ai ha-1) and flumioxazin/pyroxasulfone (240 g ai ha-1), applied PRE, resulted in 71%, 85%, 82% and 90% GR waterhemp control, respectively. The same PRE herbicides, followed by glyphosate/dicamba (1800 g ae ha-1) POST, improved control to greater than 96%. This study concludes that a two-pass program of an effective soil applied residual herbicide followed by glyphosate/dicamba POST controlled GR waterhemp in glyphosate/dicamba-resistant soybean.

Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

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 cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. 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.

Rapid and convenient transformation of cotton (Gossypium hirsutum L.) using in planta shoot apex via glyphosate selection

Cotton plants are recalcitrant with regards to transformation and induced regeneration.In the present study,5-enolpyruvylshikimate-3-phosphate（EPSPS）,a glyphosate resistant gene from the bacterium Agrobacterium sp.strain CP4,was introduced into an elite Bt transgenic cotton cultivar with a modified technique involving in planta Agrobacteriummediated transformation of shoot apex.Primary transformants were initially screened using a 0.26%glyphosate spray and subsequently by PCR analysis.Five out of 4 000 transformants from T_1 seeds were obtained resulting in an in planta transformation rate of 0.125%.Four homozygous lines were produced by continuous self-fertilization and both PCR-based selection and glyphosate resistance.Transgene insertion was analyzed by Southern blot analysis.Gene transcription and protein expression levels in the transgenic cotton lines were further investigated by RT-PCR,Western blot,and ELISA methods.Transgenic T_3 plants were resistant to as much as 0.4% of glyphosate treatments in field trials.Our results indicate that the cotton shoot apex transformation technique which is both tissue-culture and genotype-independent would enable the exploitation of transgene technology in different cotton cultivars.Since this method does not require sterile conditions,the use of specialized growth media or the application of plant hormones,it can be conducted under the greenhouse condition.

Ultrasensitive Detection of Glyphosate Using CdTe Quantum Dots in Sol-Gel-Derived Silica Spheres Coated with Calix[6]arene as Fluorescent Probes

We have developed a simple method for the preparation of highly fluorescent and stable, water-soluble CdTe quantum dots in sol-gel-derived composite silica spheres which were coated with calix[6]arene. The resulting nanoparticles (NPs) were characterized in terms of UV, fluorescence and FT-IR spectroscopy and TEM. The results show that the new NPs display more intense fluorescence intensity and are more stable than its precursors of the type SiO2/CdTe. Under the optimum, the novel NPs exhibit a higher selectivity and ultrasensitive fluorescence probes for the determination of gly-phosate over other pesticides, the fluorescence intensity increase with the concentration of glyphosate in the range from 1.0 to 25.0 nmol/L and the detection limit is low to 0.0725 nmol/L. A mechanism is suggested to explain the inclusion process by a Langmuir binding isotherm.

Development of Transgenic Glyphosate-Resistant Rice with G6 Gene Encoding 5-Enolpyruvylshikimate-3-Phosphate Synthase

Glyphosate-resistant crops have been a huge economic success for genetic engineering. The creating of new glypbosateresistant plants would increase the available choices for planting and lower the price of genetically modified crop seeds. A novel G6 gene from Pseudomonas putida that encoded 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） was previously isolated. The G6 gene was transfected into rice via Agrobacterium-mediated transformation. The transgenic rice obtained was confirmed by PCR, Southern, and Western blots. The lab experiment and field trials further confirmed that the transgenic rice can survive glyphosate spraying at a dose of 8 g L^-1. In contrast, conventional rice was killed at a weed control glyphosate spray dose of 1 g L^-1. Altogether, the present study showed that the G6 gene works well in rice in vivo for glyphosate-resistance.