Search Results (14)

Maize is highly sensitive to water deficit but has high transpiration and biomass production, leading to a substantial water demand. Genetic engineering can overcome reproductive isolation and utilize drought-tolerant genes from distant species. Ammopiptanthus nanus is a relic of the Tertiary glaciation that can adapt to harsh environmental conditions. In our previous study, five maize homozygous T₈ lines overexpressing the AnVP1 gene from Ammopiptanthus nanus were generated and showed the enhancement of drought tolerance. However, the recipient inbred line Zh-1 was poor in yield and agronomic performance. In the present study, the AnVP1 gene was backcrossed from donor parent L10 (one of the five T₈ lines) into recurrent parent Chang 7-2 (one of the elite parents of the commercial hybrid Zhengdan 958). In total, 103 InDel markers were developed and used for assisted background selection. After two generations of foreground selection through glufosinate spraying, the detection of CP4 EPSP MAb1 strips, and the PCR amplification of the AnVP1 gene, along with the similarity of agronomic traits to the recurrent parent, and background selection assisted by these InDel markers, the transgenic AnVP1 gene became homozygous in the BC₂ lines. The average recovery rate of the genetic background of the recurrent parent reached 74.80% in the BC₁ population and 91.93% in the BC₂ population, respectively. The results of RT-PCR and RT-qPCR indicated the stable expression of the AnVP1 gene in the two ultimately selected BC₂F₃ lines, BC₂-36-12 and BC₂-5-15. The drought tolerance of these two BC₂F₃ lines were significantly improved compared to the recurrent parent Chang 7-2, as revealed by their wilting phenotype and survival rate of seedlings. This improvement was related to the enhancement of water-retention ability, as indicated by higher RWC and the reduction in damage, as shown by the decrease in REL, MDA, and H₂O₂ under drought stress. The result of field evaluation in two arid and semi-arid environments indicated that the drought tolerance of Chang 7-2 was significantly improved. This study suggests that the improved Chang 7-2 can be crossed with Zheng 58 to develop the transgenic commercial hybrid Zhengdan 958. Full article

Sugarcane (Saccharum spp.), a major cash crop that is an important source of sugar and bioethanol, is strongly influenced by the impacts of biotic and abiotic stresses. The intricate polyploid and aneuploid genome of sugarcane has shown various limits for conventional breeding strategies. Nonetheless, biotechnological engineering currently offers the best chance of introducing commercially significant agronomic features. In this study, an efficient Agrobacterium-mediated transformation system that uses the herbicide-resistant CP4-EPSPS gene as a selection marker was developed. Notably, all of the plants that were identified by PCR as transformants showed significant herbicide resistance. Additionally, this transformation protocol also highlighted: (i) the high yield of transgenic lines from calli (each gram of calli generated six transgenic lines); (ii) improved selection; and (iii) a higher transformation efficiency. This protocol provides a reliable tool for a routine procedure for the generation of resilient sugarcane plants. Full article

The commercial cultivation of herbicide-resistant (HR) transgenic soybeans (Glycine max L. Merr.) raises great concern that transgenes may introgress into wild soybeans (Glycine soja Sieb. et Zucc.) via pollen-mediated gene flow, which could increase the ecological risks of transgenic weed populations and threaten the genetic diversity of wild soybean. To assess the fitness of hybrids derived from transgenic HR soybean and wild soybean, the F₂ and F₃ descendants of crosses of the HR soybean line T14R1251-70 and two wild soybeans (LNTL and JLBC, which were collected from LiaoNing TieLing and JiLin BaiCheng, respectively), were planted along with their parents in wasteland or farmland soil, with or without weed competition. The fitness of F₂ and F₃ was significantly increased compared to the wild soybeans under all test conditions, and they also showed a greater competitive ability against weeds. Seeds produced by F₂ and F₃ were superficially similar to wild soybeans in having a hard seed coat; however, closer morphological examination revealed that the hard-seededness was lower due to the seed coat structure, specifically the presence of thicker hourglass cells in seed coat layers and lower Ca content in palisade epidermis. Hybrid descendants containing the cp4-epsps HR allele were able to complete their life cycle and produce a large number of seeds in the test conditions, which suggests that they would be able to survive in the soil beyond a single growing season, germinate, and grow under suitable conditions. Our findings indicate that the hybrid descendants of HR soybean and wild soybean may pose potential ecological risks in regions of soybean cultivation where wild soybean occurs. Full article

Various proteins introduced into living modified organism (LMO) crops function in plant defense mechanisms against target insect pests or herbicides. This study analyzed the antifungal effects of an introduced LMO protein, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 (CP4-EPSPS). Pure recombinant CP4-EPSPS protein, expressed in Escherichia coli, inhibited the growth of human and plant fungal pathogens (Candida albicans, C. tropicalis, C. krusei, Colletotrichum gloeosporioides, Fusarium solani, F. graminearum, and Trichoderma virens), at minimum inhibitory concentrations (MICs) that ranged from 62.5 to 250 µg/mL. It inhibited fungal spore germination as well as cell proliferation on C. gloeosporioides. Rhodamine-labeled CP4-EPSPS accumulated on the fungal cell wall and within intracellular cytosol. In addition, the protein induced uptake of SYTOX Green into cells, but not into intracellular mitochondrial reactive oxygen species (ROS), indicating that its antifungal action was due to inducing the permeability of the fungal cell wall. Its antifungal action showed cell surface damage, as observed from fungal cell morphology. This study provided information on the effects of the LMO protein, EPSPS, on fungal growth. Full article

To regulate the degradation of transgenic DNA and lay theoretical foundations for the rational utilization of genetically modified (GM) products, variations in copy numbers and structural characteristics of DNA from GM soybean event GTS 40-3-2 during soybean protein concentrate (SPC) preparation were evaluated. Results showed that defatting and the first ethanol extraction were key procedures inducing DNA degradation. After these two procedures, copy numbers of the lectin and cp4 epsps targets decreased by more than 4 × 10⁸, occupying 36.88–49.30% of the total copy numbers from raw soybean. Atomic force microscopy images visually revealed the degradation of DNA that thinned and shortened during SPC preparation. Circular dichroism spectra suggested a lower helicity of DNA from defatted soybean kernel flour and a conformation transition of DNA from B-type to A-type after ethanol extraction. The fluorescence intensity of DNA decreased during SPC preparation, verifying the DNA damage along this preparation chain. Full article

Sea Island cotton is the best quality tetraploid cultivated cotton in the world, in terms of fiber quality. Glyphosate is a widely used herbicide in cotton production, and the improper use of herbicides has led to pollen abortion in sea island cotton and, consequently, to a dramatic decrease in yield; however, the mechanism remains unclear. In this study, different concentrations (0, 3.75, 7.5, 15, and 30 g/L) of glyphosate were applied to CP4-EPSPS transgenic sea island cotton Xinchang 5 in 2021 and 2022 at Korla, with 15 g/L glyphosate chosen as the suitable concentration. By comparing the paraffin sections of 2–24 mm anthers in the 15 g/L glyphosate treatment group with those in the water control group, we showed that the key period of anther abortion after glyphosate treatment was the formation and development of tetrads, which corresponded to 8–9 mm buds. Transcriptome sequencing analysis of the treated and control anthers revealed a significant enrichment of differentially expressed genes in phytohormone-related pathways, in particular abscisic acid response and regulation pathways. Additionally, after treatment with 15 g/L of glyphosate, there was a significant increase in the amount of abscisic acid in the anthers in the 8–9 mm buds. Further analysis of the differential expression of abscisic acid response and regulatory genes, an abscisic acid response gene GbTCP14 (Gbar_A11G003090) was identified, which was significantly upregulated in buds with 15 g/L glyphosate treatment than the control, and it could be a key candidate gene for the subsequent research involving male sterility induced by glyphosate in sea island cotton. Full article

Insect infestation and weed interference have a seriously negative impact on the growth, yield, and grain quality of maize. In this study, transgenic maize plants harboring three exogenous genes, cry2Ab, vip3A, and cp4epsps, that were constructed into a single T-DNA were developed for protection against insects and weeds. The transgene integration sites on the chromosomes in two transgenic maize events, CVC-1 and CVC-2, were determined using whole genome sequencing and specific PCR detection. As revealed by laboratory insect bioassays, these two transgenic events exhibited strong insecticidal toxicity against three major species of Lepidoptera insects, including Mythimna separata, Helicoverpa armigera, and Spodoptera frugiperda, with mortality rates exceeding 96%, 100%, and 100%, respectively, after six days of infestation. In addition, CVC-1 exhibited a high tolerance to glyphosate under field conditions. The successful expressions of cry2Ab, vip3A, and cp4epsps in various tissues at different developmental stages of CVC-1 were validated at the transcriptional and translational levels using quantitative real-time reverse transcription PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. These findings demonstrated that the transgenic maize CVC-1 developed using this triple gene construct has excellent insect resistance and herbicide tolerance, which may provide a valuable germplasm resource and data support for future maize breeding of insect and weed control. Full article

Genetically modified (GM) crops tolerant to glyphosate have delivered significant economic benefits in farm management. However, the evolution of glyphosate resistance in weeds due to prolonged intensive use of glyphosate poses a serious threat to this weed management system. It is highly desirable in China to deploy dual herbicide-tolerant corn at the very beginning of GM corn release to delay the development of weed resistance to herbicides. Here, we report the creation and characterization of a herbicide-tolerant corn event SCB-29 that expresses both cp4 epsps and bar genes. This transgenic maize is tolerant to glyphosate up to 3600 g a.e. ha⁻¹ and glufosinate up to 3600 g a.i. ha⁻¹, which are quadruple the recommended rates for the two herbicides, respectively. SCB-29 is an event with only a single copy of T-DNA inserted into chromosome 10 of the maize genome. An event-specific PCR detection method was established and three generations of SCB-29 were detected by event-specific PCR suggesting that the transgenes are stably integrated into the maize genome. Analysis of the expression levels of the transgenes among plants of multiple generations by enzyme-linked immunosorbent assays suggested that the expressions are stable over different generations. Moreover, the major agronomic performances of SCB-29 appear to be similar to those of non-transgenic maize, suggesting that SCB-29 is not likely to have yield drag. Therefore, SCB-29 is an excellent herbicide-tolerant candidate to be developed into a commercial herbicide tolerance transgenic event. Full article

ZDAX5 is a variety of herbicide-tolerant maize that contains the modified P450-N-Z1 gene isolated from Cynodon dactylon (L.) Pers. and the cp4 epsps gene isolated from the Agrobacterium tumefaciens strain CP4 and exhibits high tolerances to flazasulfuron and glyphosate under field conditions. Once ZDAX5 corn is available on the market, the evolution of herbicide-resistant weeds will be delayed by applying glyphosate and flazasulfuron to corn fields. Prior to commercialization, it is critical to assess the safety of ZDAX5 maize. Compositional analysis and feed consumption studies in rodents are an important consideration in the safety assessment of genetically modified crops. The nutritional components of ZDAX5 were analyzed and compared with those of its non-transgenic counterpart. The data showed that all the analyzed components in the herbicide-tolerant maize plants were substantially equivalent to those of its non-transgenic counterpart. Furthermore, most of the measured values from ZDAX5 were within the range of values reported for other commercial maize varieties. The sub-chronic feeding trial was carried out with grains from GM, and non-GM maize were independently added into rodent diets at concentrations of 12.5%, 25% and 50%. As a control, another set of rats was fed with a marketed diet. At the end of the 90-day feeding study, no negative effects associated with the consumption of GM maize were found. These results indicate that the herbicide-tolerant maize ZDAX5 is as nutritious and safe as non-transgenic maize. Full article

CP4-EPSPS (Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase) protein showed remarkable thermostability and was highly resistant to proteases, such as trypsin. In order to eliminate the pollution of CP4-EPSPS from the accumulated straws to the surrounding environment during the winter, the present study investigated the extracellular proteases of 21 psychrophilic strains isolated from the south polar region. The results indicated that Stenotrophomonas maltophilia 780 was able to degrade CP4-EPSPS at 18 °C efficiently. Further study indicated that it was able to grow in the extract of Roundup Ready soybean at 18 °C, with CP4-EPSPS degraded to an undetectable level within 72 h. The extracellular proteases of Stenotrophomonas maltophilia 780 are thermo-sensitive, with an optimal temperature of 65 °C. The genomic sequencing result indicated that this strain had more than a hundred putative protease and peptidase coding genes, which may explain its high capability in decomposing CP4-EPSPS. Full article

Due to the overdependence on glyphosate to manage weeds in fallow conditions, glyphosate resistance has developed in various biotypes of several grass weeds, including Chloris virgata Sw. The first case of glyphosate resistance in C. virgata was found in 2015 in Australia, and since then several cases have been confirmed in several biotypes across Australia. Pot studies were conducted with 10 biotypes of C. virgata to determine glyphosate resistance levels. The biotypes were identified as either susceptible, moderately resistant or highly resistant based on the glyphosate dose required to kill 50% of plants. Two glyphosate-susceptible (GS) and two glyphosate-resistant (GR) biotypes were identified by the dose-response study and analyzed for the presence of target-site mutation in the 5–enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. Performance of alternative herbicides to glyphosate as well as the double-knock herbicide approach was evaluated on the two GS (Ch and SGM2) and two GR (SGW2 and CP2) biotypes. Three herbicides, clethodim, haloxyfop and paraquat, were found to be effective (100% control) against all four biotypes when applied at the 4–5 leaf stage. All the sequential herbicide treatments, such as glyphosate followed by paraquat and glufosinate-ammonium followed by paraquat, provided 100% control of all four biotypes of C. virgata. This study identified effective herbicide options for the control of GR C. virgata and showed that target-site mutations were involved in the resistance of two biotypes to glyphosate (SGW2 and CP2). Results could aid farmers in selecting herbicides to manage C. virgata in their fields. Full article

Several analyses of allergen levels have been reported as part of the safety assessment of genetically modified (GM) soybean; however, few comprehensive analyses have included new allergens. Thus, in this study the levels of eight major soybean allergens, including Gly m 7 (a newly reported soybean allergen), were semi-quantitatively detected in six GM soybeans and six non-GM soybeans using antigen-immobilized ELISA and immunoblotting. We also analyzed the IgE-reactivity to these soybeans through immunoblotting, using sera from three soybean-allergic patients. The results showed that there were no significant differences in the levels of the major soybean allergens in the GM and non-GM soybeans. Moreover, there were no significant differences in the serum IgE-reactive protein profiles of the patients, as analyzed using immunoblotting. These results indicate that, in general, CP4-EPSPS-transfected GM soybeans are not more allergenic than non-GM soybeans. Full article

Glyphosate is the most important herbicide globally, and horseweed (Conyza canadensis) has been one of the most commonly encountered weed species that has developed resistance to it in various parts of the world, including Greece. After glyphosate application, horseweed populations show a wide range of phenotypic plasticity in response to selection pressure. In previous work, we have proposed a herbicide resistance mechanism that is not due to a point mutation at the codon 106 of EPSP synthase but most likely due to a synchronized overexpression of EPSPS and the ABC transporter genes. In the current study, it is hypothesized that the observed phenotypic alterations and differential expression of the EPSPS gene could be attributed to epigenetic changes. DNA methylation plays a pivotal role in many biological procedures such as gene expression, differentiation, and cellular proliferation. Sodium bisulfite sequencing was used to detect epigenetic changes that occur at the C5 position of cytosine residues within CpGdi nucleotides in two horseweed populations (resistant vs. susceptible). Results show differential methylation pattern between the two populations. This work will elucidate the naturally increased resistance of C. Canadensis to glyphosate and set the bases for future development of techniques that restrict weed resistance to herbicides. Full article

With the widespread use of Roundup Ready soya (event 40-3-2) (RRS), the traceability of transgenic components, especially protein residues, in different soya-related foodstuffs has become an important issue. In this report, transgenic components in commercial soya (including RRS) protein concentrates were firstly detected by using polymerase chain reaction (PCR) and western blot. The results illustrated the different degradation patterns of the cp4-epsps gene and corresponding protein in RRS-derived protein concentrates. Furthermore, western blot was applied to investigate the single factor of food processing and the matrix on the disintegration of CP4-EPSPS protein in RRS powder and soya-derived foodstuffs, and trace the degradation patterns during the food production chain. Our results suggested that the exogenous full length of CP4-EPSPS protein in RRS powder was distinctively sensitive to various heat treatments, including heat, microwave and autoclave (especially), and only one degradation fragment (23.4 kD) of CP4-EPSPS protein was apparently observed when autoclaving was applied. By tracing the protein degradation during RRS-related products, including tofu, tou-kan, and bean curd sheets, however, four degradation fragments (42.9, 38.2, 32.2 and 23.4 kD) were displayed, suggesting that both boiling and bittern adding procedures might have extensive effects on CP4-EPSPS protein degradation. Our results thus confirmed that the distinctive residues of the CP4-EPSPS component could be traced in RRS-related foodstuffs. Full article