Herbicide-resistant Plants Research Articles

Herbicide-resistant plants produced by precision adenine base editing in plastid DNA.

CRISPR-free, protein-only cytosine base editors (CBEs) or adenine base editors, composed of DNA-binding proteins such as zinc finger proteins or transcription activator-like effectors (TALEs) and nucleobase cytosine or adenine deaminases, respectively, enable organellar DNA editing in cultured cells, animals and plants1-4. TALE-linked double-stranded DNA deaminase toxin A (DddAtox)-derived CBEs (DdCBEs) and TALE-linked adenine deaminases (TALEDs) install C-to-T and A-to-G single-nucleotide conversions, respectively, in mitochondria and chloroplasts5-9. Interestingly, whereas TALEDs exclusively induce A-to-G conversions without C-to-T conversions in mammalian mitochondrial DNA10, they often install unwanted C-to-T edits in addition to intended A-to-G edits in plastid DNA7,9,11,12. Here we show that uracil DNA glycosylase (UDG)-fused TALEDs (UDG-TALEDs) minimize C-to-T conversions without reducing the A-to-G editing efficiency and install a mutation in the chloroplast psbA gene that encodes a single-amino-acid substitution (S264G), which confers herbicide resistance in the resulting plants.

Transport of herbicides by PIN-FORMED auxin transporters.

Auxins are a group of phytohormones that control plant growth and development 1. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides 2. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate 3,4. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane and we characterize the molecular determinants of this process using a variety of different substrates as well as protein mutagenesis to control substrate specificity. Finally, we present Cryo-EM structures of Arabidopsis thaliana PIN8 with 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-chlorophenoxyacetic acid (4-CPA) bound. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with substrate binding and transport across the membrane. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results enable development of novel synthetic auxins and for guiding precision breeding of herbicide resistant crop plants.

Identifying mutation region of herbicide-resistant Mutant Quinoa (Chenopodium quinoa Willd.) lines by molecular characterization

Quinoa can be considered as an alternative plant with its features being compatible with unfavorable climate and soil conditions. Moreover, the nutrient content of its seed is great. But there is not a selective herbicide for weeds in quinoa fields. This is a significant problem in the quinoa cultivation. Therefore, it is necessary to develop herbicide-resistant quinoa lines In the study, Tititcaca, a variety of quinoa was used. Sodium azide was used 1.5 mM for mutagenesis. Herbicide-resistant plants were determined by applying herbicide belonging to the IMI group in the M3 generation. These were named ET-6, ET-7, OT-11 and T-103. The 4 mutant lines obtained as a result of seed mutagenesis, and mutant line OT-11 plants occurred in the form of replacement of the cytosine nucleotide with the adenine nucleotide (CA) in the ALS gene. Line ET-6 plants, the thymine nucleotide was replaced with a guanine nucleotide (TG). The mutation occurring in the OT-11 and ET-6 genotypes was evaluated as a transversion-type mutation. A transition type mutation occurred in mutant line T-103 plants as well, and this mutation occurred in the form of substitution of thymine nucleotide with cytosine nucleotide (TC) at nucleotide 1114. According to the results, weed control in agriculture can be done effectively by developing varieties resistant to IMI group herbicides in quinoa plants. Herbicide resistance studies should be continued in the next generations with the ET-6, OT-11, and T-103 lines.

Discrimination between protoporphyrinogen oxidase–inhibiting herbicide-resistant and herbicide-susceptible redroot pigweed (Amaranthus retroflexus) with spectral reflectance

AbstractThe current assays to confirm herbicide resistance can be time- and labor-intensive (dose–response) or require a skill set/technical equipment (genetic sequencing). Stakeholders could benefit from a rapid assay to confirm herbicide-resistant weeds to ensure sustainable crop production. Because protoporphyrinogen oxidase (PPO)-inhibiting herbicides rapidly interfere with chlorophyll production/integrity; we propose a new, rapid assay utilizing spectral reflectance to confirm resistance. Leaf disks were excised from two PPO-inhibiting herbicide-resistant (target-site [TSR] and non–target site [NTSR]) and herbicide-susceptible redroot pigweed (Amaranthus retroflexus L.) populations and placed into a 24-well plate containing different concentrations (0 to 10 mM) of fomesafen for 48 h. A multispectral sensor captured images from the red (668 nm), green (560 nm), blue (475 nm), and red edge (717 nm) wavebands after a 48-h incubation period. The green leaf index (GLI) was utilized to determine spectral reflectance ratios of the treated leaf disks. Clear differences of spectral reflectance were observed in the red edge waveband for all populations treated with the 10 mM concentration in the dose–response assays. Differences of spectral reflectance were observed for the NTSR population compared with the TSR and susceptible populations treated with the 10 mM concentration in the green waveband and the GLI in the dose–response assay. Leaf disks from the aforementioned A. retroflexus populations and two additional susceptible populations were subjected to a similar assay with the discriminating concentration (10 mM). Spectral reflectance was different between the PPO-inhibiting herbicide-resistant and herbicide-susceptible populations in the red, blue, and green wavebands. Spectral reflectance was not distinctive between the populations in the red edge waveband and the GLI. The results provide a basis for rapidly (∼48 h) detecting PPO-inhibiting herbicide-resistant A. retroflexus via spectral reflectance. Discrimination between TSR and NTSR populations was possible only in the dose–response assay, but the assay still has utility in distinguishing herbicide-resistant plants from herbicide-susceptible plants.

Metabolic resistance to acetolactate synthase inhibitors in Beckmannia syzigachne: identification of CYP81Q32 and its transcription regulation.

Frequent herbicide use selects for herbicide resistance in weeds. Cytochrome P450s are important detoxification enzymes responsible for herbicide resistance in plants. We identified and characterized a candidate P450 gene (BsCYP81Q32) from the problematic weed Beckmannia syzigachne to test whether it conferred metabolic resistance to the acetolactate synthase-inhibiting herbicides mesosulfuron-methyl, bispyribac-sodium, and pyriminobac-methyl. Transgenic rice overexpressing BsCYP81Q32 was resistant to the three herbicides. Equally, rice overexpressing the rice ortholog gene OsCYP81Q32 was more resistant to mesosulfuron-methyl. Conversely, an OsCYP81Q32 gene knockout generated using CRISPR/Cas9 enhanced mesosulfuron-methyl sensitivity in rice. Overexpression of the BsCYP81Q32 gene resulted in enhanced mesosulfuron-methyl metabolism in transgenic rice seedlings via O-demethylation. The major metabolite, demethylated mesosulfuron-methyl, was chemically synthesized and displayed reduced herbicidal effect in plants. Moreover, a transcription factor (BsTGAL6) was identified and shown to bind a key region in the BsCYP81Q32 promoter for gene activation. Inhibition of BsTGAL6 expression by salicylic acid treatment in B. syzigachne plants reduced BsCYP81Q32 expression and consequently changed the whole plant response to mesosulfuron-methyl. Sequence polymorphisms in an important region of the BsTGAL6 promoter may explain the higher expression of BsTGAL6 in resistant versus susceptible B. syzigachne plants. Collectively, the present study reveals the evolution of an herbicide-metabolizing and resistance-endowing P450 and its transcription regulation in an economically important weedy plant species.

Effect of rhizosphere bacteria on antioxidant enzymes and some biochemical characteristics of Medicago sativa L. subjected to herbicide stress

Abiotic stresses such as herbicides can affect plant growth and yield. Using herbicide-resistant plant growth-promoting bacteria is a new approach to mitigate these side effects. This study was conducted to evaluate the effect of three native plant growth-promoting bacteria isolated from the Medicago sativa rhizosphere, including Serratia rubidaea (A) and Pseudomonas putida (B), Serratia sp. (C) plus Synorhizobium meliloti (R) and their combinations (AB, AC, BC, ABC, AR, BR, CR, ABR, ACR, BCR, and ABCR) on microbial population, plant biomass, antioxidant enzymes (CAT, APX, and GPX) activities, and hydrogen peroxide and malondialdehyde contents at the presence and absence of imazethapyr herbicide. The results indicated that herbicide application decreased plant biomass but increased microbial population, antioxidant enzymes activities, and the concentrations of hydrogen peroxide and malondialdehyde of all inoculated and non-inoculated plants. Bacterial inoculation in most cases increased microbial population, plant biomass, and antioxidant activities. These increases were more evident under herbicide application. The highest increase in these attributes was achieved by AB, AR, and ABR inoculums in the presence and absence of the herbicide. The microbial population, plant biomass and antioxidant activities were decreased under BC, CR, BCR, and ABCR inoculations. It can be concluded that in addition to growth promotion, these bacteria increase resistance against herbicide stress by controlling free-radical induced oxidative stress and lipid peroxidation through antioxidant enzymes. These findings create new visions in biofertilizer preparation for reducing environmental stresses.

Гибрид подсолнечника Суринат с устойчивостью к трибенурон-метилу

A simple interlinear hybrid of sunflower Surinat was obtained as a part of the breeding and genetic program for developing herbicide-resistant plants. It is highly resistant to the herbicide tribenuron-methyl from the sulfonylurea class. Both parental forms, i.e., the maternal line VA 76 Sur, and the paternal line VA 830 Sur, as well as the hybrid, are homozygous by the Sur gene. The Surinat hybrid belongs to the middle maturity group, has a high yield, resistance to broomrape (races A-E) and downy mildew (race 730), toler-ance to Phomopsis. The period from seedlings ap-pearance to physiological maturity is 120 days, the oil content of seeds 0 47.9 %, yield – 3.41 t / ha. The Surinat hybrid is intended for cultivation in Sumo and ExpressSunтм production systems.

P-Methoxycinnamic acid disturbs cellular respiration and increases the lignification of Euphorbia heterophylla roots

Herbicide-resistant plants evolved rapidly due to the recurrent use of active principles with only a few mechanisms of action. Today, discovering new herbicides is a pressing challenge. Here, the effects of p-methoxycinnamic acid were evaluated on root growth, cellular respiration, and lignification of the wild poinsettia weed (Euphorbia heterophylla L.). In primary roots, p-methoxycinnamic acid decreased root length and the mitotic index, followed by the loss of cell viability. At the cellular level, p-methoxycinnamic acid stimulated KCN-insensitive and KCN- and SHAM-insensitive respiration. Furthermore, it decreased KCN-sensitive respiration and ATP generation, a limiting factor for root growth. Disturbances in cellular respiration increased the H2O2 content and superoxide dismutase, catalase, and soluble peroxidase activity. p-Methoxycinnamic acid increased the activities of phenylalanine ammonia-lyase and cell wall-bound peroxidase. It also increased lignin, ferulic and p-coumaric acids, and cell wall-esterified p-methoxycinnamic acid. Collectively, these findings reveal the phytotoxic potential of p-methoxycinnamic acid on wild poinsettia. Field tests may point to new clues for using this compound as a potential herbicide.

Enzymatic antioxidant defense system and ALA-D enzyme activity in soybean Enlist™ line

Enlist™ technology confers resistance to 2,4-D herbicides, glyphosate, and ammonium glufosinate for soybeans. However, the application of herbicides to plants can generate stress, even in resistant crops. Thus, here we evaluated whether the application of herbicides in transgenic soybean farms, resistant to these herbicides, adversely affects the activity of antioxidant enzymes and porphobilinogen formation by delta-aminolevulinate dehydratase (ALA-D) activity. At seven days after herbicide application, the aerial part of the plants was collected and used to determine the activity of antioxidant enzymes as catalase, ascorbate peroxidase, and guaiacol peroxidase, such as lipid peroxidation levels and the activity of the ALA-D enzyme. At seven days after herbicide application, the aerial part of plants was collect and used to determine the activity of antioxidant enzymes, such as catalase, ascorbate peroxidase and guaiacol peroxidase, as well as the levels of lipid peroxidation and the activity of the ALA-D enzyme. The activity of important antioxidant enzymes involved in the scavenging of reactive oxygen species (ROS) was increased in the soybean cultivar with the Enlist™ technology, while no severe damage to lipids was detected. However, the activity of ALA-D was inhibited, which could potentially impair the formation of porphobilinogen and decrease photosynthetic efficiency. Thus, these results indicate that herbicides can affect the activity of ROS-scavenging enzymes even in transgenic, herbicide-resistant plants.

A Brief Overview for the Development of Herbicide-Resistant Sugarcane Transformation Approaches


 Weeds are the undesirable and unwanted plants in the wrong place in a situation that can significantly decrease the yield of desirable plant such as sugarcane. One solution to tackle this problem is the development of herbicide resistant crops like sugar cane that are highly resistant to several herbicides or broad-spectrum herbicides which is done in most cases by genetic transformation, site-directed mutagenesis, and plant breeding. Sugarcane (Saccharum officinarum) is the 2nd vital cash crop of Pakistan, placed at 6th position in world cane acreage and 15th in sugar production. Hence, sugarcane occupies a crucial position in the economy of Pakistan and acts like a backbone in economic development of country. There are several reasons for the lower sugarcane yield in Pakistan but the most important is the large number of weeds that covered most of the area of sugarcane cultivated fields which results in major loss of sugarcane yield in terms of quality and quantity. Broad spectrum herbicide resistant plants are produced to tackle this issue by the insertion of CP4 EPSPS (Glyphosate tolerant gene; 5-enolpyruvulshikimate-3-phosphate synthase from Agrobacterium tumefaciens strain CP4) into sugarcane which provides them the ability to survive after the spray of glyphosate but weeds do not survive in this condition. The most common herbicide resistant technology is Glyphosate resistant technology which gives broad-spectrum weed control feasibility along with the flexibility in the application time of herbicide. There are numerous methods of transformation which are available now-a-days for insertion of foreign DNA into plant cells like Agrobacterium-mediated transformation, micro-projectile bombardment (gene gun) and protoplast transformation. Glyphosate tolerant gene expression is showed by three sugarcane cultivars (CPF-213, SPF-234 and HSF-240). Transgenic sugarcane plants showed these expressions are most stable against herbicide to control weeds. Several farmers in many countries have rapidly and extensively utilized herbicide-tolerant crops due to low production and labour costs, huge profit, increased weed control and many environmental benefits.

Potential of Biotechnology in Phytoremediation

The accumulation of toxic substances involving the inorganic and organic contaminants in the soil is a global problem. Status of the World's Soil Resources Report (SWSR) recognized soil pollution as one of the main reasons affecting global soils and the ecosystem services provided by them. However, transgenic approaches utilizing the biodegradation capabilities of microbes and mammals into plants pledge an efficient and eco-friendly approach to renewing the environment. An effective method of phytoremediation involves an enhanced rate of pollutant uptake by the plant, followed by the detoxification of the chemicals absorbed or translocated. It also involves the production of genetically modified herbicide-resistant plants for herbicide remediation and exploits the principles of biotechnology and molecular biology for the introduction and improvement of potentially superior genes into plants. This review discusses the various transgenic approaches involved in the phytoremediation of persistent organic pollutants, metals, metalloids, and explosives. Besides, it also focuses on the limitations of transgenics and provides an insight into the future potential of emerging biotechnological tools and techniques in this field.

Potential of Biotechnology in Phytoremediation

The accumulation of toxic substances involving the inorganic and organic contaminants in the soil is a global problem. Status of the World's Soil Resources Report (SWSR) recognized soil pollution as one of the main reasons affecting global soils and the ecosystem services provided by them. However, transgenic approaches utilizing the biodegradation capabilities of microbes and mammals into plants pledge an efficient and eco-friendly approach to renewing the environment. An effective method of phytoremediation involves an enhanced rate of pollutant uptake by the plant, followed by the detoxification of the chemicals absorbed or translocated. It also involves the production of genetically modified herbicide-resistant plants for herbicide remediation and exploits the principles of biotechnology and molecular biology for the introduction and improvement of potentially superior genes into plants. This review discusses the various transgenic approaches involved in the phytoremediation of persistent organic pollutants, metals, metalloids, and explosives. Besides, it also focuses on the limitations of transgenics and provides an insight into the future potential of emerging biotechnological tools and techniques in this field.

The Development of Herbicide Resistance Crop Plants Using CRISPR/Cas9-Mediated Gene Editing.

The rapid increase in herbicide-resistant weeds creates a huge challenge to global food security because it can reduce crop production, causing considerable losses. Combined with a lack of novel herbicides, cultivating herbicide-resistant crops becomes an effective strategy to control weeds because of reduced crop phytotoxicity, and it expands the herbicidal spectrum. Recently developed clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing techniques enable efficiently targeted modification and hold great potential in creating desired plants with herbicide resistance. In the present review, we briefly summarize the mechanism responsible for herbicide resistance in plants and then discuss the applications of traditional mutagenesis and transgenic breeding in cultivating herbicide-resistant crops. We mainly emphasize the development and use of CRISPR/Cas technology in herbicide-resistant crop improvement. Finally, we discuss the future applications of the CRISPR/Cas system for developing herbicide-resistant crops.

Trp548Met mutation of acetolactate synthase in rice confers resistance to a broad spectrum of ALS-inhibiting herbicides

Herbicide resistance in crop plants is valuable for integrated weed management in agriculture. Herbicide resistant rice, in particular, is important to management of weedy rice, a close relative of cultivated rice and a noxious weed prevalent in rice fields that remains challenging to farmers worldwide. Herbicide resistant plants can be obtained through transgenic approach or by mutagenesis of regular plant and screening of mutants with elevated resistance to herbicide. In this study, we conducted ethyl methyl sulfonate mutagenesis (EMS) to elite indica cultivar Huanghuazhan (HHZ) and screened for mutants resistant to imazapic, a herbicide that can inhibit the acetolactate synthase (ALS) in plants. We obtained three mutants of OsALS gene that have not been reported previously in rice. One of the mutants, with Trp548 changed to Met (W548M), was analyzed in more details in this study. This mutation had no negative effect on the plant physiology and morphology as well as rice yield. Compared with the imidazolinone-resistant mutant S627N (Ser627 changed to Asn) that has been deployed for Clearfield rice development, W548M mutant showed high levels of resistance to a broad spectrum of five families of ALS-inhibiting herbicides, in addition to a higher level of resistance to herbicides of the imidazolinone family. The herbicide-resistance was stably inherited by crossing into other rice lines. Thus, the W548M mutation provides a valuable resource for breeding of herbicide resistant rice and weed management.

Transcriptome analysis identified the mechanism of synergy between sethoxydim herbicide and a mycoherbicide on green foxtail

Certain synthetic herbicides can act synergistically with specific bioherbicides. In this study, a sethoxydim herbicide at 0.1× label rate improved biocontrol of herbicide-sensitive green foxtail (Setaria viridis, GFT) by Pyricularia setariae (a fungal bioherbicide agent), but did not change the efficacy on a herbicide-resistant GFT biotype. Reference transcriptomes were constructed for both GFT biotypes via de novo assembly of RNA-seq data. GFT plants treated with herbicide alone, fungus alone and herbicide + fungus were compared for weed-control efficacy and differences in transcriptomes. On herbicide-sensitive GFT, sethoxydim at the reduced rate induced ABA-activated signaling pathways and a bZIP transcription factor 60 (TF bZIP60), while improved the efficacy of biocontrol. The herbicide treatment did not increase these activities or improve biocontrol efficacy on herbicide-resistant plants. An exogenous application of ABA to herbicide-sensitive plants also enhanced bZIP60 expression and improved biocontrol efficacy, which supported the results of transcriptome analysis that identified the involvement of ABA and bZIP60 in impaired plant defense against P. setariae. It is novel to use transcriptome analysis to decipher the molecular basis for synergy between a synthetic herbicide and a bioherbicide agent. A better understanding of the mechanism underlining the synergy may facilitate the development of weed biocontrol.

Identification of resistant clones of Eurasian (Myriophyllum spicatum) and hybrid (Myriophyllum spicatum × Myriophyllum sibiricum) watermilfoil to an operational rate of fluridone

AbstractGenetic assays to identify herbicide-resistant plants are a promising tool to reduce herbicide failures. However, the genetic basis of herbicide resistance is frequently unknown. In clonal weed species, DNA fingerprinting could be a useful tool to identify known resistant versus susceptible genets (clones) that occur in multiple locations, without an immediate need for understanding the genetic mutation(s) conferring resistance. Eurasian watermilfoil (Myriophyllum spicatum L.) and hybrids with native northern watermilfoil (Myriophyllum spicatum × Myriophyllum sibiricum Kom.) are mostly clonal invasive aquatic plants, and the same clones can be found in multiple waterbodies. Previously, a clone was confirmed as resistant to the commonly used herbicide fluridone, and a recent genetic survey in Michigan identified this genotype (MG-237) in at least seven other lakes. We hypothesized that MG-237 collected from different lakes would also exhibit fluridone resistance. However, MG-237 may have accumulated resistance mutations at different times during its spread across Michigan, resulting in fluridone-resistant and fluridone-susceptible MG-237 clones distributed in different lakes. We used a herbicide assay to test the response of several accessions, including MG-237 accessions from multiple lakes, to the Michigan operational rate of 6 µg L−1 fluridone. We found that all accessions of MG-237 exhibited resistance to 6 µg L−1 fluridone. A second genotype (MG-377) was also resistant to 6 µg L−1 fluridone. The rest of the accessions were found to be significantly injured by 6 µg L−1 fluridone. Our results suggest that 6 µg L−1 fluridone would not effectively control waterbodies dominated by MG-237 or MG-377, whereas waterbodies dominated by the other genotypes in our study would likely be controlled. Although more studies are needed to identify the variation in sensitivity of the accessions tested here and the genetic basis of fluridone resistance in Myriophyllum, our results suggest that multilocus genotype data may be an effective tool to identify and track herbicide-resistant genotypes of Myriophyllum in the short term.

Potential Use of Biological Herbicides in a Circular Economy Context: A Sustainable Approach

In recent years, synthetic herbicides’ intense and disordered use has triggered severe contamination of soils and water bodies, causing damage to living organisms, including vegetal herbicide-resistance. In-part, biological herbicides, offer promise in addressing these problems, especially for sustainable weed management in both agricultural and natural resource management contexts. The search for culture media to serve as alternatives to the existing conventional synthetic media has focused on microalgae biomass. The natural properties of these organisms make them ideal raw materials for fermentative processes. Therefore, this study aimed to integrate bioprocesses producing bioherbicidal extract using a submerged fermentation process. For this, a substrate of microalgae recycled biomass to replace commercially used media for fungal growth for relatively cheap biomass obtained from wastewater phycoremediation after anaerobic treatment (UASB). We also verified its potential for weed control. Preliminary tests of microalgae fermentation were conducted in the raw and lyophilized form, using the fungus Trichoderma koningiopsis. To evaluate their phytotoxic potential, the biocompounds were applied to Bidens pilosa, Conyza bonariensis, Urochloa plantaginea, and Euphorbia heterophylla weeds. A central composite rotatable design (CCRD) was used to evaluate the influence of temperature (20–45oC), agitation (0–200 RPM), and pH (3–14) on the improvement of enzyme activities, fungal growth and the effects on Cucumis sativus germination tests. We found that microalgae could be used as a substrate for obtaining bioherbicides. The biocompounds produced in this study were relevant for pre- and post-emergence control of model plants (Cucumis sativus ), with vigor indexes of up to 8.05% and herbicide-resistant plants (Conyza bonariensis) with phytotoxic damage of up to 100% after 15 days. From the experiments’ design, we found that agitation was the variable that had the most significant effect on enzyme and biomass production, followed by pH and temperature. The enzymes also showed synergistic action when acting together with microalgae and fungi, potentiating the control effect on weeds. Finally, the system integrated bioprocesses through agro-industrial effluent recovery and reuse, considered one of the essential economic viability mechanisms by enhancing the development of more sustainable technologies that embrace the principles of a circular economy.

Evaluation of quinclorac toxicity and alleviation by salicylic acid in rice seedlings using ground-based visible/near-infrared hyperspectral imaging

BackgroundTo investigate potential effects of herbicide phytotoxic on crops, a major challenge is a lack of non-destructive and rapid methods to detect plant growth that could allow characterization of herbicide-resistant plants. In such a case, hyperspectral imaging can quickly obtain the spectrum for each pixel in the image and monitor status of plants harmlessly.MethodHyperspectral imaging covering the spectral range of 380–1030 nm was investigated to determine the herbicide toxicity in rice cultivars. Two rice cultivars, Xiushui 134 and Zhejing 88, were respectively treated with quinclorac alone and plus salicylic acid (SA) pre-treatment. After ten days of treatments, we collected hyperspectral images and physiological parameters to analyze the differences. The score images obtained were used to explore the differences among samples under diverse treatments by conducting principal component analysis on hyperspectral images. To get useful information from original data, feature extraction was also conducted by principal component analysis. In order to classify samples under diverse treatments, full-spectra-based support vector classification (SVC) models and extracted-feature-based SVC models were established. The prediction maps of samples under different treatments were constructed by applying the SVC models using extracted features on hyperspectral images, which provided direct visual information of rice growth status under herbicide stress. The physiological analysis with the changes of stress-responsive enzymes confirmed the differences of samples under different treatments.ResultsThe physiological analysis showed that SA alleviated the quinclorac toxicity by stimulating enzymatic activity and reducing the levels of reactive oxygen species. The score images indicated there were spectral differences among the samples under different treatments. Full-spectra-based SVC models and extracted-feature-based SVC models obtained good results for the aboveground parts, with classification accuracy over 80% in training, validation and prediction set. The SVC models for Zhejing 88 presented better results than those for Xiushui 134, revealing the different herbicide tolerance between rice cultivars.ConclusionWe develop a reliable and effective model using hyperspectral imaging technique which enables the evaluation and visualization of herbicide toxicity for rice. The reflectance spectra variations of rice could reveal the stress status of herbicide toxicity in rice along with the physiological parameters. The visualization of the herbicide toxicity in rice would help to provide the intuitive vision of herbicide toxicity in rice. A monitoring system for detecting herbicide toxicity and its alleviation by SA will benefit from the remarkable success of SVC models and distribution maps.

Application of CRISPR/Cas9-mediated gene editing for the development of herbicide-resistant plants

Herbicide resistance is one of the most important traits for plant biotechnology, which is widely used to improve agricultural efficiency by controlling weeds and also used as a selectable marker during genetic engineering by the transformation. For these reasons, new technologies for conferring herbicide resistance to plants are continuously developing, in which CRISPR/Cas9-mediated gene editing methods enable precise modifications of DNA sequences and offer a great promise for the improvement of crops. Here, we review recent advances in developing herbicide-resistant plants by the CRISPR/Cas9-mediated gene editing technology, targeting on endogenous genes such as acetolactate synthase (ALS), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), cellulose synthase A catalytic subunit 3 (CESA3), and splicing factor 3B subunit 1 (SF3B1). In addition, we also introduce possible candidate genes to develop herbicide-resistant plants by generating loss-of-function mutations using the CRISPR/Cas9 system.

Guidelines for C to T base editing in plants: base-editing window, guide RNA length, and efficient promoter

The Cas9 protein fused with a cytidine deaminase can induce C to T substitutions at a specific site when directed by a guide RNA. Here, we compared the substitution activity and the substitution range of two base-editing systems, APOBEC1-nCas9 and nCas9-PmCDA1, in the protoplasts of Glycine max, Brassica napus, and Nicotiana tabacum. To prevent unwanted nucleotide substitution, we manipulated the length of guide RNA and found the change of nucleotide substitution activity in the target window of nCAS9-PmCDA1. Based on these results, the specific C to T conversion in the acetolactate synthase gene of N. tabacum was induced to generate herbicide-resistant plants. During the screening of herbicide-resistant plants, we found that ubiquitin promoter-driven base-editor system was much efficient than 35S promoter-driven base-editor system. This study provides guidelines on which a base editor to use and describes how to fine-tune a guide RNA for precise substitutions in plants.