A new method to identify and improve the purity of hybrid rice with herbicide resistant gene

There is a close relationship between the hy—brid rice production and seed purity.Two-linehybrid rice with higher heterosis is producedthrough the hybridization between a photo-thermo sensitive genetic male sterile(GMS) rice line and a paternal variety.But the fertili-

Recent Advances in Development of Herbicide Resistant Transgenic Hybrid Rice in China

In addition to weed control in direct seeding field of hybrid rice, herbicide resistance genes were used by Chinese scientists to increase and identify the purity of hybrid seeds, and to realize the mechanization of hybrid seed production. The elite restorer lines, such as Minghui 63, R752, T461, R402, D68 and E32 were transformed directly with herbicide resistance genes, in which D68 and E32 are restorer lines of two-line system and the others are of three-line system. Because almost all of important restorer lines are indica varieties and are recalcitrant in transformation, many herbicide resistant near-isogenic restorer lines were developed by sexual hybridization of indica and japonica varieties and backcross with indica restorer lines later, such as Ce 64, Minghui 63, Teqing, Milyang 46, R402 and 9311, in which 9311 is a restorer line of two-line system. The elite photoperiod-sensitive/thermo-sensitive genic male sterile lines, such as Pei＇ai 64S, P88S, 4008S and 7001S, were transformed with herbicide resistance genes. A few herbicide resistant male sterile lines were developed through sexual hybridization and subsequently systemic selection, such as Bar1259S, Bar2172S, 05Z221A and 05Z227A. With the employment of herbicide resistant male sterile lines or herbicide resistant restorer lines, a few herbicide resistant hybrid rice combinations were developed, such as Xiang 125S/Bar 68-1 and Pei＇ai 64S/Bar 9311. Based on herbicide resistance, the research was marching on to investigate the parental lines of hybrid rice with insect resistance, drought tolerance, etc.

Scarabaeid Larvae- and Herbicide-Resistant Transgenic Perennial Ryegrass (Lolium perenne L.) Obtained by Agrobacterium tumefaciens-Mediated Transformation of cry8Ca2, cry8Ga and bar Genes

Insect pest and weeds are two major problems for forage and turf grasses. In this study, scarab larvae- and herbicide-resistant transgenic perennial ryegrass （Lolium perenne L.） was obtained by transforming it with cry and bar genes simultaneously via the Agrobacterium-mediated method. To optimize the callus induction and plant regeneration conditions, various concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine were assayed. The transformation efficiencies of different Agrobacterium suspension media, used during Agrobacterium-mediated transformation, were compared. Then, plasmids of pCAMBIA3301 containing cry gene （cry8Ca2 or cry8Ga） and bar gene, driven by ubiquitin promoter, were transformed into perennial ryegrass. The transformants were generated and confirmed by both Southern hybridization analysis and Western hybridization analysis. Further, the resistance of transgenic perennial ryegrass plants to scarab larvae and herbicide were analyzed. After 30 d of co-cultivation with scarab larvae, the damage to the root system of transgenic plants was less than that of non-transgenic control plants. Additionally, the leaves of transgenic plants were resistant to Basta, while leaves of the wild plants wilted after Basta spraying. These results show that cry gene and bar gene were successfully transferred into perennial ryegrass by the Agrobactgerium-mediated method, and convey resistance to scarab larvae and herbicide in transgenic perennial ryegrass plants.

Developing herbicide resistant Sri-lankan rice(Oryza sativa L.)varieties:An application of Self Organizing Map

Application of high concentrations of post-emergent broad spectrum systemic herbicide,glyphosate is prevalently used to control rice weeds in Asian countries including Sri Lanka.Off target movements of glyphosate adversely affect growth of the rice plant reducing the yield.Inducing herbicide resistance(HR)in cultivated rice is a novel approach to enhance selectivity and crop safety.Studies on induced HR in Sri Lankan rice varieties are limited and studies are required to include HR rice in a cropping program.Ethyl Methyl Sulfonate(EMS),a chemical mutagen is used for functional mutations.The present study is an attempt of raising HR rice lines through conventional breeding methods.A suitable glyphosate concentration was determined by a preliminary study using five rice varieties(Bg300,Bg352,At362,Bg379-2 and H4)and varying glyphosate concentrations(0.25 g/l,0.5 g/l,1 g/l,2 g/l and 3 g/l).Resistance percentage
50%was arbitrary considered as resistant varieties.Complete Randomized Design with three replicates was used in the experiment.Agromorphological characters were recorded for the survived plants.Twelve varieties(Bg359,At362,Bw364,Ld365,Bg366,Bg369,Bg379-2,Bg403,Bg454“Pachcha perumal”,“Kalu heenati”and“Kurulu thuda”)showed natural resistance to glyphosate and fourteen varieties have increased resistance after mutagenesis by EMS(S0-First generation).HR resistance percentage of S1(Second generation)plants was similar to S0 plants indicating HR was transferred to new generation.Conventional statistics was supplemented with Self-Organizing Maps(SOM)to visualize variation of agro-morphological characters under treatment.The result proved that EMS application is an effective method in breeding new rice germplasm with HR and SOM is an important tool for visualizing the multi-dimensional dataset in lower dimensions.

Pyramiding of multiple genes generates rapeseed introgression lines with clubroot and herbicide resistance, high oleic acid content, and early maturity

Clubroot and herbicide resistance,high oleic acid(OA)content,and early maturity are targets of rapeseed(Brassica napus L.)breeding.The objective of this study was to develop new male-fertility restorer lines by pyramiding favorable genes to improve these traits simultaneously.Seven elite alleles for the four traits were introduced into the restorer line 621R by speed breeding with marker-assisted and phenotypic selection.Six introgression lines(ILs)were developed with four-to seven-gene combinations and crossed with two elite parents to develop hybrids.All ILs and their corresponding hybrids displayed high resistance to both clubroot pathotype 4 and sulfonylurea herbicides.Three ILs and their hybrids showed large increases in OA contents and four showed earlier maturity.These new ILs may be useful in rapeseed hybrid breeding for the target traits.

Development of herbicide resistance genes and their application in rice

Rice is one of the most important food crops in the world.Weeds seriously affect the rice yield and grain quality.In recent years,there are tremendous progresses in the research and application of herbicideresistant genes in rice worldwide.This article reviews the working mechanisms of six herbicides(glyphosate,glufosinate,acetolactate synthase inhibitor herbicides,acetyl-Co A carboxylase inhibitor herbicides,hydroxyhenylpyruvate dioxygenase(HPPD)inhibitor herbicides and dinitroaniline herbicides),the resistance mutations of the corresponding herbicide-target genes,and the herbicide detoxification mechanisms by non-target genes.Examples are provided on herbicide-resistant rice materials obtained by transformation of exogenous resistance genes,by artificial mutagenesis and mutant screening,and by modifying the target genes through gene editing.This paper also introduces the current application of herbicide-resistant rice,points out problems that may be caused by utilization of herbicide resistant rice and solutions to the problems,and discusses the future prospects for the development of herbicideresistant rice.

Functional and numerical responses of Cyrtorhinus lividipennis to eggs of Nilaparvata lugens are not affected by genetically modified herbicide-tolerant rice

To safely and sustainably utilize genetic breeding techniques for crop production, greater understanding of the potential effects of genetically modified herbicide-tolerant（GMHT） crops on the ecological functions of predators is required. In the laboratory, we examined the functional and numerical responses of Cyrtorhinus lividipennis Reuter to eggs of brown planthopper（BPH）, Nilaparvata lugens（St？l）, which were reared on GMHT rice Bar68-1; the untransformed parental cultivar, D68; or a BPH-susceptive rice variety, Taichung Native 1. All stages of nymphs and female adult of C. lividipennis, either on GMHT rice or control plants, exhibited typical type II functional responses when fed on BPH eggs; the attacking rate and handling time of C. lividipennis on GMHT rice Bar68-1 was not significantly different from that on D68. The numerical responses of C. lividipennis on GMHT rice or controls fit Beddington＇s model; there were no significant differences in the parameters of numerical responses between GMHT rice Bar68-1 and D68. The results indicated that the functional and numerical responses of C. lividipennis to BPH eggs are not affected by GMHT rice Bar68-1.

Inheritance and molecular characterization of resistance to AHAS- inhibiting herbicides in rapeseed

Rapeseed is a very important oil crop in China; however, its production is challenging due to the absence of effective weed management strategies. This is predominantly because of a shortage of herbicide resistance genes. Acetohydroxyacid synthase （AHAS） herbicides inhibit AHAS, a key enzyme involved in branched-chain amino acid synthesis that is required for plant growth. A rapeseed line designated M342 with AHAS herbicide resistance was developed through seed muta- genesis and was studied to assess the level and mode of inheritance of the resistance and to identify the molecular basis of resistance. M342 possessed a high level of cross-resistance to sulfonylureas （SUs） and imidazolinones （IMIs）. This resistance was due to AHAS insensitivity to these herbicides and was inherited as a dominant trait conferred by a single nuclear-encoded gene. Molecular analysis revealed the presence of a Trp574Leu mutation in M342, and an allele-specific cleaved amplified polymorphic sequence （AS-CAPS） marker was developed and cosegregated with herbicide resistance in the F2, BC1, and BC2 populations. This mutation altered the transcript levels of BnAHAS1 and BnAHAS3 in M342 compared with those in the wild type, but it did not affect the agronomic or quality traits. The simple genetic inheritance of this mutation and the availability of the cleaved amplified polymorphic sequence （CAPS） marker and herbicide resistance gene should facilitate the development of herbicide-resistant rapeseed cultivars for effective weed control in China.

SYBR~ Green qPCR Screening Methods for Detection of Anti-herbicide Genes in Genetically Modified Processed Products

The use of genetically modified organisms （GMOs） as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of GMOs are necessary in order to verify compliance with labelling requirements. There are few effective screening methods for processed GM （genetically modified） products. Three anti-herbicide genes （CP4- EPSPS, BAR and PAT） are common exogenous genes used in commercialized transgenic soybean, maize and rice, In the present study, a new SYBR Green qPCR screening method was developed to simultaneously detect the three exogenous anti-herbicide genes and one endogenous gene in a run. We tested seven samples of representative processed products （soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, maize protein powder, maize starch, and maize jam） using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products, and the sensitivity was 0.1%. These results indicated that SYBR Green qPCR screening method was appropriate for qualitative detection of transgenic soybean, maize and rice in processed products.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

Genetics, Development, and Application of Cytoplasmic Herbicide Resistance in Foxtail Millet

The effect of cytoplasmic herbicide resistant gene in millet plants was studied. The heterozygous populations and isogenic lines with homocaryotic alloplasmic genes were obtained by crossing and reciprocal crossing of cytoplasmic herbicide resistant plants with susceptive plants of foxtail millet. The characters of F1, F2, backcross and composite cross groups, and the growth and development of isogenic lines were compared. The cytoplasmic herbicide resistant gene slowed the development of seedling, delayed heading, and shortened the milking stage in the foxtail millet plant. Yield capacity and main agronomic characters were all affected by the cytoplasmic herbicide resistant gene in most of the backcross, composite cross, and F2 populations. However, there was stronger hybrid vigor in F1. The backcrosses, composite crosses, and F2 populations were widely separated and some of them had good characters similar to those of susceptive groups. The plant characters and development of foxtail millet were negatively affected by the cytoplasmic herbicide resistant gene. The authors proposed a method of using hybrid vigor to obtain high yield and avoid the negative effects of herbicide resistance cytoplasm in plant growth. The expected results could be obtained by selecting individuals in separate populations of fast developed seedlings, well-developed roots, and with capacities of early heading and fast milking. Guided by the principal mentioned above, many high yield lines and hybrid crosses of foxtail millet with herbicide resistant cytoplasm were obtained.

Control of Glyphosate-Resistant Giant Ragweed (Ambrosia trifida L.) with Isoxaflutole and Metribuzin Tankmix

Five field trials were conducted over a two-year period (2013, 2014) to determine the control of glyphosate-resistant (GR) giant ragweed with isoxaflutole (IFT) and metribuzin (MTZ) applied alone and in combination. Treatments were designed to assess the dose response of an IFT plus MTZ tank-mix as well as each chemical applied alone to classify the response using Flint’s adaptation of Colby’s equation. Two factor factorial experiments were performed in the growth room to ascertain the response of IFT versus glyphosate, IFT versus MTZ, and IFT plus MTZ versus glyphosate on single plants. Field experiments evaluated the control of GR giant ragweed with IFT plus MTZ in tank-mix in a 1:4 ratio. The rate of IFT plus MTZ for 80% control of GR giant ragweed at 4 and 8 weeks after application (WAA) was 518 (104 g a.i. ha-1 IFT + 414 g a.i. ha-1 MTZ) and 631 g a.i. ha-1 (126 g a.i. ha-1 IFT + 505 g a.i. ha-1 MTZ), respectively. A rate of 668 and 467 g a.i. ha-1 was required to reduce GR giant ragweed density and biomass by 80%, respectively. Field experiments evaluating the control of GR giant ragweed with tank-mixes of IFT plus MTZ, where glyphosate was a constant tank-mix partner, were mostly synergistic. However, the low tank-mix rate (52.5 + 210 g a.i. ha-1) had an additive response for GR giant ragweed biomass reduction. When tested in the greenhouse and growth room, glyphosate susceptible (GS) giant ragweed showed some antagonism with glyphosate and isoxaflutole tank-mixes at rates less than commercial field rates. GR giant ragweed showed an additive response across all treatments in the growth room. Greenhouse experiments evaluating IFT versus MTZ and IFT plus MTZ versus glyphosate revealed all tank-mix treatments to be synergistic at 2 WAA.

Analysis of a Critical Residue Determining Herbicide Efficiency Sensitivity in Carboxyltransferase Domain of Acetyl-CoA Carboxylase from Poaceae by Homology Modeling and Free Energy Simulation

Carboxyltransferase domain（CT） of acetyl-coenzyme A carboxylase（ACCase, EC 6.4.1.2） from a family of Poaceae is an important target of commercial herbicide APPs for controlling grass weed growth. As the abuse of APPs herbicides, the resistant ACCase due to the mutation of a single residue（Ile→Leu）, which is located in CT active site, is emergent in many populations and species of Poaceae. So it is urgent to understand the resistant mechanism so as to design new effect herbicides. Herein lies the complex of CT dimmer from Lolium rigidum and herbicide haloxyfop successfully constructed for wild type enzyme and Ile/Leu mutant, respectively, providing a basis for explaining the resistance from microscopic structure. Moreover, the binding free energy difference between wild type and mutant enzymes was predicted in good agreement with the known observation, and the various contributions to it were analyzed, by Molecular mechanics-Poisson-Boltzmann surface area（MM-PBSA） method. The results indicate the van der Waals interaction difference between the protein and inhibitor, -22.94 kJ/mol of CT wild type lower than that of mutant, is the major reason for resistance. Structure analysis further suggests that van der Waals interaction difference is originated from the steric hindrance between the side chain of mutated residue Leu and the chiral methyl group of haloxyfop. All these findings enhance the understanding of resistant mechanism of ACCase to herbicide by Ile/Leu mutation and provide an important clue for the rational design of high effective herbicides.

Allelopathic Potential of Wheat on Sourgrass Resistant to Glyphosate

We aimed with this study to assay the allelopathic potential of wheat genotypes in inhibiting germination and initial growth of sourgrass (Digitaria insularis) resistant to the herbicide glyphosate. Two experiments were installed under greenhouse conditions: the first one aimed to assay the potential of wheat genotypes in inhibiting emergence and initial growth of two test plants, and the second aimed to evaluate the potential of those wheat genotypes which performed better at the first experiment, inhibiting the emergence and initial growth of sourgrass biotypes resistant to the herbicide glyphosate. Five doses of aqueous cool-extracted extract from 32 wheat genotypes were applied to tomato and cucumber (Experiment 1) aiming to identify the ones with higher inhibitory effect. The five most inhibitory wheat genotypes were selected for Experiment 2, where the same doses of extract were applied on sourgrass. For both experiments, shoot fresh and dry mass, as well as water content, were assessed. Sourgrass germination is affected by wheat extracts, but initial seedling growth seems not to be affected;wheat genotypes differ in terms of their allelopathic potential in inhibiting sourgrass germination;wheat presents little inhibitory effect on sourgrass.

Inheritance study on the stable herbicide resistance of transgenic rice

The transgene technology showed a potentiali-ty in crop improvement such as disease and in-sect resistance,anti-adversity,and grain qual-ity.The inheritance of bar gene for herbicideBASTA resistance in stable transformed ricelines was studied for an understanding of theforeign gene inheritance pattern. BASTA resistant transgenic rice plantletswere regenerated from the embryos of rice va-rieties JY119 bombarded with bar gene.A sta-ble BASTA resistant TRline was consecutiv-

Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity

The developing trends of livestock production are efficiency availability of phytate phosphorus and abuse of antibiotics. safety and sustainability, which face two major challenges： low As a solution phytases and antimicrobial peptides are applied as feed additives. However, phytases and antimicrobial peptides are susceptible to proteases, costly by fermentation and potential toxic to production hosts. We transformed an optimized phytase-lactoferricin fusion gene PhyLfdriven by an en- dosperm-specific promoter Gt13aP and Bar （bialaphos resistance） gene as a selection maker into rice. The Bar and PhyLf genes were integrated into the rice genome, stably inherited and expressed. Their phosphinothricin acetyl transferase （PAT） protein content oftransgenic plants with glufosinate resistance varied between 50.45-93.39 IJg g-l. Fusion protein expressed especially in the seeds of transgenic rice had a summit phytase activity at 32.30 U g-l, which increased by 61.71-fold com- pared to the control/check group （CK） and 7.54-fold compared to un-optimized transgenic plant. The highest inorganic phosphorus （Pi） content of the transgenic seeds reached 13.15 mg g-~, increased by 12.77-fold compared to that of CK. Preliminary antibacterial experiments showed that the enterokinase hydrolysate product of fusion protein could inhibit the growth of Escherichia coil DH5a. These results indicated that the protein PhyLf has the potential to increase availability of feed phytate phosphorus, improve consumer＇s immunity and reduce the use of antibiotics.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

The EPSPS Pro106Ser substitution solely accounts for glyphosate resistance in a goosegrass(Eleusine indica) population from Tennessee, United States

Previous studies have documented the occurrence of glyphosate-resistant （GR） goosegrass （Eleusine indica （L.） Gaertn.） and, in at least some cases, resistance is due to an altered target site. Research was performed to determine if an altered target site was responsible for GR in a Tennessee, United States goosegrass population （TennGR）. DNA sequencing revealed a mutation in TennGR plants conferring the Prol06Ser 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） substitution previously identified in other GR populations. F2 populations were derived from TennGR plants crossed with plants from a glyphosate-susceptible population （TennGS） and analyzed for their response to glyphosate and genotyped at the EPSPS locus. Plants from the F2 populations segregated 1：2：1 sensitive：intermediate：resistant in response to a selec- tive dose of glyphosate, and these responses co-segregated with the EPSPS genotypes （PP106, PS106, and SS106）. To separately investigate the effect of the Prol06Ser substitution on GR, glyphosate dose-response curves and 50% effective dose （EDso） values were compared among the three genotypes and the two parental populations. The SS106 genotype was 3.4-fold resistant relative to the PP106 genotype, identical to the resistance level obtained when comparing the resistant and susceptible parental populations. We conclude that the mutation conferring a Prol06Ser EPSPS mutation is solely responsible for GR in the TennGR goosegrass population.

Alleles contributing to acetyl coenzyme A carboxylase(ACCase)resistance in keng stiffgrass(Pseudosclerochloa kengiana)populations from China

Keng stiffgrass is a grass weed that affects wheat-rice cropping systems in China. The extensive reliance on acetyl coen- zyme A carboxylase （ACCase）-inhibiting herbicides has resulted in keng stiffgrass developing resistance to these herbicides. The objective of this research was to evaluate the resistance level of the putative resistant keng stiffgrass populations to ACCase-inhibiting herbicides and to identify their molecular resistance mechanism. Whole-plant dose-response experi- ments demonstrated that SD-4 （R）, SD-11 （R）, and JS-25 （R） populations were highly resistant to fenoxaprop, clodinafop, and fluazifop, moderately resistant to diclofop, had low resistance to sethoxydim and pinoxaden, but were sensitive to clethodim. Partial chloroplasticACCase sequences showed that there were two copies ofACCase gene in keng stiffgrass, and all homoeologous genes were expressed. The results of sequence analyses of the ACCase CT domain revealed an isoleucine-to-asparagine substitution at position 2041 in SD-4 （R） and SD-11 （R） populations, and a tryptophan-to-cysteine substitution at position 2027 in the JS-25 （R） population. To our knowledge, this is the first report of Ile-2041-Asn and Trp- 2027-Cys mutations in ACCase-resistant keng stiffgrass. In addition, three robust （derived） cleaved amplified polymorphic sequence （（d）CAPS） markers have been developed to rapidly identify these mutations in the ACCase gene of keng stiffgrass.

CRISPR/Cas9-based Editing of Acetyl-CoA Carboxylase (ACC1) Gene in Barley

Plastid localized acetyl-CoA carboxylase(ACCase;EC 6.4.1.2)is a target for aryloxyphenoxypropionates(APPs)and cyclohexanediones(CHDs),two groups of selective herbicides used in controlling grassy weeds.Wheat and barley are important cereal crops in the grass(Poaceae or Gramineae)family,and thus sensitive for those herbicides.Characterization of this form of ACCase(or ACC1)in wheat and barley is essential if these agents are used in the sustainable agriculture.In this study,it was confirmed that a single ACC1 gene presented on the second chromosome per homologous group in common wheat,wild emmer wheat,goat grass and barley.Using CRISPR/Cas9,the barley ACC1 gene was edited,specifically in the carboxyl transferase(CT)domain that was critical for herbicide responses in grass species.Two new alleles were generated,one with a 3-bp deletion leading to ACC1:p.Ile1878del and one with a 26-bp deletion causing ACC1:p.Ser2099_Lys2311del.Both were recovered as heterozygotes in the T0 generation.All the seven T0 plants harboring the 3-bp deletion grew normally,but the only T0 plant with 26-bp deletion died at the extension stage(Zadoks 32),probably because there was inadequate ACC1 activity when the plant was big.In the T1 generation,the 3-bp deletion(or Ile1878del)did not impact the edited plants in tiller numbers,tiller height,spike length and spikelet numbers,when compared to the wild-type allele in the non-edited segregants.This study demonstrated that CRISPR/Cas9 was practical to generate single amino acid deletions in the ACC1 protein and the Ile1878 deletion did not compromise plant growth.Unfortunately,the ACC1:p.Ile1878del protein did not confer resistance to the currently tested APP herbicides,including clethodim,haloxyfop,quizalofop-Pethyl and sethoxydim.