A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

Background Herbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds.Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches.At present,no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism(GMO)regulation law.We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate(EMS)mutagenesis,offering an alternative choice for weed management.Results Seeds of an elite cotton cultivar Lumianyan 37(Lu37)were treated with EMS,and a mutant Lu37-1 showed strong tolerance to imidazolinone(IMI)herbicides was identified.A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase(ALS)(Gh_D10G1253)in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton.The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species.Cleaved amplified polymorphic sequence(CAPS)markers were developed to identify the ALS mutant allele.The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.Conclusion The nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identi-fied mutation in cotton.This substitution mutation has also been identified in the orthologous ALS genes in other crops.This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.

Trp_(548)Met 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 Os ALS gene that have not been reported previously in rice. One of the mutants, with Trp_(548) changed to Met(W_(548)M), 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 S_(627)N(Ser_(627) changed to Asn) that has been deployed for Clearfield rice development, W_(548)M mutant showed high levels of resistance to a broad spectrum of five families of ALSinhibiting 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 W_(548)M mutation provides a valuable resource for breeding of herbicide resistant rice and weed management.

Response of Underseeded Red Clover(Trifolium pratense L.)to Winter Wheat(Triticum aestivum L.)Herbicides as Affected by Application Timing

Underseeding red clover in winter wheat is a beneficial agronomic practice. Still, many growers tend to forgo this approach. One reason is that herbicides used on winter wheat may injure underseeded red clover, reducing its biomass and the subsequent benefits it provides. Therefore, the effect of winter wheat herbicides on underseeded red clover needs to be evaluated. The objectives of this research were to assess the crop tolerance of underseeded red clover to ten winter wheat herbicides used in Ontario, Canada and determine if red clover tolerance differed when the herbicides were applied at various winter wheat growth stages. Experiments were conducted in 2009 and 2010 at four different Ontario locations. Each herbicide treatment was either applied at an early, normal or late timing. Overall, red clover was not affected by herbicides applied at the early timing. The likelihood of herbicides causing injury and reducing biomass of underseeded red clover increased when they were applied at the more advanced winter wheat growth stages. If timing is a constraint, the three herbicides bromoxynil/MCPA, tralkoxydim, and fenoxaprop-pethyl are the safest to use on red clover underseeded to winter wheat. The remaining herbicides 2,4-D, dicamba/MCPA/mecoprop, dichlorprop/2,4-D, thifensulfuron/ tribenuron + MCPA, fluroxypyr + MCPA, pyrasulfotole/bromoxynil, and prosulfuron + bromoxynil are more injurious, with the last two being the most harmful. By having identified the least damaging herbicides on underseeded red clover in winter wheat and the optimal timing for herbicide application, growers are more likely to adopt this beneficial agronomic practice, save on fertilizer costs and improve soil quality.

HTolerance of Pima and Upland cotton to trifloxysulfuron (Envoke) herbicide under field conditions

Trifloxysulfuron(Envoke)is an acetolactate synthase-inhibitor herbicide and can be used to control many broadleaf weeds and nutsedges in cotton production.However,there is a lack of information on genotypic variation in response to the herbicide.In this field study,60 Pima(Gossypium barbadense L.)lines,122 Upland(G.hirsutum L.)lines,and 9 Upland×Pima segregating populations were divided into five tests(18A,18B,18G,18RB,and 18HQ)to evaluate tri-floxysulfuron tolerance at the 7-true leaf stage(42 days after planting)under the same field conditions in 2018.Across the five tests,Pima cotton genotypes tested in this study did not show any visual crop injury based on percentage of plants with chlorosis at 6 days after treatment(DAT),indicating consistent and high levels of trifloxysulfuron toler-ance.However,the response to trifloxysulfuron within Upland cotton is highly variable.While Upland cotton is overall more sensitive to trifloxysulfuron with crop injury up to 80%than Pima cotton,19 lines had injury below 5%including one line with no visual injury,and 19 lines had injury between 5%and 10%.In test 18HQ with 15 transgenic Upland cultivars and 17 non-transgenic Upland lines,the analysis of variance detected a significant genotypic difference.The broad-sense heritability estimates for trifloxysulfuron tolerance based on crop injury at 6 DAT was 0.555,suggesting that trifloxysulfuron tolerance in Upland cotton is moderately heritable.This study represents the first report that Pima cotton and many Upland cotton lines are highly tolerant to trifloxysulfuron with no or little crop injury.

Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing

Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.

Agronomic Efficiency of Herbicide Tolerant Crops in Peninsular India——A Review

Crops made resistant to herbicides by biotechnology are being widely adopted in various parts of the world and several herbicide resistant crops have become available in many countries for commercial cultivation.But in India,the technology of herbicide tolerant crops is in initial stage of field evaluation.Hence,field trials have been carried out to evaluate and consolidate the agronomic advantages of herbicide tolerant transgenic cotton and maize.Herbicide tolerant stacked traits of maize and cotton have been evaluated under Bio-safety Research Level(BRL I)as confined field trials for its agronomic efficiency on weed control and enhanced crop productivity at Tamil Nadu Agricultural University(TNAU),Coimbatore and Punjab Agricultural University(PAU),Ldhiana for many years.In both crops,potassium salt formulation of glyphosate was sprayed at different doses(900,1,350,1,800,2,700,3,600 and 5,400 g a.e./ha twice at 25 days after sowing(DAS)and 60 DAS in cotton and 900,1,800 and 3,600 g a.e./ha at 25 DAS in maize).Evaluation was made on weed control efficiency,phyto-toxicity on crops,yield and economics and carry over effects on the succeeding crops.Application of glyphosate at 2,700 g a.e./ha recorded lower weed density,dry weight and higher weed control efficiency(WCE)in cotton.Post-emergence(POE)glyphosate at 900,1,800 and 3,600 g a.e./ha registered lower weed density,dry weight and higher WCE in transgenic Hishell and 900 M Gold and in 30V92 and 30B11 corn hybrids.Post-emergence application of glyphosate in transgenic maize hybrids did not affect the germination percent,vigour and yield of succeeding green gram in the transgenic maize trials and sunflower,soybean and pearl millet in cotton trials.Phytotoxicity symptoms were not observed in cotton with glyphosate at lower doses viz.,900,1,350,1,800 and 2,700 g a.e./ha.Higher doses viz.3,600 g a.e./ha and 5,400 g a.e./ha were noticed with phytotoxicity symptoms at early stages of herbicide application.Glyphosate applied at 900,1,350,1,800 and 2,700 g a.e./ha recorded more number of bacteria,fungi and actinomycetes compared to atrazine treatments.Higher grain yield was recorded with POE application of glyphosate at 900,1,800 and 3,600 g a.e./ha in Hishell and 900 M Gold transgenic hybrids and higher net return and benefit cost ratio were recorded in glyphosate at 1,800 g a.e./ha in transgenic 900 M Gold in all the four seasons.Post-emergence application of glyphosate at 900 g a.e./ha and 1,800 g a.e./ha registered higher grain yield in transgenic 30V92 and 30B11 corn hybrids.In maize and cotton transgenic crops,post-emergence weed management with glyphosate proved to be the better management option for the control of weeds.

Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

Prime editing(PE)technology enables precise alterations in the genetic code of a genome of interest.PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants,ideally targeting multiple loci simultaneously to realize the collective effects of the edits.Here,we report the development of a modular assembly-based multiplex PE system in rice and demon-strate its efficacy in editing up to four genes in a single transformation experiment.The duplex PE(DPE)system achieved a co-editing efficiency of 46.1%in the T0 generation,converting TFIIAg5 to xa5 and xa23 to Xa23SW11.The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae(Xoo)strains in the T1 generation.In addition,we success-fully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele,achieving a co-editing rate of 57.14%.Furthermore,with the quadruple PE(QPE)system,we edited four genes—two for herbicide tolerance(OsEPSPS1 and OsALS1)and two for Xoo resistance(TFIIAg5 and OsSWEET11a)—using one construct,with a co-editing efficiency of 43.5%for all four genes in the T0 gen-eration.We performed multiplex PE usingfive more constructs,including two for triplex PE(TPE)and three for QPE,each targeting a different set of genes.The editing rates were dependent on the activity of pegRNA and/or ngRNA.For instance,optimization of ngRNA increased the PE rates for one of the targets(OsSPL13)from 0%to 30%but did not improve editing at another target(OsGS2).Overall,our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents,making it feasible for more labs to utilize PE for their editing experiments.Thesefindings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.