Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or sulfonylurea herbicides in sunflower

Wild biotypes of cultivated sunflower (Helianthus annuus L.) are weeds in corn (Zea mays L.), soybean (Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides—imidazolinones (IMIs) or sulfonylureas (SUs)—have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes (AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 (Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 (AHAS3), 6 (AHAS2), and 9 (AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant × wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.     

Inheritance and molecular characterization of broad range tolerance to herbicides targeting acetohydroxyacid synthase in sunflower

Ahasl1 is a multilallelic locus where all the induced and natural mutations for herbicide tolerance were described thus far in sunflower (Helianthus annuus L.). The allele Ahasl1-1 confers moderate tolerance to imidazolinone (IMI), Ahasl1-2, and Ahasl1-3 provides high levels of tolerance solely to sulfonylurea (SU) and IMI, respectively. An Argentinean wild sunflower population showing plants with high level of tolerance to either an IMI and a SU herbicide was discovered and used to develop an inbred line designated RW-B. The objectives of this work were to determine the relative level and pattern of cross-tolerance to different AHAS-inhibiting herbicides, the mode of inheritance, and the molecular basis of herbicide tolerance in this line. Slight or no symptoms observed after application of different herbicides indicated that RW-B possesses a completely new pattern of tolerance to AHAS-inhibiting herbicides in sunflower. Biomass response to increasing doses of metsulfuron or imazapyr demonstrated a higher level of tolerance in RW-B with respect to Ahasl1-1/Ahasl1-1 and Ahasl1-2/Ahasl1-2 lines. On the basis of genetic analyses and cosegregation test, it was concluded that tolerance to imazapyr in the original population is inherited as a single, partially dominant nuclear gene and that this gene is controlling the tolerance to four different AHAS-inhibiting herbicides. Pseudo-allelism test permitted us to conclude that the tolerant allele present in RW-B is an allelic variant of Ahasl1-1 and was designated as Ahasl1-4. Nucleotide and deduced amino acid sequence indicated that the Ahasl1-4 allele sequence of RW-B has a leucine codon (TTG) at position 574 (relative to the Arabidopsis thaliana AHAS sequence), whereas the enzyme from susceptible lines has a tryptophan residue (TGG) at this position. The utilization of this new allele in the framework of weed control and crop rotation is discussed.     

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.     

Cross-resistance to short residual sulfonylurea herbicides in transgenic tobacco plants

Transgenic Nicotiana tabacum plants, produced by Agrobacterium tumefaciens-mediated transformation with a mutant gene (csr1-1) coding for acetohydroxyacid synthase (AHAS) from a chlorsulfuron resistant Arabidopsis thaliana line GH50 (GW Haughn et al. [1988] Mol Gen Genet 211: 266-271; GW Haughn, C Somerville [1986] Mol Gen Genet 204: 430-434), were selected directly on 80 micrograms per liter (225 nanomolar) chlorsulfuron. The expression of csr-1 in two separate transgenic lines CHL-1 and CHL-2 was confirmed by biochemical and genetic analyses. The AHAS activity of GH50 and the equivalent component of AHAS activity in CHL-2 was resistant to three short residual sulfonylurea herbicides, DPX-M6316, DPX-A7881, and DPX-L5300, in addition to chlorsulfuron but not to the sulfonylurea CGA 131′036. Cross-resistance to the imidazolinones AC 263, 499, AC 252, 214, and AC 243,997 was not observed. Parallel observations were made on the inhibition of seedling growth in soil or on culture medium. The relevance of these findings for the application of transgenic plants in agriculture is discussed.     

Herbicide Resistance in Datura innoxia: Cross-Resistance of Sulfonylurea-Resistant Cell Lines to Imidazolinones

Cells resistant to the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl were isolated from a predominantly haploid cell suspension culture of Datura innoxia P. Mill. Exponentially growing cell colonies (aggregates of about 40 cells) were mutagenized with ethyl methane sulfonate, subcultured for 10 days to allow growth recovery and plated on a medium containing either chlorsulfuron or sulfometuron methyl at a concentration (10⁻⁸ molar) which killed wild type cells. Surviving clones were picked up after 3 to 4 weeks, further proliferated as callus or cell suspension cultures, and tested for their resistance to both the sulfonylureas and imidazolinones, a chemically different class of herbicides. The variants were stable and showed high (100- to 1000-fold) resistance to the sulfonylureas. While some also exhibited cross resistance to imidazolinones, others showed no cross-resistance at all or, as in one case, greater sensitivity than wild type cells to the imidazolinones. Both classes of herbicides tested inhibited acetolactate synthase activity isolated from wild type cells. The acetolactate synthase of the resistant variants, however, was found to be resistant to the sulfonylureas and also to the imidazolinone(s) in those cells showing cross-resistance to the latter. The lack of cross-resistance observed in some cases provides evidence that the two groups of herbicides have slightly different sites on the acetolactate synthase molecule.     

Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides

The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).Greenhouse pot studies were performed to assess the occurrence of inhibitory effects on bacterial growth in field conditions. Maize seedlings were bacterized with the two strains which had shown in vitro sensitivity to sulfonylureas. Following the application to the soil of a commercial formulation of rimsulfuron at rates of 0, 0.2 and 0.5 mol a.i. kg^–1, significative differences in the resulting degree of bacterial root colonization were found. Moreover, upon co-inoculation with two strains, one tolerant and one sensitive to the herbicide, the presence of rimsulfuron significantly enhanced root occupancy by resistant bacteria, suggesting that shifts in the microbial community structure of crop rhizosphere could indeed result as a consequence of weed control by AHAS inhibitors.Abbreviations AHAS acetohydroxyacid synthase - CETAB cetyltrimethylammonium bromide - ID₅₀ concentration causing 50% inhibition of enzyme activity - LD₅₀ concentration causing 50% decrease of growth constant value     

Response to imazapyr and dominance relationships of two imidazolinone-tolerant alleles at the <Emphasis Type="Italic">Ahasl1</Emphasis> locus of sunflower

Imisun and CLPlus are two imidazolinone (IMI) tolerance traits in sunflower (Helianthus annuus L.) determined by the expression of different alleles at the same locus, Ahasl1-1 and Ahasl1-3, respectively. This paper reports the level of tolerance expressed by plants containing both alleles in a homozygous, heterozygous and in a heterozygous stacked state to increasing doses of IMI at the enzyme and whole plant levels. Six genotypes of the Ahasl1 gene were compared with each other in three different genetic backgrounds. These materials were treated at the V2–V4 stage with increasing doses of imazapyr (from 0 to 480 g a.i. ha^–1) followed by an assessment of the aboveground biomass and herbicide phytotoxicity. The estimated dose of imazapyr required to reduce biomass accumulation by 50% (GR₅₀) differed statistically for the six genotypes of the Ahasl1 gene. Homozygous CLPlus (Ahasl1-3/Ahasl1-3) genotypes and materials containing a combination of both tolerant alleles (Imisun/CLPlus heterozygous stack, Ahasl1-1/Ahasl1-3) showed the highest values of GR₅₀, 300 times higher than the susceptible genotypes and more than 2.5 times higher than homozygous Imisun materials (Ahasl1-1/Ahasl1-1). In vitro AHAS enzyme activity assays using increasing doses of herbicide (from 0 to 100 μM) showed similar trends, where homozygous CLPlus materials and those containing heterozygous stacks of Imisun/CLPlus were statistically similar and showed the least level of inhibition of enzyme activity to increasing doses of herbicide. The degree of dominance for the accumulation of biomass after herbicide application calculated for the Ahasl1-1 allele indicated that it is co-dominant to recessive depending on the imazapyr dose used. By the contrary, the Ahasl1-3 allele showed dominance to semi dominance according to the applied dose. This last allele is dominant over Ahasl1-1 over the entire range of herbicide rates tested. At the level of enzymatic activity, however, both alleles showed recessivity to semi-recessivity with respect to the wild-type allele, even though the Ahasl1-3 allele is dominant over Ahasl1-1 at all the herbicides rates used.     

Multiple allelic forms of acetohydroxyacid synthase are responsible for herbicide resistance in <Emphasis Type="Italic">Setaria viridis</Emphasis>

In weed species, resistance to herbicides inhibiting acetohydroxyacid synthase (AHAS) is often conferred by genetic mutations at one of six codons in the AHAS gene. These mutations provide plants with various levels of resistance to different chemical classes of AHAS inhibitors. Five green foxtail [Setaria viridis (L.) Beauv.] populations were reported in Ontario with potential resistance to the AHAS-inhibiting herbicide imazethapyr. The objectives of this study were to confirm resistance, establish the resistance spectrum for each of the five populations, and determine its genetic basis. Dose response curves were generated for whole plant growth and enzyme activity, and the AHAS gene was sequenced. Resistance was confirmed by determining the resistance factor to imazethapyr in the five resistant green foxtail populations for whole plant dose response experiments (21- to 182-fold) and enzyme assays (15- to 260-fold). All five imazethapyr-resistant populations showed cross-resistance to nicosulfuron and flucarbazone while only three populations had cross-resistance to pyrithiobac. Sequence analyses revealed single base-pair mutations in the resistant populations of green foxtail. These mutations were coded for Thr, Asn, or Ile substitution at Ser₆₅₃. In addition, a new mutation was found in one population that coded for an Asp substitution at Gly₆₅₄. There is an agreement between the spectra of resistance observed and the type of resistance known to be conferred by these substitutions. Moreover, it indicates that, under similar selection pressure (imazethapyr), a variety of mutations can be selected for different populations, making the resistance pattern difficult to predict from herbicide exposure history.     

Imazaquin and chlorsulfuron resistance and cross resistance in mutants of Chlamydomonas reinhardtii

Summary Chlorsulfuron and/or imazaquin resistant mutants of Chlamydomonas reinhardtii strain CW15 have been obtained and shown to have actolactate synthase (ALS) with altered sensitivity to one or both of these herbicides. Herbicide resistance in the three mutants described is allelic, and resistance appears to result from a dominant or semidominant mutation in a single, nuclear gene. Imazaquin and chlorsulfuron resistant ALS from imazaquin and chlorsulfuron resistant mutants, together with single-gene Mendelian inheritance of these phenotypes, suggests that ALS is the sole site of action of the two herbicides in Chlamydomonas. A high degree of cross resistance between the two herbicides was found in only one mutant. This mutant (IM-13) was selected for resistance to imazaquin and has a high level of in vitro resistance to both imazaquin (270-fold increased I₅₀) and chlorsulfuron (900-fold increased I₅₀). In another mutant selected for resistance to imazaquin (IMR-2), hyper-sensitivity to chlorsulfuron was found. A mutant selected for resistance to chlorsulfuron (CSR-5), had a substantial degree of resistance of chlorsulfuron (80-fold increased I₅₀), but not to imazaquin (7-fold increased I₅₀).     

Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia

Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I₅₀ to susceptible I₅₀) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [¹⁴C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K_m values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.     

Selection and molecular characterization of imidazolinone resistant mutation-derived lines of Tritordeum HT621

Imidazolinone herbicides resistant varieties, induced by mutations at the AHAS gene (acetohydroxyacid synthase), have been developed in many crops. Hexaploid tritordeum (Tritordeum Asch. & Graebn.) is the amphiploid derived from the cross between Hordeum chilense (H^chH^ch) and durum wheat Triticum turgidum L. (Thell) (AABB). Tritordeums have the potential to become a new crop with high added-value for food or feed. Mutagenesis with EMS was conducted to obtain imidazolinone resistant lines derived of the tritordeum HT621. Eleven M₃ plants were selected after imidazolinone treatment and five descendants of two of these lines (HT621-M3R1-3 and HT621-M3R10-1) were analyzed at the molecular level. Partial sequences of the three homologous AHAS loci in genomes A, B, and H^ch were obtained as well as those of HT621. A partial sequence of the AHAS gene in Hordeum chilense is first described in this work, and the designation ahasL-H ^ch 1 is proposed. A single Ser-Asn₆₂₇ substitution at the AHAS locus in the B genome is responsible of resistance in both lines. We propose the name AhasL-B2 for this resistance allele. This is the first report of the selection of imidazolinone resistant lines of tritordeum and the molecular characterization of the mutation conferring this resistance.     

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 TrpLeu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.     

In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

Summary Genes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.     

Chlorsulfuron resistance in Daucus carota cell lines and plants:Involvement of gene amplification

Daucus carota L. cell lines stably resistant to the herbicide chlorsulfuron (CS) have been isolated according to a stepwise selection. Studies carried out during different selection steps show that the specific activity of the target enzyme acetohydroxyacid synthase (AHAS) increases along with CS resistance. Southern hybridization analysis performed with aBrassica napus AHAS probe in a CS highly-resistant cell line reveals the presence of a greatly amplifiedEcoRI fragment of genomic DNA. This indicates that AHAS overproduction induced by stepwise selection is due to gene amplification. Regenerants from some resistant cell lines maintained the CS-resistant trait at the whole plant level.     

Lack of Cross-Resistance of Imidazolinone-Resistant Cell Lines of Datura innoxia P. Mill. to Chlorsulfuron : Evidence for Separable Sites of Action on the Target Enzyme

Two cell lines of Datura innoxia resistant to two imidazolinone herbicides, imazapyr and imazaquin, were isolated from mutagenized, predominantly haploid cell suspension cultures. Both of the resistant variants were >1000-fold more resistant than the wild-type to the two imidazolinones. The variant resistant to imazapyr showed cross-resistance to imazaquin and vice versa, but no cross-resistance to a structurally different inhibitor, chlorsulfuron, a sulfonylurea herbicide, was observed. The target enzyme, acetolactate synthase, extracted from imidazolinone-resistant cell lines was not inhibited by imazapyr or imazaquin but was sensitive to chlorsulfuron indicating separable sites of action for these inhibitors. The variation in resistance and cross-resistance of chlorsulfuron-resistant (PK Saxena, J King [1988] Plant Physiol 86: 863-867) and imidazolinone-resistant cell lines of Datura innoxia demonstrates the possibility of separate mutations of acetolactate synthase gene resulting in specific phenotypes.     

Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L.

A biotype of Sonchus oleraceus L. (Compositae) has developed resistance to herbicides inhibiting acetolactate synthase (ALS) following field selection with chlorsulfuron for 8 consecutive years. The aim of this study was to determine the inheritance and mechanism of resistance in this biotype. Determination of ALS activity and inhibition kinetics revealed that K_m and V_max did not vary greatly between the resistant and susceptible biotypes. ALS extracted from the resistant biotype was resistant to five ALS-inhibiting herbicides in an in vitro assay. ALS activity from the resistant biotype was 14 19, 2, 3 and 3 times more resistant to inhibition by chlorsulfuron, sulfometuron, imazethapyr, imazapyr and flumetsulam, respectively, than the susceptible biotype. Hybrids between the resistant and a susceptible biotype were produced, and inheritance was followed through the F₁, F₂ and F₃ generations. F₁ hybrids displayed a uniform intermediate level of resistance between resistant and susceptible parents. Three distinct phenotypes, resistant, intermediate and susceptible, were identified in the F₂ generation following chlorsulfuron application. A segregation ratio of 121 was observed, indicative of the action of a single, nuclear, incompletely dominant gene. F₃ families, derived from intermediate F₂ individuals, segregated in a similar manner. Resistance to herbicides inhibiting ALS in this biotype of S. oleraceus is due to the effect of a single gene coding for a resistant form of the target enzyme, ALS.     

Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase), which catalyses the first reaction common to the biosynthesis of the branched-chain amino acids, L-valine, L-leucine and L-isoleucine, and is the target of several classes of herbicides, has been studied in hydroponically-grown seedlings of wheat (Triticum aestivum L. cv. Vulcan). Enzyme activity was greater in leaves than roots, reaching a maximum between 4 and 6 days after germination. AHAS was associated with the chloroplasts after centrifugation in a density gradient. A preparation of the enzyme was obtained from wheat leaves which gave a single band after electrophoresis in native gels but was resolved by denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis into three polypeptide bands of molecular mass 58, 57 and 15 kDa. The native molecular mass was approximately 128 kDa. AHAS had optimum activity at pH 7 and did not require the addition of flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP) and MgCl₂ for activity. The enzyme did not display typical hyperbolic kinetics, in that the double reciprocal plot of activity against pyruvate concentration was non-linear. The concentration of pyruvate that gave half of the maximum activity was 4 mM. Sulfonylurea and imidazolinone herbicides were potent inhibitors of wheat leaf AHAS, with 50% inhibition being observed at concentrations of 0.6 and 0.3 μM for chlorsulfuron and metsulfuron methyl, respectively, and at 2.5, 5 and 10 μM for imazaquin, imazethapyr and imazapyr. Inhibition by both classes of compounds was reversed by removal of the inhibitor. Progress curves of product formation against time in the presence of the herbicides were non-linear, and based on the assumption that inhibition by the sulfonylureas was of the slow, tight-binding type, estimates of 0.17 and 0.1 nM were obtained for the dissociation constants of chlorsulfuron and metsulfuron methyl, respectively, from the steady-state enzyme-inhibitor complex.     

Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the A19 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phenotype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the A19 gene. In these transformation experiments the mutated A19 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A19 transgene in one of the biolistically transformed R₀ plants, and a single copy in one of the R₀ plants transformed with Agrobacterium. As expected. progeny seedlings derived from outcrosses involving the R₀ plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 g/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.Abbreviations AHAS acetohydroxyacid synthase - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - FW fresh weight - GUS -glucuronidase - IC₅₀ herbicide concentration that produces a 50% reduction in the fresh weight growth of cells - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962)     

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

Summary Three corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.     

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum)

Summary Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 151 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25–260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha^-1 chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6–4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha^-1) and full (4.5 g ha^-1) rates of metsulfuron methyl.