A Sulfonylurea Herbicide Resistance Gene from Arabidopsis thaliana as a New Selectable Marker for Production of Fertile Transgenic Rice Plants

A mutant acetolactate synthase (ALS) gene, csr1-1, isolated from sulfonylurea herbicide-resistant Arabidopsis thaliana, was placed under control of a cauliflower mosaic virus 35S promoter (35S). Rice protoplasts were transformed with the 35S/ALS chimeric gene and regenerated into fertile transgenic rice (Oryza sativa) plants. The 35S/ALS gene was expressed effectively as demonstrated by northern blot hybridization analysis, and conferred to transformed calli at least 200-fold greater chlorsulfuron resistance than nontransformed control calli. Effective selection of 35S/ALS-transformed protoplasts was achieved at extremely low chlorsulfuron concentrations of 10 nm. The results demonstrated that the 35S/ALS gene is an alternative selectable marker for rice protoplast transformation and fertile transgenic rice production. The results also suggest that the mutant form of Arabidopsis ALS enzyme operates normally in rice cells. Thus, the mechanism of protein transport to chloroplast and ALS inhibition by chlorsulfuron is apparently conserved among plant species as diverse as Arabidopsis (dicotyledon) and rice (monocotyledon).     

Use of a Mutated Protoporphyrinogen Oxidase Gene as an Effective In Vitro Selectable Marker System that Also Conveys <Emphasis Type="Italic">in planta</Emphasis> Herbicide Resistance in Sugarcane

Genetic engineering can be used to introduce economically important traits in sugarcane cultivars. Part of any transformation process involves the selection of genetically transformed cells. In this study, an efficient sugarcane in vitro selection system was developed using mutated protophorhyrinogen oxidase (PPO) genes as selectable markers. Two PPO genes, that encode proteins targeted either to the mitochondria or plastid, were isolated from tobacco and maize. Site-directed mutagenesis was used to alter the nucleotide sequence of these genes so that the resulting proteins are less sensitive to diphenylether type herbicides. Sugarcane callus was genetically transformed through particle bombardment with constructs allowing expression of either transgene, and putative transgenic calli were selected on fomesafen. It took approximately 4 weeks to select herbicide resistant calli clones on 10 mg/l fomesafen in the presence of light, which increased the selection pressure, and a further 8 weeks to regenerate resistant plantlets. PCR analysis confirmed that all regenerated putative transgenic sugarcane plants contained the transgene. All transgenic plants showed levels of herbicide resistance when planted in soil.     

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.     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer

We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines.     

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.     

Production of fertile transgenic lentil (Lens culinaris Medik) plants using particle bombardment

Summary A reproducible system for gene transfer in lentil through particle bombardment is presented. Lentil cotyledonary nodes excised from germinated seedlings were bombarded with a plasmid containing a mutant acetolactate synthase gene (ALS) from tobacco conferring resistance to sulfonylurea herbicides. Putative transgenic shoots regenerated on Murashige and Skoog medium supplemented with 6-benzylaminopurine (BA) and chlorsulfuron (5 nM for first 4 wk followed by 2.5 nM for the remainder of the culture period) were micrografted and successfully transferred to soil. T₀ and selfed progeny plants were screened using metsulfuron herbicide leaflet painting. The non-transformed escapes died and transformed plants survived the test. The surviving plants were phenotypically normal and produced viable seeds. The presence and stable transmission of the transgene into genomic DNA of screened T₁ transformants was confirmed by PCR and Southern hybridization. This method for producing transformed plants will allow new opportunities for lentil breeding to produce improved cultivars.     

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.     

Physiological and photosynthetic characteristics of indica Hang2 expressing the sugarcane PEPC gene

Phosphoenolpyruvate carboxylase (PEPC) is known to play a key role in the initial fixation of CO₂ in C4 photosynthesis. The PEPC gene from sugarcane (a C4 plant) was introduced into indica rice (Hang2), a process mediated by Agrobacterium tumefaciens. Integration patterns and copy numbers of the gene was confirmed by DNA blot analysis. RT-PCR and western blotting results showed that the PEPC gene was expressed at both the mRNA and protein levels in the transgenic lines. Real-time PCR results indicated that expression of the sugarcane PEPC gene occurred mostly in green tissues and changed under high temperature and drought stress. All transgenic lines showed higher PEPC enzyme activities compared to the untransformed controls, with the highest activity (11.1 times higher than the controls) being observed in the transgenic line, T34. The transgenic lines also exhibited higher photosynthetic rates. The highest photosynthetic rate was observed in the transgenic line, T54 (22.3 μmol m^?2 s^?1; 24.6 % higher than that in non-transgenic plants) under high-temperature conditions. Furthermore, the filled grain and total grain numbers for transgenic lines were higher than those for non-transgenic plants, but the grain filling (%) and 1,000-grain weights of all transgenic lines remained unchanged. We concluded that over-expression of the PEPC gene from sugarcane in indica rice (Hang2) resulted in higher PEPC enzyme activities and higher photosynthesis rates under high-temperature conditions.     

Mutant acetolactate synthase gene is an efficient in vitro selectable marker for the genetic transformation of Brassica juncea (oilseed mustard)

We report in this study, the successful deployment of a double mutant acetolactate synthase gene (ALSdm, containing Pro 197 to Ser and Ser 653 to Asn substitutions) as an efficient in vitro selection marker for the development of transgenic plants in Brassica juncea (oilseed mustard). The ALS enzyme is inhibited by two categories of herbicides, sulfonylureas (e.g. chlorsulfuron) and imidazolinones (e.g. imazethapyr), while the mutant forms are resistant to the same. Three different selection agents (kanamycin, chlorsulfuron and imazethapyr) were tested for in vitro selection efficiency in two B. juncea cultivars, RLM198 and Varuna. For both the cultivars, higher transformation frequencies were obtained using chlorsulfuron (3.8 +/- 0.6% and 4.6 +/- 0.9% for RLM198 and Varuna, respectively) and imazethapyr (10.2 +/- 0.7% for RLM198 and 7.8 +/- 1.2% for Varuna) as compared to that obtained on kanamycin (3.1 +/- 0.2% and 2.8 +/- 0.5% for RLM198 and Varuna, respectively). Additionally, transformation frequencies were higher on imazethapyr than on chlorsulfuron for both the cultivars indicating that imidazolinones are better selective agents than sulfonylureas for the selection of mustard transgenics.     

Site of action of chlorsulfuron: inhibition of valine and isoleucine biosynthesis in plants

The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I₅₀ values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.     

Resistance to Acetolactate Synthase-Inhibiting Herbicides in Annual Ryegrass (Lolium rigidum) Involves at Least Two Mechanisms

WLR1, a biotype of Lolium rigidum Gaud. that had been treated with the sulfonylurea herbicide chlorsulfuron in 7 consecutive years, was found to be resistant to both the wheat-selective and the nonselective sulfonylurea and imidazolinone herbicides. Biotype SLR31, which became cross-resistant to chlorsulfuron following treatment with the aryloxyphenoxypropionate herbicide diclofop-methyl, was resistant to the wheat-selective, but not the nonselective, sulfonylurea and imidazolinone herbicides. The concentrations of herbicide required to reduce in vitro acetolactate synthase (ALs) activity 50% with respect to control assays minus herbicide for biotype WLR1 was greater than those for susceptible biotype VLR1 by a factor of >30, >30, 7,4, and 2 for the herbicides chlorsulfuron, sulfometuron-methyl, imazapyr, imazathapyr, and imazamethabenz, respectively. ALS activity from biotype SLR31 responded in a similar manner to that of the susceptible biotype VLR1. The resistant biotypes metabolized chlorsulfuron more rapidly than the susceptible biotype. Metabolism of 50% of [phenyl-U-¹⁴C]chlorsulfuron in the culms of two-leaf seedlings required 3.7 h in biotype SLR31, 5.1 h in biotype WLR1, and 7.1 h in biotype VLR1. In all biotypes the metabolism of chlorsulfuron in the culms was more rapid than that in the leaf lamina. Resistance to ALS inhibitors in L. rigidum may involve at least two mechanisms, increased metabolism of the herbicide and/or a herbicide-insensitive ALS.     

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.     

Establishment of multiple shoot clumps from maize (Zea mays L.) and regeneration of herbicide-resistant transgenic plantlets

A kind of quick, efficient and season-free inducing embryoid and multiple shoot clumps system from shoot tip meristems that derived from elite inbreds of maize was established. The herbicide-resistant gene als (coding Acetolactate synthase) isolated from a mutant of Arabidopsis thaliana was transferred to tissue pieces of maize multiple shoot clumps by microprojectile bombardment. Herbicide-resistant tissue and regenerants were obtained through selections with herbicide chlorsulfuron. PCR analysis and Southern blot hybridization indicated that gene als has been transferred to some regenerants. The test of spraying chlorsulfuron displayed that the transgenic plantlets and R₁ plants had favorable herbicide-resistant trait. We have established a new genotype-free system of maize which could rapidly and efficiently produce large quantities of transgenic plantlets.     

Characterization and molecular mechanism of a naturally occurring metsulfuron-methyl resistant strain of Pseudomonas aeruginosa

Strain L36, naturally resistant to the herbicide metsulfuron-methyl (SM), was isolated and characterized with respect to the molecular mechanism of resistance. The isolate was identified as Pseudomonas aeruginosa based on bacterial morphology, physiology, cellular fatty acid, and 16S rRNA gene sequence. Minimal inhibitory concentrations of metsulfuron-methyl against the growth of L36 and wild type isolate PAO1 were 6.03 and 1.33 mM, respectively. L36 carried a nucleotide base change in the acetolactate synthase (ALS) gene that coded for a single amino acid mutation (Ala29 → Val29). The mutated ilvIH gene was functionally expressed, purified, and the kinetic properties of the purified ALS were tested. The mutant enzyme had K _m for pyruvate fourfold higher than the wild type enzyme, and K _i ^app for sulfonylureas some 30-fold higher. The A29 V mutation in the ALS resulted in the resistance of P. aeruginosa to sulfonylurea herbicides but not to imidazolinone herbicides.     

New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin

The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyruvate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, an initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute assay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of [¹⁴C]chlorsulfuron and [¹⁴C]imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency.     

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.     

RNA virus accumulation is inhibited by ribonuclease activity of 3D8 scFv in transgenic Nicotiana tabacum

A mannose selection system was adapted for Agrobacterium-mediated transformation of plum (Prunus domestica L.) hypocotyl explants and the recovery of transgenic plants. Adventitious regeneration from non-transformed hypocotyl sections was inhibited when 3 mg/l mannose, combined with 10 mg/l sucrose, was added to the medium. Mature seed hypocotyl slices from the cultivar ‘Claudia Verde’ were infected with A. tumefaciens AGL1, carrying the pNOVgus vector, and placed onto different selective media with mannose. A low mannose selection (1.5 g/l, regeneration below the inhibitory concentration) applied for 16 weeks led to the regeneration of escapes. However, when mannose at 1.5 g/l or at 3 g/l (the regeneration-inhibiting concentration) was applied for 6 weeks from the beginning of the experiments and, after that, was increased to 5 g/l, several independent transgenic lines were obtained. The transformation events were monitored by detection of the GUS enzymatic activity at different stages of the process. Nevertheless, stable integration of transgenes into the genome of the plum plants was confirmed by PCR and Southern blot analysis. The transformed shoots were rooted on a medium supplemented with 10 g/l sucrose and 4 g/l mannose. The transformation procedure described here, using the pmi/mannose system for selection of transgenic plum plants, represents an alternative for the production of transgenic plum plants under conditions that are safe regarding human health and the environment, and would permit the insertion of more transgene/s in a pre-existing transgenic line.