Sulfonylurea Resistance Research Articles

Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a filamentous ascomycete that is pathogenic towards a broad host range of insect targets and is increasingly serving as a model for examining fungal development and host-pathogen interactions. B. bassiana displays a prohibitive level of resistance against many current fungal and/or yeast selection markers including hygromycin, neomycin, and zeocin. A genetic transformation system for B. bassiana based upon the use of a sulfonylurea resistance cassette derived from the Magnaporthe grisea, acetolactate synthase gene (sur) was developed. The transformation frequency ranged from 100-150 transformants per microgram DNA/10(8) cells and Southern blot analysis indicated that the plasmid vector was randomly integrated into the genome of B. bassiana. In addition, a construct bearing the sur gene and the enhanced green fluorescent protein gene egfp as a visual marker was used to successfully transform B. bassiana. Over 95% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. The described transformation method increases opportunities for the genetic manipulation of B. bassiana.

Inheritance of sulfonylurea resistance in Monochoria vaginalis

SummaryThe inheritance of sulfonylurea (SU) resistance in Monochoria vaginalis was investigated based on the bensulfuron‐methyl response phenotypes of F1 plants between SU‐resistant (R) and ‐susceptible (S) and segregation analysis in F2 progenies. Differences of SU resistance between SU‐R biotypes and F1 plants at the recommended field dose were also investigated by comparing shoot dry weight. All F1 plants survived the treatment with 25 g a.i. ha−1 bensulfuron‐methyl, one‐third of the recommended field dose, and showed similar responses to SU‐R plants. Conversely, all F1 plants died or showed extreme necrosis at 225 g a.i. ha−1, three times the recommended field dose, as SU‐S plants. F2 plants were classified as either R or S phenotype. Segregation for resistance to bensulfuron‐methyl in F2 families did not differ from the expected 3:1 (R:S) ratio at 25 g a.i. ha−1. At 225 g a.i. ha−1, the F2 families segregated in a 1:3 (R:S) ratio. These results suggest that SU resistance in M. vaginalis is controlled by a single nuclear allele with resistance being dominant at low dose and susceptibility dominant at high dose. Moreover, F1 plants died or were extremely injured after application of bensulfuron‐methyl at the recommended field dose, although SU‐R biotypes grew normally.

Genetic diversity of sulfonylurea‐resistant and ‐susceptible Monochoria vaginalis populations in Japan

Summary Monochoria vaginalis is one of the most serious weeds of rice fields in Asia. The species is predominantly selfing. To reveal the potential for multiple mutational events, outcrossing and gene flow in the sulfonylurea‐resistant (SU‐R) M. vaginalis populations, we investigated (i) if each SU‐R population was a single SU‐R biotype or a mixture of several SU‐R biotypes using restriction analysis or direct sequencing of acetolacatate synthase (ALS) genes and (ii) genetic diversity of SU‐R and ‐susceptible (S) populations using amplified fragment length polymorphism (AFLP) analysis. Nineteen or 20 individuals were sampled from four SU‐R and five SU‐S populations respectively. Amino acid substitutions conferring resistance in the SU‐R populations were Pro197Ser in the ALS1 or ALS3, or Asp376Glu in the ALS1 and each SU‐R population was composed of a single SU‐R biotype. In cluster analysis each SU‐R individual formed a cluster, whereas the individuals from a SU‐S population belonged to different clusters. Some SU‐R populations showed polymorphic AFLP loci. The results indicated that these SU‐R biotypes emerged from a single mutational event and any gene flow of SU‐R genes from adjacent populations did not occur. A low level of outcrossing and recombinations of SU‐R genes occurred within some SU‐R populations of M. vaginalis.

Nucleotide substitutions in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Monochoria vaginalis (Pontederiaceae)

Some point mutations in acetolactate synthase (ALS) confer resistance to ALS-inhibiting herbicides in weeds. To clarify the evolution of the herbicide resistance of Monochoria vaginalis, a weed in rice fields in Japan, the nucleotide sequences of four genes encoding ALS were surveyed in five sulfonylurea-resistant (SU-R) and five sulfonylurea-susceptible (SU-S) biotypes. In the ALS1 gene, two SU-R biotypes showed nucleotide substitutions changing Pro197 to Ser and Leu, respectively. In a different gene, ALS3, three other SU-R biotypes showed either of the two nonsynonymous nucleotide substitutions seen in ALS1. Only two biotypes geographically located distantly from each other shared the same mutation conferring SU resistance in the same gene. These patterns of nucleotide substitutions indicate that the SU-R phenotype was acquired independently by different biotypes. Nucleotide diversity values of the genes showing SU-R mutations were higher than those of ALS2 lacking any SU-R mutation and of a putative pseudogene, ALS4. This result suggests that the maintenance of nucleotide variability within target genes provides an opportunity for the evolution of SU-R phenotypes by herbicide-driven selection for mutations conferring resistance.

Weeding Efficacy of Sulfonylurea Resistance Weed, Monochoria (Monochoria vaginalis) with Brown Leaf Blight Caused by BWC01-54

A summer annual weed of monochoria (Monochoria vaginalis) grows in the edges of rice paddies, ditches, and moist upland throughout Korea. It is very difficult to control with herbicide because of its sulfonylurea resistance. It is very competitive with fast growing pattern, that can cause reducing yields of rice. Brown leaf blight of monochoria (Monochoria vaginalis) occurred naturally in rice paddy, is first reported in Korea. The fungal isolate BWC01-54 was successfully isolated from the diseased leaves of monochoria. The fungus BWC 01-54 was grown well at , conidia of the greysh black brown mycelia were abundant produced on PDA at 15 days. The fungus was grown well in potato dextrose broth at and fully grown within 10 days in 250 ml of flask. In host and pathogenicity test, conidia suspension of BWC01-54 was the most effective to control of monochoria compare to others isolates. Typical symptoms having pin point brown lesions were formed on stem and leaf and which severely affected the whole plants ware blighted within two weeks, respectively. Under paddies field condition, conidial suspension of the fungus BWC01-54 gave around 90% control. Therefore, we conclude that the fungus may have a potential as a biological control agent against sulfonylurea resistance weed in rice paddy.

Molecular basis of resistance to sulfonylureas in Papaver rhoeas

A Papaver rhoeas population resistant to several acetolactate synthase (ALS) inhibiting herbicides, called 25/98, was found in Catalonia (Northeastern of Spain). This population has an altered form of the enzyme that showed cross-resistance to several herbicides of this group. The highest resistance was found with tribenuron-methyl and sulfometuron-methyl. Studies were conducted to define the molecular basis of this resistance. Two regions of the ALS gene were amplified using degenerated universal primers and sequenced. Population 25/98 contained a single nucleotide substitution in domain A changing Pro 197 by Ser (using the nomenclature of Arabidopsis thaliana) that confers sulfonylurea resistance. Another change was detected in a region located outside of any conserved domains described to date, but its implication in the resistance remains unclear. We analyze the putative role of the found mutations in relation to the observed resistance using a putative three-dimensional model of the Papaver ALS enzyme.

Properties of acetolactate synthase from sulfonylurea-resistant Scirpus juncoides Roxb. var. ohwianus T. Koyama

Properties of acetolactate synthase (EC 4.1.3.18; ALS) from sulfonylurea-resistant (SUR) Scirpus juncoides Roxb. var. ohwianus T. Koyama were studied biochemically and physiologically in comparison with those from sulfonylurea-susceptible weed (SUS). GR 50 values for growth inhibition and I 50 values for ALS inhibition by imazosulfuron were determined for both SUR and SUS. Imazosulfuron controlled the SUS above 80% at the dosage more than 10 g a.i./ha but did not control the SUR at the even great dosage of 1000 g a.i./ha. The rates required for 50% growth inhibition of the SUR relative to the SUS (R/S ratio) were 271-fold. The I 50 value for inhibition of ALS from the SUS was 15 nM, compared to I 50 of >3000 nM for inhibition of ALS from the SUR. These results suggest that a resistance may due to an altered ALS that is insensitive to imazosulfuron. The K m (pyruvate) value of ALS from the SUR was similar to the K m for ALS from the SUS, suggesting that a mutation resulting in resistance does not change the affinity of the enzyme for pyruvate. The specific activity of the SUR ALS was similar to that of the SUS ALS, which indicates that resistance is not an over-expression of the enzyme. ALS activity from both biotypes was inhibited by isoleucine, valine, and leucine in this order. However, the SUR ALS was less sensitive to inhibition by valine than the SUS ALS.

Engineering a genetic transformation system for Colletotrichum acutatum, the causal fungus of lime anthracnose and postbloom fruit drop of citrus.

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.

Engineering a genetic transformation system for Colletotrichum acutatum, the causal fungus of lime anthracnose and postbloom fruit drop of citrus

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene ( sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0–17.9 per μg circular DNA using 10 7 protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.

Light Requirement in Rapid Diagnosis of Sulfonylurea-Resistant Weeds of Lindernia spp. (Scrophulariaceae)

Sulfonylurea resistance has been found in Japan, and four of the nine resistant weeds are in the genus Lindernia (Scrophulariaceae). To develop appropriate control methods for these resistant weeds, a rapid method to diagnose sulfonylurea-resistant weeds was improved and applied to common falsepimpernel (L. procumbens), two varieties of low falsepimpernel (L. dubia var. dubia and var. major), and azetogarashi (L. micrantha). Illumination is crucial for diagnosis in Lindernia spp., and treatment with 7.5 or 75 ng ai/ml thifensulfuron-methyl produced clear results.

Sulfonylurea resistance in Lindernia micrantha, an annual paddy weed in Japan

Resistance to sulfonylurea herbicides, including bensulfuron‐methyl, pyrazosulfuron‐ethyl, imazosulfuron and ethoxysulfuron, was discovered in naturally occurring populations of Lindernia micrantha D. Don in rice fields that had been treated with sulfonylurea‐based herbicides for 3–7 consecutive years. The resistant biotype was approximately 80≈300 times more resistant than the susceptible one to the above four sulfonylurea herbicides. This is the second confirmed occurrence of herbicide resistance resulting from the use of sulfonylurea herbicides in Japan. Several herbicides with different modes of action, including pretilachlor, cafenstrole, bifenox, naproanilide, thiobencarb + simetryn + MCPB, MCPA‐thioethyl + simetryn and cyhalofop‐butyl + bentazone, effectively controlled the resistant biotype in pot trials.

In vitro and whole-plant magnitude and cross-resistance characterization of two imidazolinone-resistant sugarbeet (Beta vulgaris) somatic cell selections

Acetolactate synthase (ALS)-inhibiting herbicide carryover in soil can severely affect sugarbeets grown in the year(s) following application. Two newly developed imidazolinone-resistant (IMI-R) sugarbeet somatic cell selections (Sir-13 and 93R30B) were examined for magnitude of resistance and extent of cross-resistance to other classes of ALS inhibitors and compared to a previously developed sulfonylurea-resistant (SU-R) selection, Sur. In vitro shoot culture tests indicated Sir-13 resistance was specific to imidazolinone (IMI) herbicides at approximately a 100-fold resistance compared to the sensitive control sugarbeet. Sur was 10,000-fold resistant to the sulfonylurea (SU) herbicide, chlorsulfuron, and 40-fold resistant to the triazolopyrimidine sulfonanilide (TP) herbicide, flumetsulam, but not cross-resistant to the IMI herbicides. 93R30B was selected for IMI-R from a plant homozygous for the SU-R allele,Sur, and displayed similar in vitro SU-R and TP-R as Sur, but also displayed a very high resistance to various IMI herbicides (400- to 3,600-fold). Compared to the sensitive control, Sir-13 was 300- and > 250-fold more resistant to imazethapyr and imazamox residues in soil, respectively. Response by whole plants to postemergence herbicide applications was similar to that observed in shoot cultures. Sir-13 exhibited > 100-fold resistance to imazethapyr as well as imazamox, and 93R30B showed > 250-fold resistance to both herbicides. 93R30B showed great enough resistance to imazamox to merit consideration of imazamox for use as a herbicide in these sugarbeets. Sir-13 showed a two- to threefold higher level of resistance in the homozygous vs. heterozygous state, indicating that like most ALS-inhibitor resistance traits, it was semidominantly inherited.

Germination, Growth, and Development of Herbicide Resistant and Susceptible Populations of Rigid Ryegrass (Lolium rigidum)

Rate of seedling emergence, relative growth rate (RGR), and phenological development were compared in several accessions of rigid ryegrass belonging to three distinct resistance classes. The aryloxyphenoxypropionate resistant (AOPP-R) class had a faster and less variable seedling emergence than the sulfonylurea resistant (SU-R) and susceptible (S) classes. However, even the fastest of the AOPP-R accessions was within the range of the S and SU-R classes. No significant differences were detected among the resistant classes in seed dormancy, RGR, and the rate of phenological development. The rate of spike emergence, irrespective of the resistance class, was related to the latitude of the origin of the accessions, suggesting adaptation to the local climates since introduction. Due to considerable variation among weed populations for most biological attributes, the need to include several R and S accessions, in studies similar to the one reported here, is of vital importance. Because of the means and variances of the three resistance classes, at least four accessions from each resistance class would have been required to detect the observed differences between emergence rates of the AOPP-R and S classes with a confidence of 95%.

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 Trp-->Leu) 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.

Development of herbicide resistance in annual ryegrass populations (Lolium rigidum Gaud.) in the cropping belt of Western Australia

Annual ryegrass (Lolium rigidum) samples from the cropping belt of Western Australia were screened for herbicide resistance in 1992 and 1993. There was a strong relationship between the number of applications of a herbicide group and development of resistance in ryegrass populations. Resistance was detected in all populations that received >7 applications of aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides or >4 applications of sulfonylurea (SU) herbicides. Some AOPP-resistant populations had also developed crossresistance to SU herbicides, a group with a different mode of action. Inclusion of pasture in the rotation had little effect on the relationship between the number of applications of the AOPP and SU herbicides and development of resistance. A subset of 33 populations was chosen to determine the response of triasulfuron-resistant populations to sulfometuron, a nonselective SU herbicide which has been shown to be effective against metabolic-type resistance. All triasulfuron-resistant populations were found to be resistant to sulfometuron, possibly due to insensitive acetolactate synthase (ALS) in these ryegrass populations. Some of these SU-resistant populations were also resistant to the imidazolinone herbicide imazethapyr, another ALS inhibitor. However, there were several populations with a high level of SU resistance that were still susceptible to imazethapyr.

Identification of Two Mechanisms of Sulfonylurea Resistance Within One Population of Rigid Ryegrass (Lolium rigidum) Using a Selective Germination Medium

A biotype of rigid ryegrass (Lolium rigidumGaudin biotype VLR69) resistant to some ALS inhibitors was characterized to determine the mechanisms of resistance to the sulfonylurea herbicides. The biotype had a high level of resistance to chlorsulfuron (20×) and triasulfuron (25×), and an intermediate level of resistance to imazaquin (7×) and sulfometuron (7.5×) but exhibited a low level of resistance to imazapyr (2.5×). At 60 to 90 g ai ha-1sulfometuron, 4% of the population survived without apparent herbicidal effect The same response to sulfometuron was also observed when seeds of the resistant biotype VLR69 were germinated on agar containing sulfometuron. At 27 nM sulfometuron, 4% of the seeds germinated and grew normally while the growth of the bulk of the population was retarded. This differential response to sulfometuron at the seedling stage allowed selection of sulfometuron-resistant individuals from the population. Activity of ALS extracted from these sulfometuron-resistant plants was less sensitive to inhibition by chlorsulfuron than ALS extracted from plants considered sulfometuron susceptible in the same system. Unselected plants from the VLR69 population were able to detoxify chlorsulfuron more rapidly than susceptible VLR1 plants. It is apparent that there are at least two mechanisms of sulfonylurea resistance in the resistant biotype VLR69 which occur at different frequencies within the population. These data show that more than one mechanism or resistance may develop in response to herbicide selection pressure and that the resulting populations are not necessarily homogeneous.

A Comparison of Two Genes for Sulfonylurea Herbicide Resistance in Transgenic Tobacco Seedlings

Previous work in tobacco (Nicotiana tabacum L.) showed the csr1‐1 gene for sulfonylurea resistance was inadequate for use in conjunction with a new, low residual sulfonylurea herbicide, DPX‐R9674 {a mixture of (methyl 2[[[[N‐4‐methoxy‐6‐methyl‐1,3,5‐triazin‐2‐yl)methylamino]‐carbonyl]amino]sulfonyl) benzoate) and (methyl 1‐3‐[[[[(4‐methoxy‐6‐methyl‐1,3,5‐triazin‐2‐yl)amino] carbonyl]amino]sulfonyl]‐2‐thiophenecarboxylate}. This study compared a new sulfonylurea resistance gene, ahas3r, with csr1‐1 in order to determine if ahas3r confers superior resistance to DPX‐R9674. The genetic material used in the study consisted of transgenic tobacco seedlings carrying either one of the two genes alone or both genes together. These genotypes were compared with each other and untransformed controls using acetohydroxyacid synthase (AHAS) assays and contrast comparisons of absolute growth rates of tobacco seedlings 10 and 20 d following herbicide application. Both the AHAS assays and the growth analyses of tobacco seedlings showed the genes to confer similar levels of resistance to chlorsulfuron {2‐chloro‐N‐[(4‐methoxy‐6‐methyl‐1,3,5‐triazin‐2‐yl)aminocarbonyl] benzenesulfonamide}. However, the ahas3r gene clearly conferred a greater degree of resistance to DPX‐R9674 than the csr1‐1 gene and may therefore be a good candidate for use with this herbicide.

Agronomic Performance of Sulfonylurea‐Resistant Transgenic Flue‐Cured Tobacco Grown under Field Conditions

Field testing of transgenic crops is an essential step towards commercialization. This study was conducted to assess the agronomic performance of herbicide‐resistant transgenic tobacco (Nicotiana tabacum L.) lines relative to untransformed controls and to evaluate their sensitivity to sulfonylurea herbicides in a field situation. Two transgenic flue‐cured tobacco lines harboring the csr1‐1 gene for sulfonylurea resistance were evaluated after application of three rates of two sulfonylurea herbicides [chlorsulfuron (2‐chloro‐N[(4‐methoxy‐6‐methyl‐1,3,5 triazin‐2‐yl) aminocarbonyl]‐benzenesulfonamide) and DPXR9674, a 2:1 mixture of thifensulfuron (methyl‐3‐[[4‐methoxy‐6‐methyll, 3,5‐triazin‐2‐yl aminocarbonyl]aminosulfonyl]‐2‐thiophenecarboxylate) and tribenuron (methyl‐2[[[|4‐methoxy‐6‐methyl‐l,3,5‐triazin‐2‐yl]carbonyl]amino]sulfonyl]benzoate)]. We showed that one of the lines was resistant to 10 g a.i. ha−1 of chlorsulfuron but not to 20 g a.i. ha−1 and that both lines were susceptible to DPX‐R9674. Comparison of transgenics to an untransformed control in the absence of herbicide treatment showed that both transgenics were lower yielding than the controls. This impairment of agronomic performance could be attributed to any of a number of factors. Resistance to chlorsulfuron was adequate, but margins of safety need to be increased before any farm level use of these transgenic lines can be considered. Selection among lines for maximum expression of the transgene and selection or backcrossing to recover the parental phenotype may further improve agronomic performance.

Monogenic Dominant Sulfonylurea Resistance in Sugarbeet from Somatic Cell Selection

Injury to sugarbeet, Beta vulgaris L., from sulfonylurea herbicide residues from preceding cropping years has kindled interest in developing resistant cultivars. This study was conducted to obtain chlorsulfuron (2‐chloro‐N‐[[(4‐methoxy‐6‐methyl‐1,3,5‐triazin‐2‐yl)amino]carbonyl]benzenesulfonamide) resistance from cell cultures and to determine its inheritance and magnitude. Utilizing annual diploid sugarbeet clone REL‐1, dispersed suspension cultures were initiated from callus induced on leaf disk cultured on a modified Murashige and Skoog (MS) agar medium + 1.0 mg L−1 N6‐benzyladenine (BA) and placed in the liquid form of the same medium. Unmutagenized cell clusters were plated on solid medium containing 2.8 nM chlorsulfuron in MS + 1.0 mg L−1 BA. A single colony arose, from which shoots were regenerated. Shoots were resistant to 28 nM chlorsulfuron, a concentration that killed similar shoots of REL‐1. Resistance (designated Sur) was inherited as a monogenic dominant. In vitro shoot resistance to chlorsulfuron was 300 to 1000‐fold greater than in REL‐1. Resistance was also expressed in leaf disk expansion in vitro with MS + 1.0 mg L−1 BA.

Directed excision of a transgene from the plant genome.

The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, beta-glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALSr), were integrated into the genome of tobacco and Arabidopsis. The ALSr gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALSr gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The beta-glucuronidase gene remained active. The excision efficiency varied in F1 progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox x Cre F1 progeny were chimeric and some F2 progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.