Genotoxicity of 2,4-D and dicamba revealed by transgenic Arabidopsis thaliana plants harboring recombination and point mutation markers

The phenoxy herbicides 2,4-D and dicamba are released daily into the environment in large amount. The mechanisms of genotoxicity and mutagenicity of these herbicides are poorly understood, and the available genotoxicity data is controversial. There is a cogent need for a novel genotoxicity monitoring system that could provide both reliable information at the molecular level, and complement existing systems.We employed the transgenic Arabidopsis thaliana ‘point mutation’ and ‘recombination’ plants to monitor the genetic effects of the herbicides 2,4-D and dicamba. We found that both herbicides had a significant effect on the frequency of homologous recombination A→G mutation. Neither herbicides affected the T→G mutation frequency. Interestingly, these transgenic biomonitoring plants were able to detect the presence of phenoxy herbicides at concentrations that were lower than the guideline levels for Drinking Water Quality. The results of our studies suggest that our transgenic system may be ideal for the evaluation of the genotoxicity of herbicide-contaminated water. Moreover, the unique ability of the plants to detect both double-strand breaks (homologous recombination) and point mutations provides tremendous potential in the study of molecular mechanisms of genotoxicity and mutagenicity of phenoxy herbicides.     

Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber calli

The effects of the herbicides 1,1'-dimethyl-4,4'-bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1muM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.     

A sensitive transgenic plant system to detect toxic inorganic compounds in the environment

We describe a transgenic plant-based assay to study the genetic effects of heavy metals. Arabidopsis thaliana plants carrying a beta-glucuronidase (GUS) marker gene either with a point mutation or as a recombination substrate were used to analyze the frequency of somatic point mutations and homologous recombination in whole plants. Transgenic test plants sown on media contaminated by the salts of the heavy metals Cd2+, Pb2+, Ni2+, Zn2+, Cu2+, and As2O3 exhibited a pronounced uptake-dependent increase in the frequencies of both somatic intrachromosomal recombination and point mutation. The test was applied to monitor the genotoxicity of soils sampled in sites contaminated with several heavy metals. Our results indicate that this is a highly sensitive system for monitoring metal contamination in soils and water.     

Responses of Populus tremula to Picloram and Other Translocated Herbicides

Whole aspen plants and isolated aspen root segments were three to ten times more sensitive to the growth-inhibitory and toxic effects of picloram than to those of 2,4-D, 2,4,5-T and dicamba. The activity of picloram in the inhibition of root growth was about ten times higher than that of 2,4-D and dicamba when tbe substances were added to the nutrient solution. Epinastic responses indicated a very rapid translocation of picioram from the roots to the growing shoot parts. When the herbicides were applied to the mature leaves dicamba rapidly caused a complete inhibition of root growth indicating a rapid translocation of this compound from the leaves to the root tips. Leaf-applied picloram, 2,4-D and 2,4,5-T affected root growth considerably more slowly. Dicamba, 2,4-D and 2,4,5-T rapidly killed the directly treated leaf tissue due to high acute toxicity while picloram did not show this type of toxicity. It is concluded that the higher activity of picloram in killing the plant and in inhibition of root and shoot growth can only partly be explained as a result of greater uptake and translocation of this compound.     

Differences among auxin treatments on haploid production in durum wheat × maize crosses

Three doubled haploid lines of durum wheat [Triticum turgidum ssp. durum (Desf.) Husn.] were crossed with maize (Zea mays L.), and five hormone treatments were applied to test their effect on the production of caryopses, embryos and haploid plants. The auxin treatments consisted of 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mg/l or 50 mg/l dicamba and two combination mixtures of 95/5 mg/l and 50/50 mg/l 2,4-D plus dicamba, respectively. Hormones were added to the culture medium of the detached tillers. Differences were not observed among the four hormone treatments that contained dicamba, nevertheless, these treatments significantly increased the production of caryopses, embryos and haploid plants. On average, 8.9 caryopses, 2.6 embryos and 1.3 haploid plants per spike were obtained following the treatment with 100 mg/l 2,4-D, and 15.0 caryopses, 6.0 embryos and 3.0 haploid plants per spike were obtained following the various treatments with dicamba. We propose the application of dicamba alone, or dicamba plus 2,4-D, as a means for improving the yield of haploid plants of durum wheat through crosses with maize.     

Evaluation of the genotoxicity of 2,4-dichlorophenoxyacetic acid and its derivatives in mammalian cell cultures.

2,4-dichlorophenoxyacetic acid and its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity of an ester (2,4-D 2-butoxyethylester) and two salts (2,4-D isopropylamine and 2,4-D triisopropanolamine) was investigated in cultured mammalian cells. The end points used were the induction of chromosomal aberrations in primary cultures of rat lymphocytes and forward mutations at the HGPRT locus of Chinese hamster ovary cells. There was no evidence of genotoxicity for the test materials in the experimental systems used. These results were consistent with the general lack of genotoxic potential for 2,4-D in a number of other test systems.     

Vegetative growth and sporulation ofBipolaris sorokiniana on sequentially older infected leaves ofPoa pratensis exposed to postemergence herbicides

Poa pratensis was exposed to the postemergence herbicides 2,4-D, MCPP, and dicamba at concentrations of 10^–6 M applied to the soil surface. Sequentially older leaf blades were inoculated withBipolaris sorokiniana and detached to determine the effect of the herbicides on saprophytic hyphal growth and sporulation on the tissue of each older leaf. 2,4-D and dicamba stimulated hyphal growth in vitro from leaf tissue of all ages; MCPP had no effect on hyphal growth. All herbicides increased sporulation byB. sorokiniana on leaf tissue of all ages. Both hyphal growth and sporulation increased progressively on tissue from sequentially older leaves ofP. pratensis exposed to dicamba.Journal Paper No. J-14985 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, USA, Project No. 2616.     

Mutant analysis in Arabidopsis provides insight into the molecular mode of action of the auxinic herbicide dicamba

Herbicides that mimic the natural auxin indole-3-acetic acid are widely used in weed control. One common auxin-like herbicide is dicamba, but despite its wide use, plant gene responses to dicamba have never been extensively studied. To further understand dicamba's mode of action, we utilized Arabidopsis auxin-insensitive mutants and compared their sensitivity to dicamba and the widely-studied auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The mutant axr4-2, which has disrupted auxin transport into cells, was resistant to 2,4-D but susceptible to dicamba. By comparing dicamba resistance in auxin signalling F-box receptor mutants (tir1-1, afb1, afb2, afb3, and afb5), only tir1-1 and afb5 were resistant to dicamba, and this resistance was additive in the double tir1-1/afb5 mutant. Interestingly, tir1-1 but not afb5 was resistant to 2,4-D. Whole genome analysis of dicamba-induced gene expression showed that 10 hours after application, dicamba stimulated many stress-responsive and signalling genes, including those involved in biosynthesis or signalling of auxin, ethylene, and abscisic acid (ABA), with TIR1 and AFB5 required for the dicamba-responsiveness of some genes. Research into dicamba-regulated gene expression and the selectivity of auxin receptors has provided molecular insight into dicamba-regulated signalling and could help in the development of novel herbicide resistance in crop plants.     

Effects of artichoke (Cynara scolymus) leaf and bloom head extracts on chemically induced DNA lesions in Drosophila melanogaster

The genotoxicity of bloom head (BHE) and leaf (LE) extracts from artichoke (Cynara scolymus L.), and their ability to modulate the mutagenicity and recombinogenicity of two alkylating agents (ethyl methanesulfonate – EMS and mitomycin C – MMC) and the intercalating agent bleomycin (BLM), were examined using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Neither the mutagenicity nor the recombinogenicity of BLM or MMC was modified by co- or post-treatment with BHE or LE. In contrast, co-treatment with BHE significantly enhanced the EMS-induced genotoxicity involving mutagenic and/or recombinant events. Co-treatment with LE did not alter the genotoxicity of EMS whereas post-treatment with the highest dose of LE significantly increased this genotoxicity. This enhancement included a synergistic increase restricted to somatic recombination. These results show that artichoke extracts promote homologous recombination in proliferative cells of D. melanogaster.     

Ames assays and unscheduled DNA synthesis assays on 2, 4-dichlorophenoxyacetic acid and its derivatives.

2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes. In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D. There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays. These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats.     

三种除草剂对五爪金龙的防除作用及2,4-D丁酯对环境的影响

用3种除草剂(2,4-D丁酯、麦草畏和塔隆)对五爪金龙(Ipomoea cairica L.Sweet)进行化学防除试验。结果表明:1.00 mL L-1的2,4-D丁酯可以彻底杀灭五爪金龙。喷施1.00 mL L-12,4-D丁酯20 d后,五爪金龙茎叶枯死率接近100%;60 d后五爪金龙的总生物量显著低于其它处理及对照;90 d后未出现生长恢复,最终盖度防效为99.8%。而喷施1.00 mL L-1的麦草畏40 d后,五爪金龙的茎叶枯死率为99.0%,但仍有少量存活的根,90 d后再次萌生率为10.0%;喷施1.00 mL L-1塔隆40 d后,五爪金龙的茎叶枯死率为100%,90 d后再次萌生率为100%。土壤残留分析表明:在有机质含量较高[(10.14±1.01)g kg-1]的土壤中2,4-D丁酯降解速率较快,半衰期为14 d,施药后80 d的土壤中已检测不到2,4-D丁酯。此外,在野外喷洒1.0 mL L-1的2,4-D丁酯对其它植物是安全的,施药1年后,样地内的植物均能恢复生长。因此,实践中可用1.00 mL L-1的2,4-D丁酯来防除五爪金龙。     

Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.     

麦草畏室内生测方法建立及大豆对麦草畏耐受性分析

室内生物测定是植物对除草剂等化学物质耐受性鉴定的一种常用筛选方法,已广泛应用于大豆、稗草、棉花等植物对草甘膦、氯嘧磺隆等除草剂的耐受性研究,但麦草畏的室内生物测定方法和大豆对麦草畏耐受性相关研究尚未见报道。本研究以麦草畏对催芽大豆下胚轴伸长抑制率为评价指标,结合回归方程曲线分析和抑制中浓度分析,建立了大豆对麦草畏耐受性室内生物测定方法,确定以300μg/L麦草畏筛选浓度作为大豆室内生物测定临界筛选浓度。利用该方法对35份源自微核心种质的大豆品种进行鉴定,结果表明,随麦草畏浓度增加,不同品种对麦草畏的耐受性存在显著差异,大豆品种对麦草畏的耐受性降低,从中筛选出对麦草畏耐受性较高的大黄豆-1和什邡螺丝豆。本研究结果为培育抗麦草畏品种的亲本选配以及后代选择提供了理论依据、材料和技术支撑。     

Effect of nitrogen and phosphorus status on the translocation of three herbicides in field bindweed (Convolvulus arvensis L.)

Twenty-eight day old field bindweed plants grown in culture solutions deficient in nitrogen (N) or phosphorus (P) for the last seven days of growth translocated significantly less foliarly applied dicamba (3,6-dichloro-o-anisic acid) and 2,4-D [(2,4-dichlorophenoxy) acetic acid] to their roots than did plants grown in complete nutrient solutions. In contrast, N deficiency stimulated basipetal translocation of glyphosate [N-(phosphonomethyl) glycine] and inhibited its acropetal translocation in field bindweed. Deficiencies of both N and P decreased translocation of dicamba from the treated area, but had no influence on translocation of glyphosate or 2,4-D from the treated area.Journal Article No. 4406 of the Agric. Exp. Stn., Oklahoma State University.     

Effects of phenoxy acid herbicides and glyphosate on nitrogenase activity (acetylene reduction) in root-associated Azospirillum, Enterobacter and Klebsiella

Abstract Nitrogenase activity (C₂H₂ reduction) in root-associated Azospirillum lipoferum, Klebsiella pneumoniae, Enterobacter agglomerans and Pseudomonas sp. isolated from roots of Finnish grasses was assayed in the presence of glyphosate, the phenoxy acid herbicides 2-methyl-4-chlorophenoxy acetic acid (MCPA), 2,4-dichlorophenoxy acetic acid (2,4-D), (±)-2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) and (±)-2-(2,4-dichlorophenoxy)propionic acid (dichlorprop), and the commercial products Roundup, Nurmikko-Hedonal, Mepro, and Dipro. In the presence of the phenoxy acid herbicides the nitrogenase activity of K. pneumoniae was significantly inhibited, but that of E. agglomerans was stimulated. With the exception of Mepro and mecoprop no phenoxy acid herbicides inhibited the nitrogenase activity of A. lipoferum and none that of Pseudomonas sp. Nurmikko-Hedonal considerably stimulated the nitrogenase activity of E. agglomerans , and Pseudomanas sp. On the other hand, the nitrogenase activity of both K. pneumoniae and E. agglomerans was considerably repressed by glyphosate and Roundup, which also inhibited the growth of the bacteria. These chemicals had no effect on the growth of A. lipoferum and Pseudomonas sp., but stimulated their nitrogenase activity.     

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues

Horizontal gene transfer (HGT) is part of prokaryotic life style and a major factor in evolution. In principle, any combinations of genetic information can be explored via HGT for effects on prokaryotic fitness. HGT mechanisms including transformation, conjugation, transduction, and variations of these plus the role of mobile genetic elements are summarized with emphasis on their potential to translocate foreign DNA. Complementarily, we discuss how foreign DNA can be integrated in recipient cells through homologous recombination (HR), illegitimate recombination (IR), and combinations of both, site-specific recombination, and the reconstitution of plasmids. Integration of foreign DNA by IR is very low, and combinations of IR with HR provide intermediate levels compared to the high frequency of homologous integration. A survey of studies on potential HGT from various transgenic plants indicates very rare transfer of foreign DNA. At the same time, in prokaryotic habitats, genes introduced into transgenic plants are abundant, and natural HGT frequencies are relatively high providing a greater chance for direct transfer instead of via transgenic plants. It is concluded that potential HGT from transgenic plants to prokaryotes is not expected to influence prokaryotic evolution and to have negative effects on human or animal health and the environment.     

Radiation hazard caused by the Chernobyl accident in inhabited areas of Ukraine can be monitored by transgenic plants

The genetic impact of the 1986 accident at the Ukraine Chernobyl Nuclear Power Plant (NPP) on populations of living organisms has yet to be fully assessed. Monitoring of the genotoxicity of polluted soils is a key element in the disaster management program. We used Arabidopsis thaliana and Nicotiana tabacum plants transgenic for a reporter gene revealing homologous recombination to study the genetic effects of chronic low-dose radiation stemming from the soil in inhabited areas of Ukraine where contamination by the accident ranges from 1 to 40 Ci/km2. We noted a significant dose-dependent increase of homologous recombination in plants cultivated in the affected inhabited areas, proving the persistently high genotoxicity of the radioactively contaminated soils.     

In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase

Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 μg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 μg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide.     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid