Effects of three herbicides on soil microbial biomass and activity

Three post-emergence herbicides (2,4-D, picloram and glyphosate) were applied to samples of an Alberta agricultural soil at concentrations of 0, 2, 20, and 200 μg g⁻¹. The effects of these chemicals on certain microbial variables was monitored over 27 days. All herbicides caused enhancement of basal respiration but only for 9 days following application, and only for concentrations of 200 μg g⁻¹. Substrate-induced respiration was temporarily depressed by 200 μg g⁻¹ picloram and 2,4-D, and briefly enhanced by 200 μg g⁻¹ glyphosate. It is concluded that because changes in microbial variables only occurred at herbicide concentrations of much higher than that which occurs following field application, the side-effects of these chemicals is probably of little ecological significance.     

Influence of herbicide, 2,4-dichlorophenoxy acetic acid, on haemocyte DNA of in vivo treated mussel

The influence of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) on haemocyte DNA of in vivo treated mussels Mytilus galloprovincialis has been investigated by flow cytometry and epifluorescence microscopy. Haemocyte proliferation and atypical flow cytometric DNA histograms were observed in mussels treated with 20 and 100 μg/g of 2,4-D. The stimulation of proliferation by 2,4-D was also obvious by DNA labelling with BrdU followed by FITC conjugated anti-BrdU MoAb visualised by epifluorescence microscopy. An apoptotic sub-G₁ peak resulted in mussels that were exposed to higher doses of herbicide at 100 and 500 μg/g as well as subpopulation could be detected by flow cytometric analysis. In these experiments morphological changes characteristic for apoptotic cells were looked for by fluorescence microscopy. A low percentage of cells in S as well as in G₂M phase indicating G1 arrest were detected in haemocytes from these mussels that had survived 4 days of 20 μg/g 2,4-D exposure. In addition, sister-chromatid exchanges (SCE) could be seen with the immunolabelling BrdU method. Thus, in vivo treatment and the subsequent uptake of 2,4-D causes serious genetic consequences and raises concerns regarding the potential overall fitness and health effects in mussel populations.     

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of glucose on the fatty acid composition of Cupriavidus necator JMP134 during 2,4-dichlorophenoxyacetic acid degradation: implications for lipid-based stable isotope probing methods

Combining lipid biomarker profiling with stable isotope probing (SIP) is a powerful technique for studying specific microbial populations responsible for the degradation of organic pollutants in various natural environments. However, the presence of other easily degradable substrates may induce significant physiological changes by altering both the rate of incorporation of the target compound into the biomass and the microbial lipid profiles. In order to test this hypothesis, Cupriavidus necator JMP134, a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium, was incubated with [(13)C]2,4-D, [(13)C]glucose, or mixtures of both substrates alternatively labeled with (13)C. C. necator JMP134 exhibited a preferential use of 2,4-D over glucose. The isotopic analysis showed that glucose had only a small effect on the incorporation of the acetic chain of 2,4-D into the biomass (at days 2 and 3) and no effect on that of the benzenic ring. The addition of glucose did change the fatty acid methyl ester (FAME) composition. However, the overall FAME isotopic signature reflected that of the entire biomass. Compound-specific individual isotopic analyses of FAME composition showed that the (13)C-enriched FAME profiles were slightly or not affected when tracing the 2,4-D acetic chain or 2,4-D benzenic ring, respectively. This batch study is a necessary step for validating the use of lipid-based SIP methods in complex environments.     

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Mineralization of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and Mixtures of 2,4-D and 2,4,5-Trichlorophenoxyacetic Acid by Phanerochaete chrysosporium

Evidence is presented for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in nutrient-rich media (high-nitrogen and malt extract media) by wild-type Phanerochaete chrysosporium and by a peroxidase-negative mutant of this organism. Mass balance analysis of [U-ring-¹⁴C]2,4-D mineralization in malt extract cultures showed 82.7% recovery of radioactivity. Of this, 38.6% was released as ¹⁴CO₂ and 27.0, 11.2, and 5.9% were present in the aqueous, methylene chloride, and mycelial fractions, respectively. 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were simultaneously mineralized when presented as a mixture, and mutual inhibition of degradation was not observed. In contrast, a relatively higher rate of mineralization of 2,4-D and 2,4,5-T was observed when these compounds were tested as mixtures than when they were tested alone.     

Effect of certain herbicide treatments on pasture composition and inoculum of the take-all fungus

The improvement of pastures by the use of a range of herbicides to eliminate grasses, and their effect on populations of the take-all fungus (Gaeumannomyces graminis vartritici=Ggt) were studied in the field (at Esperance Downs, on the south-coast of Western Australia) from 1982 to 1985. Field trials were conducted to evaluate three herbicide treatments (2,4-D amine+propyzamide; 2,4-D amine+paraquat; paraquat/ diquat) and an unsprayed control. A pot trial involving these treatments with two levels of nitrogen was undertaken to confirm treatment effects observed in the field trial. All herbicide treatments resulted in reduced grass composition of pastures, in both the year of spraying and in the second year of pasture, but reduced dry matter production in the year of spraying. In the year of spraying, however, inoculum ofGgt was reduced (P<0.1) only following the 2,4-D amine+propyzamide treatment and was greater (P<0.1) after 2,4-D amine+paraquat treatment than the unsprayed treatment. Despite reduced grass levels in the herbicide-treated plots in the second year of pasture,Ggt inoculum did not differ between treatments, nor did it after a wheat crop which followed a second year pasture. There was high correlation (P<0.001) between disease levels and dry weights of grasses in the pot trial. There was significantly less (P<0.001) grass in pots treated with herbicides compared to the unsprayed control but no difference (P>0.05) was evident between treatments. Inoculum levels were lower (P<0.05) in the treated pots than the unsprayed control with no evidence of differences among treatments (P>0.05). Nitrogen level had no effect on disease (P>0.05). All herbicide treatments tested reduced grass level and total dry matter, both in the field and in pots. Whereas in the pot trial reduced grass levels resulted in reducedGgt inoculum, in the field such a reduction occurred only with the 2,4-D amine+propyzamide treatment and only in the year of spraying. Herbicide treatments had no effect onGgt inoculum in second year of pasture or crop. Unknown soil and environmental factors in the field precluded a simple relationship between grass level in pasture and subsequent level ofGgt inoculum, and where such a relationship did occur (2,4-D amine+propyzamide treatment) it appeared to be shortlived.     

Monitoring gene expression in mixed microbial communities by using DNA microarrays

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.     

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。     

Bacterial, Fungal, and Actinomycete Populations in Soils Receiving Repeated Applications of 2,4-Dichlorophenoxyacetic Acid and Trifluralin

Soil samples were collected from an untreated plot and plots receiving repeated applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and alpha,alpha,alpha-trifluoro-2, 6-dinitro-N,N-dipropyl-p-toluidine (trifluralin); they were then plated on media specific for bacteria, fungi, and actinomycetes. The actinomycete colony count in the trifluralin-treated plot was greater than the control, but the same as the control in the 2,4-D-treated plot. The bacterial count was lower in both treated plots. Fungal colonies in the trifluralin-treated plots were greater than the control, but not different from the control in the 2,4-D-treated plot.     

Effects of elicitation on the production of saponin in cell culture of Panax ginseng

This study was initiated to investigate the impacts of elicitor concentration and elicitor-adding time on the saponin synthesis and the cell growth of Panax ginseng cell suspensions. Both of the elicitors tested, yeast extract and methyl jasmonate, significantly improved saponin production. The highest additive level of the seven ginsenosides tested was 2.07% (dry weight basis), which was 28-fold higher than that in the control. The optimum time to add either elicitor was found to be on the day of inoculation. The addition of either elicitor did not show as significant an influence on cell growth as on saponin production. It was advisable to remove 2,4-dichlorophenoxyacetic acid (2,4-D) from the medium when methyl jasmonate was used as the elicitor as methyl jasmonate interacts antagonistically with 2,4-D. These results suggest that the addition of an elicitor to ginseng cell suspension cultures could stimulate saponin production.     

Microbial degradation of 2,4-dichlorophenoxyacetic acid on the Greenland ice sheet

The Greenland ice sheet (GrIS) receives organic carbon (OC) of anthropogenic origin, including pesticides, from the atmosphere and/or local sources, and the fate of these compounds in the ice is currently unknown. The ability of supraglacial heterotrophic microbes to mineralize different types of OC is likely a significant factor determining the fate of anthropogenic OC on the ice sheet. Here we determine the potential of the microbial community from the surface of the GrIS to mineralize the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Surface ice cores were collected and incubated for up to 529 days in microcosms simulating in situ conditions. Mineralization of side chain- and ring-labeled [(14)C]2,4-D was measured in the samples, and quantitative PCR targeting the tfdA genes in total DNA extracted from the ice after the experiment was performed. We show that the supraglacial microbial community on the GrIS contains microbes that are capable of degrading 2,4-D and that they are likely present in very low numbers. They can mineralize 2,4-D at a rate of up to 1 nmol per m(2) per day, equivalent to ～26 ng C m(-2) day(-1). Thus, the GrIS should not be considered a mere reservoir of all atmospheric contaminants, as it is likely that some deposited compounds will be removed from the system via biodegradation processes before their potential release due to the accelerated melting of the ice sheet.     

Impacts of 2,4-D application on soil microbial community structure and on populations associated with 2,4-D degradation

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) application rate on microbial community structure and on the diversity of dominant 2,4-D degrading bacteria in an agricultural soil was examined using cultivation-independent molecular techniques coupled with traditional isolation and enumeration methods. Fingerprints of microbial communities established under increasing concentrations of 2,4-D (0-500 mg kg-1) in batch soil microcosms were obtained using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments. While a 2,4-D concentration of at least 100 mg kg-1 was required to obtain an apparent change in the community structure as visualized by DGGE, the greatest impact of 2,4-D concentration occurred in the 500 mg kg-1 treatment, resulting in significantly reduced diversity of the dominant populations and enrichment by Burkholderia-like populations. The greatest diversity of 2,4-D degrading isolates was cultivated from the 10 mg kg-1 treatment, indicating that under these conditions, cultivation was more sensitive than DGGE for detecting changes in community structure. Most of these isolates harbored homologs of Ralstonia eutrophus JMP134 and Burkholderia cepacia tfdA catabolic genes. Results from this study revealed that agriculturally relevant application rates of 2,4-D may provide a temporary selective advantage for organisms capable of utilizing 2,4-D as a carbon and energy source.     

Influence of 2,4-D, IAA, and duration of callus induction in anther cultures of spring wheat

Culture media and environmental factors may significantly influence the yield of haploid plants from anther cultures. Our objectives were to identify a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) concentrations which produce the maximum number of haploid plants, and to evaluate the effects of duration in induction medium on calli induction, plant regeneration, and green plant production from anther cultures in spring wheat. Significant (P ≤ 0.01) plant growth regulator concentration effects (2,4-D and IAA) were observed on the number of calli, green plants and albino plants produced, and on direct plant regeneration. Addition of 2,4-D to the induction medium resulted in significantly (P ≤ 0.01) higher means for all anther culture components compared to IAA> While addition of 2,4-D significantly (P ≤ 0.01) reduced plant regeneration, it substantially increased green plant percentage at a 0.3-mg l⁻¹ concentration of IAA. Use of response functions to estimate the maximum effective 2,4-D × IAA combination implied that higher 2,4-D levels in the induction medium should be investigated, and that the optimum hormone combination differs for plant regeneration and green plant percentage. Significant (P ≤ 0.01) effects of duration on callus induction medium were observed for plant regeneration and green plant percentage.     

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.