A rapid method to screen degradation ability in chlorophenoxyalkanoic acid herbicide-degrading bacteria

AIMS: An agar medium containing a range of related chlorophenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop and racemic 2,4-DP (2-(2,4-dichlorophenoxy) propionic acid) was developed to assess the catabolic activity of a range of degradative strains. METHODS AND RESULTS: The medium was previously developed containing 2,4-D as a carbon source to visualise degradation by the production of dark violet bacterial colonies. Strains isolated on mecoprop were able to degrade 2,4-D, MCPA, racemic mecoprop, (R)-mecoprop and racemic 2,4-DP, whereas the 2,4-D-enriched strains were limited to 2,4-D and MCPA as carbon sources. Sphingomonas sp. TFD44 solely degraded the dichlorinated compounds, 2,4-D, racemic 2,4-DP and 2,4-DB (2,4-dichlorophenoxybutyric acid). However, Sphingomonas sp. AW5, originally isolated on 2,4,5-T, was the only strain to degrade the phenoxybutyric compound MCPB (4-chloro-2-methylphenoxybutyric acid). CONCLUSION: This medium has proved to be a very effective and rapid method for screening herbicide degradation by bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This method reduces the problem of assessing the biodegradability of this family of compounds to an achievable level.     

Comparative study on cerebrovascular injuries by three chlorophenoxyacetic acids (2,4-D, 2,4,5-T and MCPA)

At toxic doses in rats, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) caused injuries in blood vessels in the brain. 2. 2,4-D caused extravasations of the circulating Evans blue-albumin complex also in the spinal cord. 3. By contrast, potency in damaging cerebral vessels was almost non-existent with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). 4. None of the three chlorophenoxyacetic acids caused such cerebrovascular injuries in mice, guinea pigs, Syrian hamsters, rabbits and chickens.     

Mineralization of 2,4-dichlorophenoxyacetic acid in soil previously enriched with organic substrates

Samples of chernozem soil were enriched with vanillic acid, protocatechuic acid glucose, a mixture of glucose and (NH₄)₂SO₄ (C∶N = 5∶1), ethanol and 2,4-dichlorophenoxyacetic acid (2,4-D). After a 6-d (with 2,4-D 35-d) incubation during which primary oxidation of the introduced substrates occurred, the soil was supplied with a solution of 2-¹⁴C-2,4-D (50ppm; 6.7kBq) and production of¹⁴CO₂ (product of microbial degradation of 2,4-D) was measured. Previously enriched samples exhibited a higher degradation rate; both the lag phase and doubling time of mineralization activity in the exponential phase of the process were markedly higher. This reflected an overall proliferation of bacteria and the increased relative proportion of bacterial strains capable of mineralizing 2,4-D in enriched samples. The stimulation of 2,4-D degradation may involve specific adaptation and selection mechanisms (as in the case with samples previously enriched with 2,4-D or its structural analogues—aromatic monomers, ethanol) as well as nonspecific mechanisms. The extent of mineralization of 2,4-D was not affected by soil pretreatment, about 1/3 of introduced radioactive carbon being invariably transformed to¹⁴CO₂.     

Genetic transformation of willows (Salix spp.) byAgrobacterium tumefaciens

Anin vitro transformation method has been developed for stem explants of fast-growing willow clones (Salix spp.) usingAgrobacterium tumefaciens as a vector. Transformants obtained with the strains C58 and GV3101 (pGV3851::pLD1) were selected on hormone-free medium and on medium containing kanamycin, respectively. Transformation was confirmed by Southern blot analysis and nopaline assay. Inoculation of green-house grown plants with nopaline and octopine wildtype strains and shoot or root inducing mutant strains caused undifferentiated tumors at a frequency of 0 to 80%, depending on theSalix genotype and the bacterial strain used.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - NPT neomycin phosphotransferase     

The biological detoxication of hormone herbicides in soil

Summary (1) The results of experiments on the continuous perfusion of aerated solutions of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2, Methyl-4, chlorophenoxyacetic acid (MCPA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) through garden soil indicate that the kinetics of their breakdown are essentially similar.(2) Three phases can be distinguised: (a) an immediate initial adsorption onto soil colloids amounting to 0.167±0.035 mg per g dry soil, (b) a lag phase of varying duration in which there is little or no disappearance of herbicide, (c) a final phase of rapid complete detoxication. Such enriched soils detoxicate subsequent perfusions with the same herbicide molecule at a constant high rate. Enrichment times for 2,4-D, MCPA and 2,4,5-T are roughly of the order or 14, 70 and 270 days respectively.(3) This indirect evidence, from kinetics of detoxication, of the development of a bacterial flora adapted to herbicide breakdown, is strongly supported by experiments in which the detoxicating activity of such enriched soils was completely destroyed by low concentrations (0.01%) of the bacterial poison, sodium azide.(4) Attempts to isolate in pure culture the 2,4-D-decomposing organism were successful. Preliminary classification work relegates the organism to the Bacterium globiforme group. Further confirmatory work is required.(5) Cross perfusion experiments in which 2,4-D enriched soils were perfused with MCPA and vice versa indicate that both can detoxicate the other analogue, although at a somewhat lower rate than the soil directly enriched with that analogue.(6) 2,4-D enriched soil will not detoxicate 2,4,5-T although some activity may be temporarily induced by an intermediate perfusion with MCPA.     

Biodegradation of endosulfan and endosulfan sulfate by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO₂ from the culture media with endosulfan as sulfur source as compared to MgSO₄ suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.     

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.     

Chlorinated phenoxyacetic acids and chlorophenols in the modified Allium test

The chlorinated phenoxyacetic acids 2-methyl-4-chlorophenoxyacetic acid (MCPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4,5-T butoxyethyl ester and the chlorophenols 2,4-dichlorophenol and 2,4,5-trichlorophenol were tested for genotoxicity in the modified Allium test, which is based on exposure to the test chemicals of growing onions. The mean length of growing roots were measured and chromosome damage was recorded. Of the substances tested, MCPA was the most toxic and the chlorophenoxyacetic acids were more toxic than the chlorophenols. The lower threshold values for growth retardation were below 0.1 ppm for the acids, approx. at 0.1 ppm for the ester and less than 5 ppm for the phenols. Though a monocotyledon, Allium cepa was sensitive enough to respond to even low concentrations of these dicotyledon-selecting pesticides.     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Effect of inoculant strain and organic matter content on kinetics of 2,4-dichlorophenoxyacetic acid degradation in soil.

We monitored rates of degradation of soluble and sorbed 2,4-dichlorophenoxyacetic acid (2,4-D) in low-organic-matter soil at field capacity amended with 1, 10, or 100 micrograms of 2,4-D per g of wet soil and inoculated with one of two bacterial strains (MI and 155) with similar maximum growth rates (mu max) but significantly different half-saturation growth constants (Ks). Concentrations of soluble 2,4-D were determined by analyzing samples of pore water pressed from soil, and concentrations of sorbed 2,4-D were determined by solvent extraction. Between 65 and 75% of the total 2,4-D was present in the soluble phase at equilibrium, resulting in soil solution concentrations of ca. 8, 60, and 600 micrograms of 2,4-D per ml, respectively. Soluble 2,4-D was metabolized preferentially; this was followed by degradation of both sorbed (after desorption) and soluble 2,4-D. Rates of degradation were comparable for the two strains at soil concentrations of 10 and 100 micrograms of 2,4-D per g; however, at 1 microgram/g of soil, 2,4-D was metabolized more rapidly by the strain with the lower Ks value (strain MI). We also monitored rates of biodegradation of soluble and sorbed 2,4-D in high-organic-matter soil at field capacity amended with 100 micrograms of 2,4-D per g of wet soil and inoculated with the low-Ks strain (strain MI). Ten percent of total 2,4-D was present in the soluble phase, resulting in a soil solution concentration of ca. 30 micrograms of 2,4-D per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inoculant strain and organic matter content on kinetics of 2,4-dichlorophenoxyacetic acid degradation in soil.

We monitored rates of degradation of soluble and sorbed 2,4-dichlorophenoxyacetic acid (2,4-D) in low-organic-matter soil at field capacity amended with 1, 10, or 100 micrograms of 2,4-D per g of wet soil and inoculated with one of two bacterial strains (MI and 155) with similar maximum growth rates (mu max) but significantly different half-saturation growth constants (Ks). Concentrations of soluble 2,4-D were determined by analyzing samples of pore water pressed from soil, and concentrations of sorbed 2,4-D were determined by solvent extraction. Between 65 and 75% of the total 2,4-D was present in the soluble phase at equilibrium, resulting in soil solution concentrations of ca. 8, 60, and 600 micrograms of 2,4-D per ml, respectively. Soluble 2,4-D was metabolized preferentially; this was followed by degradation of both sorbed (after desorption) and soluble 2,4-D. Rates of degradation were comparable for the two strains at soil concentrations of 10 and 100 micrograms of 2,4-D per g; however, at 1 microgram/g of soil, 2,4-D was metabolized more rapidly by the strain with the lower Ks value (strain MI). We also monitored rates of biodegradation of soluble and sorbed 2,4-D in high-organic-matter soil at field capacity amended with 100 micrograms of 2,4-D per g of wet soil and inoculated with the low-Ks strain (strain MI). Ten percent of total 2,4-D was present in the soluble phase, resulting in a soil solution concentration of ca. 30 micrograms of 2,4-D per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in soil inoculated with Pseudomonas cepacia DBO1(pRO101), Alcaligenes eutrophus AEO106(pRO101) and Alcaligenes eutrophus JMP134(pJP4): effects of inoculation level and substrate concentration

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) by two Alcaligenes eutrophus strains and one Pseudomonas cepacia strain containing the 2,4-D degrading plasmids pJP4 or pRO101 (=pJP4::Tn1721) was tested in 50 g (wet wt) samples of non-sterile soil. Mineralization was measured as ¹⁴C-CO₂evolved during degradation of uniformly-ring-labelled ¹⁴C-2,4-D. When the strains were inoculated to a level of approximately 10⁸ CFU/g soil, between 20 and 45% of the added 2,4-D (0.05 ppm, 10 ppm or 500 ppm) was mineralized within 72 h. Mineralization of 0.05 ppm and 10 ppm, 2,4-D by the two A. eutrophus strains was identical and rapid whereas mineralization by P. cepacia DBO1(pRO101) occurred more slowly. In contrast, mineralization of 500 ppm 2,4-D by the two A. eutrophus strains was very slow whereas mineralization by P. cepacia DBO1 was more rapid. Comparison of 2,4-D mineralization at different levels of inoculation with P. cepacia DBO1(pRO101) (6×10⁴, 6×10⁶ and 1×10⁸ CFU/g soil) revealed that the maximum mineralization rate was reached earlier with the high inoculation levels than with the low level. The kinetics of mineralization were evaluated by nonlinear regression analysis using five different models. The linear or the logarithmic form of a three-half-order model were found to be the most appropriate models for describing 2,4-D mineralization in soil. In the cases in which the logarithmic form of the three-half-order model was the most appropriate model we found, in accordance with the assumptions of the model, a significant growth of the inoculated strains.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CFU colony forming units - PTYG peptone, tryptone, yeast & glucose - DPM disintegrations per minute     

Isolation and characterization of a stable 2,4-dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus, using chemostat culture

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway. © Rapid Science Ltd. 1998     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

The Effects of Cyanobacterial Exudates on Bacterial Growth and Biodegradation of Organic Contaminants

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.     

Isolation and characterization of a 2,4-dichlorophenoxyacetic acid-degrading soil bacterium

Summary A 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, Xanthobacter sp. CP, was isolated after enrichment in aerated soil columns. A limited number of chlorinated phenols and chlorinated phenoxyalkanoic acids with an even number of carbon atoms in the side chain served as substrates for growth, although whole cells exhibited oxygen uptake with a wide range of those compounds. The maximal growth rate with 2,4-D was 0.13·h^-1 at a growth yield of 0.1 g biomass/g 2,4-D. Chloride ions were released quantitatively from 2,4-D and related chlorinated aromatic compounds which served as growth substrates. No by-products of 2,4-D metabolism were detected in oxygen-sufficient cultures of Xanthobacter sp. CP and catechols were cleaved exclusively by catechol 1,2-dioxygenase.     

Retention behavior of phenoxyacetic herbicides on a molecularly imprinted polymer with phenoxyacetic acid as a dummy template molecule

Molecular imprinted polymers (MIPs) binding with phenoxyacetic acid (PA) as a dummy template molecule were synthesized via thermal initiation in aqueous medium. The retention behaviors of benzoic acid (BA), PA, 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-chlorophenoxyacetic acid (4-CPA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on this MIP column indicate that this material can selectively retain phenoxyacetic herbicides. To investigate these recognition mechanisms, the interactions between the functional monomer 4-vinylpyridine (4-VP) and PA or 2,4-D were investigated by computational modeling. (1)H NMR spectroscopy of 2,4-D titrated by 4-VP was recorded. The chemical shift of the 2,4-D acidic proton (12.15-14.32ppm) shows the existence of the ion-pair interaction. This kind of polymers could be useful as stationary phases to extract 2,4-D, 4-CPA or MCPA and avoid leakage of a trace amount of target analyte remaining in the MIPs.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium

Summary The 2-(2,4-dichlorphenoxy)propionic acid (2,4-DP)-degrading bacterial strain MH was isolated after numerous subcultivations of a mixed culture obtained by soil-column enrichment and finally identified as Flavobacterium sp. Growth of this strain was supported by 2,4-DP (maximum specific growth rate 0.2 h^–1) as well as by 2,4-dichlorophenoxyacetic acid (2,4-D), 4(2,4-dichlorophenoxy)butyric acid (2,4-DB), and 2-(4-chloro-2-methyphenoxy)propionic acid (MCPP) as sole sources of carbon and energy under aerobic conditions. 2,4-DP-Grown cells (10⁸) of strain MH degraded 2,4-dichlorophenoxyalkanoic acids, 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol at rates in the range of 30 nmol/h. Preliminary investigations indicate that cleavage of 2,4-DP results in 2,4-DCP, which is further mineralized via ortho-hydroxylation and ortho-cleavage of the resulting 3,5-dichlorocatechol. Offprint requests to: F. Streichsbier     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency