Comparative metabolism of 2,4-dichlorophenoxyacetic Acid in cotyledon and leaf callus from two varieties of soybean

Only quantitative differences were observed in the metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) by soybean cotyledon callus as influenced by variety (Acme and Amsoy), source of tissue (cotyledon, leaf and root) and age. All tissues metabolized 2,4-D to water-soluble (glycosides) and ether-soluble (primarily amino acid conjugates) metabolites.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Evidence for compartmentalization of conjugates of 2,4-dichlorophenoxyacetic Acid in soybean callus tissue

Soybean Glycine max L. Merrill var. Amsoy 71 root callus tissue labeled with [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) which was subsequently incubated for 24 hours in the absence of 2,4-D, released considerable amounts of label into the media. These results led to an examination of the efflux of 2,4-D and 2,4-D metabolites during a 6-hour time period. Fifty% of the free 2,4-D was lost in 15 minutes and 99% in 6 hours. After 6 hours, only about 48% of the ether-soluble fraction (mainly the glutamic and aspartic conjugates) and about 33% of the aqueous-soluble fraction (mainly hydroxylated glycosides) effluxed from the tissue. Neutral red efflux from stained callus tissue was enhanced only 5% above the control by treatment with 7.5% dimethylsulfoxide (DMSO) and 50% with 20% DMSO. Similar soybean callus tissue preincubated with [1-¹⁴C]2,4-D and subsequently incubated with H₂O, 7.5% DMSO, and 20% DMSO was examined for efflux of ¹⁴C label. DMSO similarly enhanced the efflux of the ether and aqueous soluble conjugates.

DMSO concentrations of less than 10% did not damage the vacuolar membranes which also has been reported with cultured tobacco cells (Delmer 1979 Plant Physiol 64: 623-629). From these data, it seems that the 2,4-D metabolites are located in a compartment of the cell and presumably the vacuole.

     

Metabolism of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Soybean Root Callus : EVIDENCE FOR THE CONVERSION OF 2,4-D AMINO ACID CONJUGATES TO FREE 2,4-D

An auxin-requiring soybean root callus metabolized [1-¹⁴C]-2,4-dichlorophenoxyacetic acid (2,4-D) to diethyl ether-soluble amino acid conjugates and water-soluble metabolites. The uptake in tissue varied with incubation time, concentration, and amount of tissue. Uptake was essentially complete (80%) after a 24-hour incubation and the percentage of free 2,4-D in the tissue fell to its lowest point at this time. At later times, the percentage of free 2,4-D increased and the percentage of amino acid conjugates decreased, whereas the percentage of water-soluble metabolites increased only slightly. Similar trends were seen if the tissue was incubated for 24 hours in radioactive 2,4-D, followed by incubation in media without 2,4-D for 24 hours. Inclusion of nonlabeled 2,4-D during the 24-hour chase period did not reduce amino acid conjugate disappearance but did reduce the percentage of free [1-¹⁴C]2,4-D. Thus, an external supply of 2,4-D does not directly prevent amino acid conjugate metabolism in this tissue. It is concluded that 2,4-D amino acid conjugates were actively metabolized by this tissue to free 2,4-D and water-soluble metabolites.     

The ambiguous role of 2,4-dichlorophenoxyacetic acid in wheat tissue culture

The very basal, highly immature regions of dissected young leaves of Triticum aestivum L. cv. Kite formed adventitious roots on a nutrient medium supplemented with comparatively low concentrations (0.16 to 0.63 μ M ) of 2,4-dichlorophenoxyacetic acid (2,4-D). Higher concentrations (up to 640 μ M ) had to be applied to stimulate growth from more mature regions higher up the leaf. Yet, already at 2.5 μ M roots were less distinct and more callus-like, and eventually (at 10 to 640 μ M ) only a subculturable callus of apparently suppressed, slowly proliferating root primordia developed. Furthermore, at the most basal, highly immature regions growth was significantly retarded when the auxin concentration was raised. The leaf culture system appears to reflect the dual action of 2,4-D known from herbicide research, namely growth stimulation from differentiating (or differentiated) cells, but growth suppression at or in the vicinity of apical meristems. Correspondingly, when the callus of apparently suppressed, slowly proliferating root primordia was transferred to media without 2,4-D or with low concentrations (0.16–2.5 μ M ) rapid proliferation commenced, leading to profuse root outgrowth. The system demonstrates the ambiguous role which this auxin appears to have, at least in wheat tissue culture.     

Uptake, transport and metabolism of 14C-2,4-dichlorophenoxyacetic acid (14C-2,4-D) in cucumber (Cucumis sativus L.) explants

The uptake, distribution and metabolism of 2,4-D using 14C-labelled 2,4-dichlorophenoxyacetic acid (14C-2,4-D) was studied in isolated hypocotyl and cotyledon explants of cucumber (Cucumis sativus L.) in vitro. Cotyledons had a higher uptake capacity than hypocotyls; the uptake in cotyledons increased linearly up to 20 h, while in hypocotyls only up to 8 h. The distribution of 14C-activity in both organs was basipetal, but more pronounced in cotyledons. The 2,4-D taken up by cotyledons was metabolized very rapidly: only 7% of 14C-activity was associated with free 2,4-D after 20 h exposure. Hypocotyls metabolized 2,4-D more slowly: after 20 h 50% of the 14C-activity was still associated with free 2,4-D. After short incubation periods (2–5 h) 2,4-D-aspartate was a predominant metabolite, after longer incubation (8–20 h) 2,4-D-glucosyl ester prevailed. Roots or callus were formed on the base of cotyledons depending on the length of exposure to 2,4-D.     

In vitro selection for 2,4-D tolerance in red clover

Summary In vitro, selection is a viable method of selecting herbicide-tolerant crops. This research was to evaluate in vitro selection techniques for enhancing 2,4-D [(2,4-dichlorophenoxy) acetic acid] tolerance in red clover (Trifolium pratense L.). In vivo and in vitro responses to 2,4-D of eight diverse red clover populations were correlated (r=0.77), justifying in vitro selection for 2,4-D tolerance. Suspension cultures of a red clover genotype capable of regeneration were plated onto agar-based nutrient media supplemented with 0.18 mM 2,4-D for selection experiments. After two cycles of selection, 16 2,4-D tolerant callus lines were identified based on visual growth assessment. These lines were evaluated for 2,4-D tolerance (based on 2,4-D content), using a 2,4-D bioassay procedure which consisted of placing selected callus tissue pieces on top of oat (Avena sativa L.) coleoptile or internode sections. The relative amount of 2,4-D in the callus tissue was estimated by the amount of oat section elongation after 24 h. Two of the more tolerant callus lines had 61% and 83% less 2,4-D in their tissues than the susceptible control tissue. These studies indicated that in vitro selection can enhance the levels of 2,4-D tolerance in red clover callus tissue.Florida Agricultural Experiment Station Journal Series No. 8943     

Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

Synchronized shoot regeneration of rice (Oryza sativa L.) calli on solid medium by adjustment of intracellular 2,4-dichlorophenoxyacetic acid concentration

The present research aimed to establish conditions for synchronized plantlet regeneration from rice callus based on a quantitative analysis of the relationship between intracellular 2,4-dichlorophenoxyacetic acid (2,4-D) concentration and shoot regeneration rate. To prepare the rice calli with different intracellular 2,4-D concentrations prior to regeneration, callus precultures were carried out in medium containing 4 mg/l 2,4-D and in 2,4-D-free medium for predetermined periods. As the critical intracellular 2,4-D concentration of the calli precultured in 2,4-D-free medium was too low to analyze precisely by conventional analytical methods, it was estimated using a kinetic model which described the behavior of 2,4-D by taking its uptake, metabolism and/or inactivation rates during the callus preculture into consideration. An experimental relationship between intracellular 2,4-D concentration and regeneration rate of rice calli revealed that the intracellular 2,4-D concentration should be controlled as low as 2.6×10^–2μg/g fresh weight to reach the same synchronization in shoot regeneration as seen with rice seed germination. This condition was realized by feeding sugar into the 2,4-D-free medium after 4 days preculture when the carbon source was exhausted. Received: 29 June 1998 / Revision received: 24 September 1998 / Accepted: 27 October 1998     

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.     

The regeneration potential of wheat shoot meristems in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Tissue cultures capable of plant regeneration were successfully initiated from extremely immature shoot meristems of 21 randomly selected genotypes of wheat on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). By means of scanning electron microscopy it was demonstrated that cultures consisted of teratomatous primordia, which were kept in a proliferating budding state by the 2,4-D. These are characteristic of cereal tissue cultures. Release of the primordia and outgrowth of normal shoots and roots occurred when the cultures were no longer exposed to 2,4-D. Shoot primordia which were clearly identifiable were always associated with root primordia in a quasi-bipolar fashion. Sometimes regions assumed the shape of zygotic embryos, but the transition from apparently normal embryos with scutellum to abnormal configurations with shoot and root regions was gradual. The differences between genotypes in shoot regeneration potential was minimal compared to cultures derived from explants which were taken from regions temporally and spatially more distant from the shoot apex. It is concluded that the ability to give rise to cultures capable of shoot regeneration was lost within a fraction of a millimeter distance from the apical meristem in many genotypes. The proliferating tissues were subcultured at regular intervals over a period of one year and the regeneration potential was monitored. Areas capable of shoot regeneration tended to deteriorate more or less rapidly and were overgrown by root-type tissue in a number of genotypes. The results are discussed in the context of the frequently observed, but largely unexplained, variability in the regeneration potential of cereal tissue cultures.     

Biological Properties of d-Amino Acid Conjugates of 2,4-D

Some d-amino acid (glutamic acid, valine, or leucine) conjugates of 2,4-dichlorophenoxyacetic acid (2,4-D) at 10⁻⁵ molar, stimulated elongation of Avena sativa L. var Mariner coleoptile sections and growth of soybean (Glycine max. L. var Amsoy) tissue as much as did the l-amino acid conjugates at 10⁻⁶ molar. The d-methionine conjugate did not stimulate growth of soybean root callus tissue but did stimulate Avena elongation. The d-aspartic acid conjugate did not stimulate elongation of Avena coleoptiles but did stimulate growth of root callus tissue.     

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Inhibition of 2,4-dichlorophenoxyacetic Acid conjugation to amino acids by treatment of cultured soybean cells with cytokinins

Kinetin, and all other cytokinins tested, inhibited the conjugation of [¹⁴C]2,4-dichlorophenoxyacetic acid (2, 4-D) to amino acids when supplied simultaneously with the 2,4-D to cultured soybean cells. Upon transfer to hormone-free medium, the cytokinin-treated cells released more of their [¹⁴C]2,4-D than did the control cells. Initial exposure to low 2,4-D and high kinetin levels resulted in the greatest release of 2,4-D upon subsequent transfer. The observed alteration in 2,4-D metabolism did not seem to be correlated with growth rate. Appropriate treatment of soybean cells with kinetin resulted in 2,4-D metabolism that resembled the 2,4-D metabolism of embryogenic carrot cells. However, no new morphological structures were observed in these soybean cultures, indicating that other factors are related to the failure of soybean cells to regenerate in culture.     

Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.)

 Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2–4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium. Received: 1 December 1997 / Revision received: 15 February 1999 / Accepted: 26 February 1999     

The relative amounts and identification of some 2,4-dichlorophenoxyacetic Acid metabolites isolated from soybean cotyledon callus cultures

Soybean (Glycine max L.) cotyledon callus grown on radioactive 2,4-dichlorophenoxyacetic acid (2,4-D-1-¹⁴C) as an auxin produced 2,4-D metabolites, which qualitatively and quantitatively changed with time. Water soluble fractions from the tissue exhibited a steady increase in radioactivity during the course of 24 days. Following β-glucosidase treatment, at least eight aglycones were obtained from the water soluble fraction of the tissue after 8 days. The metabolite, 4-hydroxy-2,5-dichlorophenoxyacetic acid was the most abundant aglycone during the entire 32 day growth period while 4-hydroxy-2,3-dichlorophenoxyacetic acid was detected as a minor metabolite. Radioactivity in the ether soluble acidic fractions reached a maximum of 82% of the total in the tissue after 2 days. The level then decreased to 44% by the end of 24 days. A total of seven ether soluble components were detected. In addition to 2,4-D glutamic acid, which was detected in high amounts after 24 hours, 2,4-D aspartic acid was found to be the most abundant ether soluble metabolite after longer time periods. Mass spectral data and a fragmentation pattern are presented for 2,4-D aspartic acid.     

Preliminary observations on organogenesis inTaraxacum officinale tissue cultures

Summary Tissue cultures ofTaraxacum officinale have been isolated from the secondary thickened root. Callus development and leaf and root formation occur on a basal medium supplemented with coconut milk and IAA or NAA, and the addition of kinetin to these media enhances callus growth and organogenesis. Cultures grown on the basal medium with coconut milk and 2,4-D show only callus growth, but organogenesis is induced by the substitution of IAA for 2,4-D. In the 2,4-D grown callus a layer of secondary meristematic tissue is present and organogenesis apparently occurs from localized regions of this tissue which have undergone de-differentiation to the primary meristematic condition.     

Morphogenetic effects of 2,4-dichlorophenoxyacetic acid on pinto bean (Phaseolus vulgaris L.) leaf explants in vitro

Roots, callus and/or globular structures were produced on primary leaf and distal cotyledon explants of pinto bean (Phaseolus vulgaris L. cv. UI 114) cultured on semisolid MS medium with a wide range of 2,4-D concentrations (0.01 to 80 mg/L) with either 0 or 1.0 mg/L kinetin. Explants rooted at lower 2,4-D concentrations than at those favoring globule formation on callus, although roots, callus and globules often developed from the same explant. Isolated opaque green globular structures developed when callus initiated on media with 3 or more mg/L 2,4-D was subcultured in liquid MS + 30 mg/L 2,4-D. These structures multiplied with a fresh weight doubling time of 8–9 days in MS + 30 mg/L 2,4-D. Although this multiplicative behavior and opaque color were reminiscent of embryoids reported for other species, no cotyledons or roots were seen.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - MS Murashige-Skoog medium Cooperative investigations of the Agricultural Research Service, U.S. Department of Agriculture and the Michigan Agricultural Experiment Station, East Lansing, Michigan 48824. Michigan Agricultural Experiment Station Journal article No. 11923     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2–3 μg ml⁻¹ for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 μg ml⁻¹. This revised version was published online in July 2006 with corrections to the Cover Date.