Meiotic and isozymic characterization of plants regenerated from euploid and selfed monosomic tall fescue embryos

Summary Tissue culture of tall fescue (Festuca arundinacea Schreb., 2n=6x=42) would be enhanced by improving the callus induction and plant regeneration efficiency, and evaluating the meiotic and isozymic variation induced by culture. Mature embryos were cultured from four lines of Kenhy tall fescue and from the progeny of three selfed monosomics. Evaluation of six media-auxin combinations showed callus initiation was greatest on SH medium with 2.5 mg/l 2,4,5-T or 7.4 mg/l pCPA, while plant regeneration was greatest on SH medium with 0.5 mg/l 2,4-D. Cytological analyses of 27 plants derived from euploid parents showed a high frequency of aneuploidy (15/27). Chromosome numbers of aneuploids ranged from 36 to 41, with one plant having 80 chromosomes and two plants being asynaptic. Two of ten monosomic-derived plants were euploid, five were monosomic, one was monosomic with a fragment and two were double monosomic. Zymograms of the parents and regenerants were obtained for the enzymes ACPH, ADH, GOT, 6-PGD and PGI. Isozyme variation was observed for two groups of plants derived from the same Kenhy embryos. One group of four monosomic-derived plants differed for the enzymes GOT and ACPH, and all four plants had a PGI pattern. different from that of the parental monosomic plant. This indicated loss of a PGI allele, probably as a result of callus culture.Contribution No. 89-3-141 of the Kentucky Agricultural Experiment Station in cooperation with the USDA-ARS. Part of thesis research for senior author's M. S. degree     

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l^-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic calli - NE nonembryogenic calli - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Plant regeneration from in vitro cultures of anthers and mature seeds of rice (Oryza sativa L.) cv. Basmati-370

Pollen plants were obtained from anther-derived calli of the indica rice variety Basmati-370. Anther-response (anthers producing pollen derived calli) and plant regeneration frequency from the pollen derived calli. was very low. Donor plants which flowered at the average max/min. temperature of 34.2°/23.3°C gave a significantly higher anther-response to in vitro techniques, than did those which flowered at 29.1°/16.4°C. Somatic callus induction and subsequent plant regeneration was readily obtained from mature seed embryos. While 2,4-D or 2,4,5-T (1 or 2 mg/l) proved highly efficient for callus induction, tryptophan (50 or 100 mg/l) induced a high frequency of green plants from the calli.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - K kinetin - BA benzyladenine - Trp tryptophan - CW coconut water     

Long-duration,high-frequency plant regeneration from cereal tissue cultures

By visual examination of calli derived from germinating seeds of wheat, oats, rice, proso millet, and pearl millet it has been possible to visually select embryogenic (E) callus which, on transfer to a regeneration medium, forms plants an average of 33 times more frequently than non-embryogenic (NE) callus of equal mass. Embryogenic callus consists of small isodiametric cells averaging 31 m in diameter; NE callus consists of long tubular cells averaging 52 m in width and 355 m in length. Production of E callus is in many cases promoted by media containing 2,4-di- or 2,4,5-trichlorophenoxyacetic acid (2,4-D or 2,4,5-T) plus indole-3-acetic acid or tryptophan+kinetin. Production on NE callus is promoted by media containing 2,4-D or 2,4,5-T alone. As a result of initial experiments to optimize both media for E callus production and media for plant regeneration, callus derived in six passages from an average of 26 seeds could produce about 1,000 regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - Trp L-tryptophan - E embryogenic - NE non-embryogenic     

Heritable somaclonal variation in wheat

Summary Efficient tissue culture and regeneration methods were established using immature wheat embryos as expiants. Genotype differences in culturability were evident, and from the ten accessions most amenable to culture, a total of 2,846 plants were regenerated. Extensive somaclonal variation for morphological and biochemical traits was observed among 142 regenerants of a Mexican breeding line, Yaqui 50E, and their progeny. Variant characters included height, awns, tiller number, grain colour, heading date, waxiness, glume colour, gliadin proteins and -amylase regulation. The variant characters were heritable through two seed generations and included traits under both simple and quantitative genetic control. Segregation data suggested that mutations both from dominance to recessiveness, and from recessiveness to dominance, had occurred. Most mutations in the primary regenerants were in the heterozygous state but some were true-breeding and presumed to be homozygous. Chromosome loss or addition did not account for the variation and none of the variant phenotypes was observed in over 400 plants from the parental seed source. The distinctive parental gliadin pattern was maintained in the somaclones thus excluding seed contamination or cross-pollination as a source of the variation.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - 2,4,5-T 2,4,5-trichlorophenoxy acetic acid - IAA indole acetic acid - BAP 6-benzyl amino purine - ABA abscisic acid - GA₃ gibberellic acid - DAP days after planting     

Plant regeneration from mesocotyl callus of Hordeum vulgare L.

Callus tissue was induced on barley mesocotyl explants of germinated seven-day-old seedlings on MS medium supplemented with 2,4-D or 2,4,5-T in high concentrations. Two morphologically different tissue cultures were maintained in vitro for a long time: a callus tissue without organogenesis and a culture with high rhizogenic capacity. Shoots and plantlets were generated when the auxin-media induced callus was transferred to medium supplemented with 3 M TIBA. In 62% of cultures, during the first five subcultures, four to twentyeight plants per single mesocotyl were obtained. Some cultures produced shoots even in the 9th subculture, being in culture for nearly 14 months. The largest number of plants obtained per one mesocotyl was forty. Plantlets rooted well on MS with 5.7 M IAA and survived transplantation to soil in high percentages.Abbreviations IAA indole-3yl-acetic acid - BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog (1962) medium     

Biodegradation of 2,4,5-trichlorophenoxyacetic acid in liquid culture and in soil by the white rot fungus Phanerochaete chrysosporium

Summary Extensive biodegradation of [¹⁴C]-2,4,5-trichlorophenoxyacetic acid ([¹⁴C]-2,4,5-T) by the white rot fungus Phanerochaete chrysosporium was demonstrated in nutrient nitrogen-limited aqueous cultures and in [¹⁴C]-2,4,5-T-contaminated soil inoculated with this fungus and supplemented with ground corn cobs. After incubation of [¹⁴C]-2,4,5-T with aqueous cultures of the fungus for 30 days, 62.0%±2.0% of the [¹⁴C]-2,4,5-T initially present was degraded to ¹⁴CO₂. Mass balance analysis demonstrated that water soluble metabolites were formed during degradation, and HPLC and thin layer chromatography (TLC) of methylene chloride-extractable material revealed the presence of polar and non-polar [¹⁴C]-2,4,5-T metabolites. It was also shown that only 5% of the [¹⁴C]-2,4,5-T initially present in cultures remained as undegraded [¹⁴C]-2,4,5-T. In incubations composed of [¹⁴C]-2,4,5-T-contaminated soil, ground corn cobs, and 40% (w/w) water, 32.5%±3.6% of the [¹⁴C]-2,4,5-T initially present was converted to ¹⁴CO₂ after 30 days of incubation. These results suggest that it may be possible to develop practical systems based on the use of this fungus to detoxify 2,4,5-T-contaminated water and soil.     

Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl^–1 kinetin, and 100 mgl^–1 kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl^–1 each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the -glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl^–1 myo-inositol, 1 mgl^–1 2,4-D, 0.5 mgl^–1 BA, and 0.5 mgl^–1 kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.Abbreviations BA 6-benzyladenine; 2,4-D,2,4-dichlorophenoxyacetic acid - DIG digoxigenine - GA₃ gibberellic acid - X-gluc X-glucuronide - GUS -glucuronidase - MS Murashige and Skoog (1962)     

Effect of synthetic auxins on callus induction from tea stem tissue

A study was initiated to establish an in vitro culture protocol for tea (Camellia sinensis). Explant sources, disinfestation methods and culture media were examined. Segments (divots) were dissected from greenwood stem (current year growth) internodes of field grown plants. Disinfestation was achieved by separate treatments of 3.75% sodium hypochlorite and 7.5% CaCl₂. MS medium with sucrose (30 g/L), inositol (100 mg/L) and thiamine-HCl (1.3 mg/L) and kinetin was used with combinations of the auxins: (2,4-dichlorophenoxy) acetic acid (2,4-D), (2,4,5-trichlorophenoxy) acetic acid (2,4,5-T), (naphthalene) acetic acid (NAA) and 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram). Picloram (10^-7M) induced the most callus proliferation without kinetin. At a constant level of kinetin (10^-5M), the concentrations inducing the most callus growth were 10^-7M for 2,4-D, 10^-6M for 2,4,5-T, 10^-7M for Picloram and 10^-8M for NAA. A factorial test of 2,4,5-T and kinetin concentrations showed the optimum for callus growth was 10^-7M and 10^-5M, respectively.Technical Contribution No. 2532 of the South Carolina Agricultural Experiment Station, Clemson University.Graduate Research Assistant and Professor, respectively.     

The influence of auxins,light and cell differentiation on solasodine production bySolanum eleagnifolium Cav. calli

The effect of auxins, light and cellular production ofSolanum eleagnifolium Cav. calli were studied. 2,4-dichlorophenoxyacetic acid (4.5 M) was the plant growth regulator used for calli initiation and this produced the highest solasodine concentration. The solasodine concentration in darkness was significantly lower than that achieved under a photoperiod of 16 h. Differentiated tissue obtained by adequate hormonal balance (several ratios of 3-indolebutyric acid to 6-benzylaminopurine) produced higher yields of solasodine than non-differentiated tissue. 3-indolebutyric acid (2.5 M) and 6-benzylaminopurine (8.8 M) increased the productivity of solasodine by 100%.Abbreviations BAP 6-benzylaminopurine - KIN Kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - NAA 1-naphtaleneacetic acid - IBA 3-indolebutyric acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - DW dry weight - GI      

Production and characterization of intergeneric somatic hybrids through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon pennellii

Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.Abbreviations MS Murashige and Skoog basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - PEG polyethylen glycol 6,000 - MES 2-(N-morpholino)ethanesulfonic acid - AC alternating current - EST esterases - PRX peroxidase - 6-PGD phosphogluconate dehydrogenase - GOT glutamate oxaloacetate transaminase - FDA fluorescein diacetate     

Biodegradation of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by dichlorophenol-adapted microorganisms from freshwater,anaerobic sediments

Reductive dechlorination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated in anaerobic sediments by non-adapted microorganisms and by microorganisms adapted to either 2,4- or 3,4-dichlorophenol (DCP). The rate of dechlorination of 2,4-D was increased by adaptation of sediment microorganisms to 2,4-DCP while dechlorination by sediment microorganisms adapted to 3,4-DCP displayed a lag phase similar to non-adapted sediment slurries. Both 2,4- and 3,4-DCP-adapted microorganisms produced 4-chlorophenoxyacetic acid by ortho-chlorine removal. Lag phases prior to dechlorination of the initial addition of 2,4,5-T by DCP-adapted sediment microorganisms were comparable to those from non-adapted sediment slurries. However, the rates of dechlorination increased upon subsequent additions of 2,4,5-T. Biodegradation of 2,4,5-T by sediment microorganisms adapted to 2,4- and/ or 3,4-DCP produced 2,5-D as the initial intermediate followed by 3-chlorophenol and phenol indicating a para > ortho > meta order of dechlorination. Dechlorination of 2,4,5-T, by either adapted or non-adapted sediment microorganisms, progressed without detection of 2,4,5-trichlorophenol as an intermediate.     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Effect of some herbicides on the production of lysine byAzotobacter chroococcum

Summary Production of lysine byAzotobacter chroococcum strain H23 was studied in chemically-defined media amended with different concentrations of alachlor, metolachlor, 2,4-D, 2,4,5-T and 2,3,6-TBA. The presence of 5, 10, and 50g/ml of alachlor or 2,3,6-TBA significantly decreased quantitative production of lysine. However, the presence 2,4-D or 2,4,5-T at concentrations of 10 and 50g/ml enhanced the production of lysine. Quantitative production of lysine was not affected as consequence of the addition of metolachlor to the culture medium, showing that the release lysine to the culture media byA. chroococcum was not affected by that herbicide.     

Somatic embryogenesis and plant regeneration of squash Cucurbita pepo L cv. YC 60

Plant regeneration from tissue cultures of summer squash, Cucurbita pepo L., cv. YC60, has been observed. Somatic embryos organized from shoot apex derived callus cultured on Murashige and Skoog (MS) medium supplemented with 1.2 mg/l 2,4,5-trichlorophenoxyacetic acid, 0.8 mg/l benzylaminopurine, and 0.1 mg/l kinetin. Embryos developed into plantlets by transfer of immature somatic embryos to MS medium with 0.05 mg/l NAA and 0.05 mg/l kinetin. Regenerated plants appeared morphologically normal and set fruits with seeds which could germinate normally.Abbreviation BAP 6-benzylaminopurine - 2,4-D 2, 4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - KN kinetin - NAA -naphthyleneacetic acid - MS Murashige and Skoog - 2,4,5-T 2, 4,5-trichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

Natural selection for 2,4,5-trichlorophenoxyacetic acid mineralizing bacteria in agent orange contaminated soil

Agent Orange contaminated soils were utilized in direct enrichment culture studies to isolate 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) mineralizing bacteria. Two bacterial cultures able to grow at the expense of 2,4,5-T and/or 2,4-D were isolated. The 2,4,5-T degrading culture was a mixed culture containing two bacteria, Burkholderia species strain JR7B2 and Burkholderia species strain JR7B3. JR7B3 was able to metabolize 2,4,5-T as the sole source of carbon and energy, and demonstrated the ability to affect metabolism of 2,4-D to a lesser degree. Strain JR7B3 was able to mineralize 2,4,5-T in pure culture and utilized 2,4,5-T in the presence of 0.01 yeast extract. Subsequent characterization of the 2,4-D degrading culture showed that one bacterium, Burkholderiaspecies strain JRB1, was able to utilize 2,4-D as a sole carbon and energy source in pure culture. Polymerase chain reaction (PCR) experiments utilizing known genetic sequences from other 2,4-D and 2,4,5-T degrading bacteria demonstrated that these organisms contain gene sequences similar to tfdA, B, C, E, and R (Strain JRB1) and the tftA, C, and E genes (Strain JR7B3). Expression analysis confirmed that tftA, C, and E and tfdA, B, and C were transcribed during 2,4,5-T and 2,4-D dependent growth, respectively. The results indicate a strong selective pressure for 2,4,5-T utilizing strains under field condition.