Effect of surfactants on pyrene degradation by Pseudomonas fluorescens 29L

The effect of surfactants on pyrene degradation in Pseudomonas fluorescens 29L was investigated. This strain produced 30.1 μM of rhamnolipid equivalents (RE) of biosurfactants on 50 mg of pyrene per liter of medium. The production of biosurfactants was significantly correlated with the water solubility (S _w) of the substrate and the growth rate on it. When chrysene, with a S _w of 2.8 × 10⁻³ mg per liter of water, was the carbon source, 13.1 μM of RE of biosurfactants were produced compared to 10.3 μM of RE of biosurfactants on acenaphthene with a S _w of 1.9 mg per liter of water. No biosurfactants were produced on salicylic acid, catechol, and citrate. All of the strain 29L mutants which grew on pyrene produced biosurfactants while among the mutants which grew on naphthalene, only 88.4% produced biosurfactants. The rhamnolipid mixture, JBR425, inhibited the growth of Strain 29L wild type (WT) and all of its mutants on pyrene. However, these mutants were able to grow in the presence of pyrene when the growth medium was supplemented with 10⁻⁶ mg of emulsan per milliliter of medium. This study implies biosurfactants are produced by Strain 29L as a physiological response to the hydrophobicity of pyrene. The combined use of indigenous biosurfactants and the added biosurfactant, emulsan, is a biotechnology to enhance pyrene degradation by Pseudomonas fluorescens 29L.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Influence of different ethylene inhibitors on somatic embryogenesis and secondary embryogenesis from <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr.

The effect of cobalt chloride, salicylic acid, and silver nitrate for embryogenesis was studied in in vitro cultures of Coffea canephora. Murashige and Skoog (in Physiol. Plant. 15:473–497, 1962) medium containing 20 and 40 μM either of cobalt chloride, silver nitrate, or salicylic acid supplemented with 1.1 μM N ⁶ benzyladenine and 2.85 μM indole-3-acetic acid was used for the study. At 20 and 40 μM silver nitrate treatment, 35–48% explants responded for embryogenesis, and 38 ± 7 and 153 ± 27 embryos were produced from each callus mass, respectively, whereas only 5% control explants responded on medium devoid of silver nitrate, cobalt chloride, or salicylic acid. Secondary embryogenesis was observed in 70–90% of the explants, and around 100–150 embryos were produced from each explant cultured on a medium containing silver nitrate, and only a 3% response was noticed in control embryo explants. Yellow friable embryogenic calluses were obtained from the cut edges of most of the tissues grown in a medium supplemented with cobalt chloride. The results clearly demonstrated that, among the tested ethylene inhibitors, silver nitrate is very effective in reprogramming the cellular machinery toward embryogenesis.     

Effect of Nitrogen Source on End Products of Naphthalene Degradation

Soil cultures, enrichment cultures, and pure culture isolates produced substantial quantities of salicylic acid from naphthalene in a mineral salts medium containing NH₄Cl as the nitrogen source. However, when KNO₃ was substituted for NH₄Cl, these same cultures failed to accumulate detectable quantities of salicylic acid but did turn the medium yellow. When an isolate identified as a Pseudomonas species was used, viable cell numbers were much greater in the medium containing KNO₃, but up to 94% of the naphthalene was utilized in both media. After 48 h of incubation in a 0.1% naphthalene-mineral salts medium, the cultures containing NH₄Cl showed irregular clumped cells, a pH of 4.7, 42 μg of salicylic acid per ml, and the production of 4.4 ml of CO₂. Under the same conditions, the cultures in the medium containing KNO₃ showed uniform cellular morphology, a pH of 7.3, no salicylic acid, the production of 29.7 ml of CO₂, and a distinct yellow coloration of the medium. The differences between nitrogen sources could not be accounted for by pH alone since results obtained using buffered media were similar. Growth with NH₄NO₃ displayed a pattern similar to that obtained when NH₄Cl was used. The yellow coloration in the medium containing KNO₃ was apparently due to more than one compound, none of which were 1,2-naphthoquinone or acidic in nature, as suggested by other investigators. Further attempts to identify the yellow compounds by high-pressure liquid chromatography, infrared analysis, and gas chromatography-mass spectrometry have been unsuccessful thus far.     

Induction of somatic embryos from cultures of <Emphasis Type="Italic">Agave vera-cruz</Emphasis> Mill.

Somatic embryogenesis from cultures of shoot apices, cotyledon and young leaves of in vitro shoots of Agave vera-cruz Mill. was studied. Embryogenic callus was obtained when explants were cultured on Murashige and Skoog’s (MS) medium (1962) supplemented with L₂ vitamins, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-d) or 5.37 μM ∝-naphthalene acetic acid (NAA). Somatic embryos differentiated from this embryogenic callus upon subculture to maturation/conversion medium containing cytokinin either alone or with auxin and l-glutamine. The best combination of growth regulators for development of somatic embryos was found to be 5.37 μM naphthalene acetic acid plus 0.91 μM zeatin and 40 g/l sucrose. The conversion frequency of somatic embryos to plantlets varied from 46–50%. Rooted plantlets were transferred directly to pots containing a soil, sand, and manure mixture without any hardening phase with 96–98% survival of the plantlets. Based on the histological observations, the potential origin of the somatic embryo is discussed.     

Stable integration and expression of β-glucuronidase and NPT II genes in mango somatic embryos

Summary Somatic proembryos of mango (Mangifera indica L. cv. Hindi) were co-cultivated withAgrobacterium tumefaciens strain A208 harboring pTiT37-Se::pMON 9749 (9749 ASE). Transformed somatic proembryos capable of growing on selection medium containing 200 μg/ml kanamycin produced the characteristic indigo blue precipitate in the presence of 5-bromo-4-chloro-3-glucuronic acid. These proembryos were chimeral consisting of transformed (blue) and nontransformed (yellow/white) cells. A stepwise selection strategy was found necessary to eliminate chimeras. a) Initial screening at 200 μg/ml kanamycin to enable growth of transformed cells, b) further screening at 400 μg/ml kanamycin to reduce chimeras, and c) recovery of pure transformed clones of proembryos in liquid selection medium with 100 μg/ml kanamycin. The integration of the NPT II and GUS genes into mango genome was confirmed by Southern hybridization.     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Micropropagation of Alocasia amazonica using semisolid and liquid cultures

An efficient, simple micropropagation method was developed for Alocasia amazonica using corms in semisolid and liquid cultures. Explants were cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–497, 1962) supplemented with different cytokinins (Benzyladenine [BA, 2.22–13.32 μM], kinetin [2.32–13.95 μM], Thidiazuron [TDZ, 0.45–4.54 μM]) and cytokinin in combination with auxins [naphthalene acetic acid (NAA, 0.54–5.37 μM)/indole acetic acid (IAA, 0.57–5.71 μM)/indole butyric acid (IBA, 0.49–4.9 μM)]. All supplementary-induced shoot proliferation and the optimal results was on the medium supplemented with 2.27 μM TDZ, which induced 5.1 shoots per explant. Among the different concentrations of sucrose (0–120 g l⁻¹) tested for shoot proliferation, 30 g l⁻¹ was found suitable for corm cultures of Alocasia amazonica. The optimal shoot proliferation and biomass values were with the plantlets grown at 30 μmol m⁻² s⁻¹ photosynthetic photon flux (PPF) and 25°C. Liquid cultures found suitable for shoot proliferation and biomass accumulation was compared to semisolid cultures. Comparative studies of bioreactor systems [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were greatest with the raft bioreactor system. Plantlets (cormlets) from the bioreactor were hydroponically cultured for 30 days, and 100% of plants were acclimatized successfully. The simple efficient method of production of plantlets (cormlets) is useful for large-scale multiplication of this important ornamental plant. An erratum to this article can be found at     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.     

Efficient regeneration of Eucalyptus urophylla from seedling-derived hypocotyls

Seedling hypocotyls were used as explants to establish a regeneration protocol for Eucalyptus urophylla and N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU), one kind of di-substituted urea, was found useful growth regulator. The hypocotyls incubated on a modified Murashige and Skoog medium (SPCa), supplemented with 6.6 μM PBU and 0.57 μM indole-3-acetic acid (IAA) dedifferentiated and form calli (100 % after 7 d). Compared with other growth regulator combinations, PBU stimulated more vigorous calli and restrained their darkening. In addition, the calli induced by PBU showed high frequency of adventitious buds formation (57%). Shoot proliferation and elongation was then stimulated on SPCa medium containing 0.44 μM 6-benzyladenine (BA), 0.54 μM naphthalene acetic acid (NAA) and 0.3 μM gibberellic acid (GA3). For rooting, shoots were cultivated on root induction medium containing 2.5 μM indole-3-butyric acid (IBA). Plantlets were then successfully transplanted to greenhouse.     

Shoot organogenesis and plantlet regeneration from hypocotyl-derived cell suspensions of a tree legume, Dalbergia sissoo Roxb

Summary A complete and efficient protocol is presented for plant regeneration from cell-suspension cultures of Dalbergia sissoo Roxb., an economically important leguminous tree. Factors influencing callus initiation, establishment of cell-suspension culture, callus formation from embredded microcolonies, and shoot organogenesis from suspension-derived callus were identified. Of the two different auxins tested, callus induction was better on a medium containing naphthalene acetic acid (NAA). The percentage of callus induction increased considerably when NAA at 2.0 mg l⁻¹ (10.8 μM) was added in conjunction with 0.5 mg l⁻¹ (2.2 μM) N⁶-benzyladenine (BA). Of the three different explants evaluated for callus induction, hypocotyl segments were most responsive. Friable hypocotyl-derived callus from the second subculture passage was used to initiate the cell-suspension culture. Optimum growth of the cell suspension was observed in MS medium supplemented with the same growth regulators as described above for callus induction, with an initial inoculum cell density of 1%. The plating efficiency of the microcolonies was greatly influenced by harvesting time and the gelling agent used for plating. Efficiency was highest (93%) with cells harvested at their exponential growth phase and plated in 1.2 g l⁻¹ Phytagel. Shoot organogenesis from callus cultures was higher on a medium supplemented with a combination of BA and NAA than on BA alone. Seventy-one per cent of cultures exhibited shoot-bud differentiation on a medium containing 3.0 mg l⁻¹ (13.3 μM) BA and 0.5 mg l⁻¹ (2.7 μM) NAA. Regenerated shoots were rooted on half-strength MS medium containing 1 mg l⁻¹ each of indole-3-acetic acid (5.7 μM), indole-3-butyric acid (4.9 μM) and indole-3-propionic acid (5.3 μM). Plantlets were acclimated and established in soil.     

Influence of explants, genotypes and culture vessels on sprouting and proliferation of pre-existing meristems of cotton (Gossypium hirsutum L. and Gossypium arboreum L.)

Summary A simple and efficient method for multiple shoot induction and proliferation was achieved in six Indian cotton cultivars from the pre-existing meristems of 21-d-old in vitro-grown seedlings. Combinations of naphthalene acetic acid (0.3–10.7 μM) and 6-benzylaminopurine (BA; 2.2 or 4.4 μM) were used for induction of shoots. The shoots proliferated and were maintained on MS (Murashige and Skoog) medium supplemented with 4.4 μM BA. Simultaneous elongation of shoots was obtained in the same medium. Optimum multiplication was observed in cv. LRK-516 (19.7±4.6), in cotyledonary nodes isolated from the adjoining node and cultured individually in 250 ml flasks. This indicates lateral inhibition of adjoining meristems. A positive influence of culture flasks as opposed to test tubes on the proliferation of multiple shoots was observed in all six cultivars tested. The morphogenic response varied with genotype and the nature of explants. Rooting of elongated shoots was achieved on MS medium devoid of growth regulators. The plantlets were transferred to the field after hardening in the greenhouse. All plants flowered and formed bolls on maturity.     

Bioproduction of Ascorbic Acid in Root Nodule and Root of the Legume Pulse Phaseolus mungo

The root nodules of Phaseolus mungo (L.), a herbaceous leguminous pulse, contain high amounts of ascorbic acid (AsA). A glucose pool present in the nodule might serve as precursor for AsA production. From root nodule, a Rhizobium sp. was isolated. The symbiont produced a large amount of AsA (290.5 μg/ml) from glucose-supplemented basal medium. The production of AsA by the symbiont was much greater than that of the control when the glucose (0.5%)-supplemented mineral medium was enriched with thiamine hydrochloride (20 μg/100 ml), biotin (20 μg/100 ml), and L-asparagine (0.2%). The possible role of the rhizobial production of AsA on rhizobia–legume symbiosis is discussed.     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp.

Summary Embryogenic callus was induced from primary leaves of Vigna unguiculata (L.) Walp. in MS medium (Murashige and Skoog, 1962) containing 2,4-dichlorophenoxyacetic acid (2,4-D). Greenish-white, friable embryogenic calluses were used to establish suspension cultures. A shaking speed of 90 rpm and 0.4 ml packed cell volume per 25 ml medium were found to be optimal for maintaining suspension cultures. Globular, heart-shaped and torpedo-shaped embryos were developed in suspension culture containing 4.52 μM 2,4-D. Maturation of cotyledonary-stage somatic embryos was achieved on 0.05 μM 2,4-D, 5 μM abscisic acid and 3% mannitol. Twenty-two percent of the embryos were converted into plants and survived; survival in the field was 8–10%.     

Effects of Thymol on Ruminal Microorganisms

Thymol (5-methyl-2-isopropylphenol) is a phenolic compound that is used to inhibit oral bacteria. Because little is known regarding the effects of this compound on ruminal microorganisms, the objective of this study was to determine the effects of thymol on growth and lactate production by the ruminal bacteria Streptococcus bovis JB1 and Selenomonas ruminantium HD4. In addition, the effect of thymol on the in vitro fermentation of glucose by mixed ruminal microorganisms was investigated. Neither 45 nor 90 μg/ml of thymol had any significant effect on growth or lactate production by S. bovis JB1, but 180 μg/ml of thymol completely inhibited growth and lactate production. In the case of S. ruminantium HD4, 45 μg/ml of thymol had little effect on growth and lactate production; however, 90 μg/ml of thymol completely inhibited growth of S. ruminantium HD4. Thymol also decreased glucose uptake by whole cells of both bacteria. When mixed ruminal microorganisms were incubated in medium that contained glucose, 400 μg/ml of thymol increased final pH and the acetate to propionate ratio and decreased concentrations of methane, acetate, propionate, and lactate. In conclusion, thymol was a potent inhibitor of glucose fermentation by S. bovis JB1 and S. ruminantium HD4. Even though thymol treatment decreased methane and lactate concentrations and increased final pH in mixed ruminal microorganism fermentations of glucose, concentrations of acetate and propionate were also reduced. Received: 13 May 2000 / Accepted: 14 June 2000     

Shoot-bud regeneration in subcultured callus of engelmann spruce

Summary Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N⁶-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N⁶-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures. This study was supported by the Natural Sciences and Engineering Research Council of Canada grant G1438 to T. A. Thorpe and D. I. Dunstan.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot multiplication through shoot tip cultures of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., a threatened plant endemic to Southern India

Summary An efficient and rapid micropropagation system was developed for a food and medicinally important endangered shrub, Decalepis hamiltonii (‘swallow root’), through shoot multiplication. The influence of 2.5–7.5 μM isopentenyladenine (2iP), 4.4–17.7 μM 6-benzyladenine, 2.3–4.7 μM kinetin, 2.8–6.8 μM thidiazuron, and 2.3–11.4 μM zeatin alone and in combination with 0.3–0.9 μM indole-3-acetic acid (IAA) on in vitro multiple shoot production was studied. The maximum number of multiple shoots (6.5±0.4) was induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.9 μM 2iP. But, both zeatin (9.1 μM) and kinetin (4.7 μM) in combination with IAA (0.6 μM) were able to produce a maximum of 5.0±0.4 and 5.1±0.4 multiple shoots, respectively. Further elongation of shoots and adventitious shoot formation was obtained on medium containing 2.5 μM 2iP and 0.3 μM gibberellic acid. Elongated shoots were separated and rooted on MS medium supplemented with 9.8μM indole-3-butyric acid (IBA) and various phenolic compounds within 5–6 wk. Phloroglucinol and salicylic acid interaction with IBA stimulated in vitro rooting of shoots. Successful field transfer was achieved in rooted plantlets.     

Humic acid effect on pyrene degradation: finding an optimal range for pyrene solubility and mineralization enhancement

The addition of humic acid (HA) to polycyclic aromatic hydrocarbon (PAH) contaminated systems has been shown to enhance, inhibit, or have no effect on the biodegradation of these PAHs. In this study, the surfactant effects of Elliott soil HA (ESHA) at two pH values were tested. At pH 7.0, ESHA did not behave as a surfactant. At pH 11.8, ESHA acted as a surfactant, as displayed by a decrease in surface tension with increasing concentrations of ESHA. The effect of ESHA on pyrene solubility was tested by adding 0 to 800 μg ESHA/g soil to soil-slurries. Enhancement of pyrene apparent solubility demonstrated a dose- and time-related effect. Broader doses from 0 to 10,080 μg ESHA/g soil and three higher doses from 3,360 to 10,080 μg ESHA/g soil were tested for their effects on pyrene mineralization by indigenous soil microorganisms and a novel PAH-degrading Mycobacterium sp. KMS in soil microcosms, respectively. ESHA amendments between 20 and 200 μg ESHA/g soil were found to consistently increase pyrene mineralization by indigenous microorganisms, while the 10,080 μg ESHA/g soil produced inhibition and all other doses presented no effects. Pyrene degradation by M. KMS was significantly inhibited by the addition of the highest dose of ESHA.