Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67 mg L⁻¹ h⁻¹), higher nitrite accumulation (47.3∼99.8 mg L⁻¹), and higher N₂O emission ratios (5∼283 mg L⁻¹/mg L⁻¹). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05 mg L⁻¹) was found, and the absence of Cu resulted in massive N₂O emission due to poor synthesis of N₂O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5 mg L⁻¹) > Cu(II) (0.5∼5 mg L⁻¹) > Ni(II) (2∼10 mg L⁻¹) > Zn(II) (25∼50 mg L⁻¹). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ≈ cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

     

An efficient callus-based in vitro regeneration protocol for Warburgia Ugandensis Sprague,an important medicinal plant in Africa

Warburgia ugandensis Sprague is a woody species in the family Canellaceae and an important source of medicines in Africa. Natural propagation of W. ugandensis is problematic due to its recalcitrant seeds and lack of an efficient in vitro regeneration system for this species. This study describes an efficient regeneration protocol. Petiole bases and shoot tips were used as explants. Callus tissue developed when the explants were cultured on Murashige and Skoog medium containing 30 g L⁻¹ sucrose and 7 g L⁻¹ agar (MS30 medium), supplemented with 1.0 mg L⁻¹ indole-3-butyric acid (IBA), 1.6 mg L⁻¹ 6-benzylaminopurine (BA), and 0.1 mg L⁻¹ thidiazuron (TDZ). Adventitious buds were efficiently induced from the callus when the MS30 medium was supplemented with 0.8 mg L⁻¹ BA and 0.2 mg L⁻¹ IBA. Root induction occurred within 7–10 d on half-strength MS30 medium supplemented with 0.8–1.0 mg L⁻¹ 1-napthalene acetic acid (NAA), 0.2 mg L⁻¹ IBA, and 0.03% (w/v) activated charcoal (AC). Roots were followed by root elongation on the same medium but lacking NAA and IBA. Approximately 50% of the plantlets cultured produced roots, while more than 80% of the plantlets survived and successfully grew to maturity.

     

Highly efficient regeneration and medicinal component determination of Phellodendron chinense Schneid

Phellodendron chinense Schneid is an important Chinese herb with berberine and phellodendrine in stems and leaves, but with little information available on in vitro culture of this species. Disinfection of explants in 75% alcohol for 45 s, sterilization in 0.1% HgCl₂ for 20 min, and submersion in 1.0 mol L⁻¹ gibberellin3 (GA₃) solution for 24 h was the optimal condition for seed germination. Murashige and Skoog’s (MS) medium supplemented with 2.0 mg L⁻¹ 6-benzylaminopurine (6-BA) in combination with 1.5 mg L⁻¹ 1-naphthylacetic acid (NAA) was optimal for callus induction. MS medium supplemented with 2.0 mg L⁻¹ 6-BA was the appropriate medium for induction of adventitious shoots, and 1/2MS medium supplemented with 2.0 mg L⁻¹ indole-3-butytric acid (IBA) and 0.5% active carbon was the optimal medium for root induction. The 15-d survival rate of regenerated plantlets after transplanting to basins containing perlite and peat moss (1:4) was greater than 80%, and the berberine and phellodendrine accumulation was lower in callus compared with regenerated plantlets. The establishment of highly efficient regeneration system provides technical support for genetic breeding of Phellodendron chinense Schneid.

     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl₂ for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L⁻¹ resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L⁻¹), along with IAA (0.5 to 2 mg L⁻¹), or Kn (0.5 to 5 mg L⁻¹), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

     

Genetic homogeneity and high shoot proliferation in banana (Musa acuminata Colla) by altering medium thiamine level and sugar type

To enhance the multiplication rate in Musa acuminata Colla (banana; ‘Grand Nain’) organogenesis, higher amounts of thiamine along with different sugar types and concentrations were evaluated at the proliferation phase. Thiamine at 1, 10, 50, 100, and 200 mg L⁻¹ was compared with 0.1 mg L⁻¹ thiamine found in conventional Murashige and Skoog (MS) medium. Maximum proliferation of banana was induced with 100 mg L⁻¹ thiamine. Additionally, 15, 30, and 45 g L⁻¹ sucrose, glucose, fructose, and sorbitol combined with regular and optimal levels of thiamine were tested. Glucose at 30 g L⁻¹ most improved shoot proliferation alone and enhanced shoot proliferation further, when combined with 100 mg L⁻¹ thiamine, followed by sucrose and fructose, whereas sorbitol completely inhibited growth and caused tissue browning. All evaluated vegetative traits were significantly affected by sugar type and concentration, and thiamine levels, unlike the photosynthetic pigments. Moreover, genetic stability of the plants recovered from the enhanced protocol was confirmed by inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. A total of 230 bands generated by both marker types were monomorphic for the randomly selected regenerated plants, compared with their mother plant. Thus, the proliferation medium supplemented with 30 g L⁻¹ glucose and 100 mg L⁻¹ thiamine could be recommended for banana organogenesis. Results herein are of great importance and helpful in enhancing the commercial in vitro propagation protocols of banana, without the need of increasing the number of subcultures, which can cause somaclonal variation.

     

Use of bioreactors for large-scale multiplication of sugarcane (Saccharum spp.), energy cane (Saccharum spp.), and related species

The objective of this study was to set up a plant micropropagation facility to mass propagate sugarcane, energy cane, and related clonally propagated species. An efficient methodology for micropropagation of energy cane and perennial grasses using temporary immersion bioreactors was developed. Several different methods of tissue culture initiation, multiplication, and rooting were evaluated for several varieties of sugarcane (Saccharum officinarum L.) and sugarcane-related species such as Erianthus spp., Miscanthus spp., and Sorghum spp. × sugarcane hybrids, all from a germplasm collection. Apical meristem cultures were initiated for all genotypes that were micropropagated, when liquid or semisolid Murashige and Skoog (MS) medium was used, which was supplemented with 0.1–0.2 mg L⁻¹ BAP, 0.1 mg L⁻¹ kinetin, 0–0.1 mg L⁻¹ NAA, and 0–0.2 μg L⁻¹ giberellic acid. These cultures produced shoots between 4 and 8 wk after initiation. Shoot regeneration from leaf rolls or immature inflorescences was observed as early as 4 wk after initiation. Shoot multiplication was successful for all genotypes cultured in MS medium with 0.2 mg L⁻¹ BAP and 0.1 mg L⁻¹ kinetin. Energy cane had a significantly higher combined multiplication rate when grown under four or five LED lamps than when grown under three LED lamps, or under fluorescent lights in a growth chamber. The addition of 2 mg L⁻¹ NAA produced faster and better rooting in all of the genotypes tested. Shoots produced well-developed roots after one cycle of 15–21 d in the bioreactors. The maximum number of plantlets produced per bioreactor was 1080. Plantlets developed a vigorous root system and were ready to be transplanted into the field after 2 mo. A protocol was standardized for different energy cane clones that were recommended for their biomass production and cell wall composition. Different tissues were used to speed up or facilitate tissue culture initiation. Visual assessment of micropropagated plants in the field did not show any off-types, based on gross morphological changes of plant morphology or disease reaction, compared to plants of the same genotype derived from a traditional propagation method (stem cuttings). This is the first report of energy cane and Miscanthus spp. micropropagation using the SETIS bioreactor.

     

Somatic embryogenesis to overcome low seed viability and conserve wild banana (Ensete superbum (Roxb.) Cheesman)

The seed viability, ex vitro germination, and percentage of in vitro zygotic embryo germination were found to be very low in Ensete superbum (Roxb.) Cheesman. Only 33.33% of seeds were viable, and the ex vitro germination percentage was only 5%, while the percentage of in vitro zygotic embryo germination was 33%. Somatic embryogenesis experiments produced competent callus on Murashige and Skoog (MS) medium supplemented with 2.5 mg L⁻¹ 2,4-D and 3 mg L⁻¹ BAP from inflorescence explants. The embryogenic callus produced the maximum number of somatic embryos on MS basal medium kept in a dark chamber for 15 wk. Half-strength MS medium supplemented with 500 mg L⁻¹ glutamine was optimal for somatic embryo germination and development of plantlets. Regenerated plants had 80 to 90% survival rate. Therefore, somatic embryogenesis can be considered as an efficient method to overcome a drastic reduction in population and to achieve germplasm conservation.

     

Somatic embryogenesis and plant regeneration in two wild cotton species belong to G genome

The present work describes the plant regeneration via somatic embryogenesis in two wild cotton species belonging to G genome: Gossypium nelsonii Fryx and Gossypium australe F Muell. The role of plant hormones and carbohydrates was also evaluated for somatic embryogenesis and somatic embryo development. Normal plants were obtained from G. nelsonii Fryx; abnormal plants and somatic embryos were obtained from G. australe F Muell. The best medium for callus induction for these G genome wild cotton species was MSB₅ supplemented with 0.1 mg L⁻¹ KT and 0.1 mg L⁻¹ 2,4-D. For embryogenic callus proliferation, the best medium used was MSB₅ supplemented with 0.2 mg L⁻¹ KT and 0.5 mg L⁻¹ IBA. The medium MSB₅ supplemented with 0.15 mg L⁻¹ KT and 0.5 mg L⁻¹ NAA was used successfully for root initiation and plant growth. In addition, adding CuSO₄ and AgNO₃ in the callus-inducing and proliferation medium resulted in a number of somatic embryos. Glucose and maltose, the carbon sources in somatic culture, were used for callus induction, but maltose worked even better than glucose for proliferation of embryogenic callus and development of somatic embryos.     

Estuarine sediment hydrocarbon-degrading microbial communities demonstrate resilience to nanosilver

Little is currently known about the potential impact of silver nanoparticles (AgNPs) on estuarine microbial communities. The Colne estuary, UK, is susceptible to oil pollution through boat traffic, and there is the potential for AgNP exposure via effluent discharged from a sewage treatment works located in close proximity. This study examined the effects of uncapped AgNPs (uAgNPs), capped AgNPs (cAgNPs) and dissolved Ag₂SO₄, on hydrocarbon-degrading microbial communities in estuarine sediments. The uAgNPs, cAgNPs and Ag₂SO₄ (up to 50 mg L⁻¹) had no significant impact on hydrocarbon biodegradation (80–92% hydrocarbons were biodegraded by day 7 in all samples). Although total and active cell counts in oil-amended sediments were unaffected by silver exposure; total cell counts in non-oiled sediments decreased from 1.66 to 0.84 × 10⁷ g⁻¹ dry weight sediment (dws) with 50 mg L⁻¹ cAgNPs and from 1.66 to 0.66 × 10⁷ g⁻¹ dws with 0.5 mg L⁻¹ Ag₂SO₄ by day 14. All silver-exposed sediments also underwent significant shifts in bacterial community structure, and one DGGE band corresponding to a member of Bacteroidetes was more prominent in non-oiled microcosms exposed to 50 mg L⁻¹ Ag₂SO₄ compared to non-silver controls. In conclusion, AgNPs do not appear to affect microbial hydrocarbon-degradation but do impact on bacterial community diversity, which may have potential implications for other important microbial-mediated processes in estuaries.     

Determination of pioglitazone hydrochloride by flow injection chemiluminescence tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex system

A novel flow injection-chemiluminescence (FI–CL) approach is proposed for the assay of pioglitazone hydrochloride (PG-HCl) based on its enhancing influence on the tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex (Ru(bipy)₃²⁺-DPA) CL system in sulfuric acid medium. The possible CL reaction mechanism is discussed with CL and ultraviolet (UV) spectra. The optimum experimental conditions were found as: Ru(bipy)₃²⁺, 5.0 × 10⁻⁵ M; sulfuric acid, 1.0 × 10⁻³ M; diperiodatoargentate(III) (DPA), 1.0 × 10⁻⁴ M; potassium hydroxide, 1.0 × 10⁻³ M; flow rate 4.0 ml min⁻¹ for each flow stream and sample loop volume, 180 μl. The CL intensity of PG-HCl was linear in the range of 1.0 × 10⁻³ to 5.0 mg L⁻¹ (R² = 0.9998, n = 10) with limit of detection [LOD, signal-to-noise ratio (S/N) = 3] of 2.2 × 10⁻⁴ mg L⁻¹, limit of quantification (LOQ, S/N = 10) of 6.7 × 10⁻⁴ mg L⁻¹, relative standard deviation (RSD) of 1.0 to 3.3% and sampling rate of 106 h⁻¹. The methodology was satisfactorily used to quantify PG-HCl in pharmaceutical tablets with recoveries ranging from 93.17 to 102.77 and RSD from 1.9 to 2.8%.     

Modification of light intensity influence essential oils content,composition and antioxidant activity of thyme,marjoram and oregano

Thymus vulgaris L. (thyme), Origanum majorana L. (marjoram), and Origanum vulgare L. (oregano) were used to determine whether light modification (plants grown under nets with 40% shaded index or in un-shaded open field) could improve the quantity and quality of essential oils (EOs) and antioxidant activity. The yield of EOs of thyme, marjoram, and oregano obtained after 120 min of hydrodistillation was 2.32, 1.51, and 0.27 mL/100 g of plant material, respectively. At the same time under shading conditions plants synthetized more EOs (2.57, 1.68, and 0.32 mL/100 g of plant material). GC/MS and GC/FID analyses were applied for essential oils determinations. The main components of the thyme essential oil are thymol (8.05–9.35%); γ-terpinene (3.49–4.04%); p-cymene (2.80–3.60%) and caryophyllene oxide (1.54–2.15%). Marjoram main components were terpinene 4-ol (7.44–7.63%), γ-terpinene (2.82–2.86%) and linalool (2.04–2.65%) while oregano essential oil consisted of the following components: caryophyllene oxide (3.1–1.93%); germacrene D (1.17–2.0%) and (E)-caryophyllene (1.48–1.1%). The essential oil from thyme grown under shading (EC₅₀ value after 20 min of incubation) have shown the highest antioxidant activity – 0.85 mg mL⁻¹ in comparison to marjoram and oregano (shaded plants EC₅₀ 19.97 mg mL⁻¹ and 7.02 mg mL⁻¹ and unshaded, control plants EC₅₀ 54.01 mg mL⁻¹ and 7.45 mg mL⁻¹, respectively). The medicinal plants are a good source of natural antioxidants with potential application in the food and pharmaceutical industries. For production practice, it can be recommended to grow medicinal plants in shading conditions to achieve optimal quality parameters.     

In vitro propagation of Trichosanthes kirilowii Maxim. through nodal segment shoot proliferation

Trichosanthes kirilowii Maxim. is a vital traditional herbal medicinal plant found in northeastern Asia. Its roots, fruits, and seeds are used as food and medicine. Roots harvested for medicinal use take over 3 yr to mature when the plant is grown in a traditional way through cultivation in the field. This coupled with uncertainty in identification of the plant when collected from the wild calls for a standard in vitro propagation system to meet the increasing demand for it. The purpose of this study was to develop a standard protocol for the in vitro micropropagation of T. kirilowii. Ten different media supplemented with different concentrations of plant growth regulators were evaluated. At 5 wk, De Greef and Jacobs medium supplemented with 0.1 mg L⁻¹ kinetin led to optimal shoot growth, while the same medium supplemented with 0.5 mg L⁻¹ indole 3-butyric acid induced optimal root growth, also at 5 wk. The micropropagated plants that were acclimatized for 8 wk in the greenhouse produced mature root tubers after planted in the field for 3 mo. Therefore, these findings provide a basis for future large-scale in vitro propagation of T. kirilowii.

     

Effect of molybdenum on secondary metabolic process of glycyrrhizic acid in Glycyrrhiza uralensis Fisch.

A pot experiment was conducted to investigate into effects of molybdenum (Mo) on the secondary metabolic process of glycyrrhizic acid (GA). One-year-old seedlings were grown in pots with washed vermiculite and sand. Hoagland nutrition solution was irrigated with four concentrations: 0, 0.52, 5.2 and 10.4 mg L⁻¹. The accumulations of GA and its biosynthetic precursors (β-amyrin and squalene) and then expression of the key synthase (β-amyrin synthase, β-AS) were studied on 35, 70 and 105 d. In the early stage, that was on the 35 and 70 d, the contents of squalene and GA, and the expression of β-AS gene under 0.52 and 5.2 mg L⁻¹ Mo treatments were significantly higher than that under 0 and 10.4 mg L⁻¹ Mo. There was a contrary result of β-amyrin. However, the content of squalene under 0 mg L⁻¹ Mo was the highest on 105 d. Thus, it suggested an appropriate concentration of Mo could promote the accumulation of GA, by affecting the biosynthetic process of GA at a certain time. Practically, the time and amount of application of Mo on Glycyrrhiza uralensis should be the noted.     

Azo dyes decolorization under high alkalinity and salinity conditions by Halomonas sp. in batch and packed bed reactor

Biodecolorization and biodegradation of azo dyes are a challenge due to their recalcitrance and the characteristics of textile effluents. This study presents the use of Halomonas sp. in the decolorization of azo dyes Reactive Black 5 (RB5), Remazol Brilliant Violet 5R (RV5), and Reactive Orange 16 (RO16) under high alkalinity and salinity conditions. Firstly, the effect of air supply, pH, salinity and dye concentration was evaluated. Halomonas sp. was able to remove above 84% of all dyes in a wide range of pH (6–11) and salt concentrations (2–10%). The decolorization efficiency of RB5, RV5, and RO16 was found to be ≥ 90% after 24, 13 and 3 h, respectively, at 50 mg L⁻¹ of dyes. The process was monitored by HPLC-DAD, finding a reduction of dyes along the time. Further, Halomonas sp. was immobilized in volcanic rocks and used in a packed bed reactor for 72 days, achieving a removal rate of 3.48, 5.73, and 8.52 mg L⁻¹ h⁻¹, for RB5, RV5 and RO16, respectively, at 11.8 h. The study has confirmed the potential of Halomonas sp. to decolorize azo dyes under high salinity and alkalinity conditions and opened a scope for future research in the treatment of textile effluents.

     

Cellulolytic enzyme production from agricultural residues for biofuel purpose on circular economy approach

This study evaluated the production of cellulolytic enzymes from different agricultural residues. The crude enzyme extract produced was characterized and applied for saccharification of some agricultural residues. Maximum cellulolytic activities were obtained using soybean hulls. All enzymatic activities were highly stable at 40 °C at a pH range of 4.5–5.5. For stability at low temperatures, the enzyme extract was stored at freezing temperature and cooling for about 290 days without major loss of activity. The K_m values found for total cellulase (FPase), endoglucanase (CMCase), and xylanase were 19.73 mg ml⁻¹, 0.65 mg ml⁻¹, and 22.64 mg ml⁻¹, respectively, and V_max values were 0.82 mol min⁻¹ mg⁻¹, 0.62 mol min⁻¹ mg⁻¹, and 104.17 mol min⁻¹ mg⁻¹ to cellulose, carboxymethyl cellulose, and xylan, respectively. In the saccharification tests, the total amount of total reducing sugars (TRS) released from 1 g of soybean hulls catalyzed by the enzymes present in the crude enzyme extract was 0.16 g g⁻¹ dry substrate.

     

应用稀土调控藏红花胚性愈伤组织的生长与分化

为提高藏红花(Crocus sativus)胚性愈伤组织的繁殖系数与出芽率, 以建立藏红花离体快繁体系, 解决其资源短缺问题, 采用两步法, 用稀土调控其胚性愈伤组织的生长与分化。结果表明, 在添加了0.25 mg·L^–1 NAA、3 mg·L^–1 6-BA和400 mg·L^–1 CH的B₅固体培养基中, 0.04 mmol·L^–1 La³⁺促进胚性愈伤组织生长的效果最佳, 繁殖系数为12, 是不添加稀土处理组的1.48倍; 在添加了0.25 mg·L^–1 NAA、3 mg·L^–1 6-BA和400 mg·L^–1 CH的1/2 B₅固体培养基中, 0.06 mmol·L^–1 Ce³⁺促进胚性愈伤组织分化出芽的效果最佳, 出芽率高达84.5%, 是不添加稀土处理组的1.81倍, 且高于国外报道的出芽率(40%)。初步解决了藏红花胚性愈伤组织生长慢和出芽率低等问题, 为建立高效稳定的藏红花离体快繁体系奠定了基础。     

Mycoremediation of oil contaminant by Pleurotus florida (P.Kumm) in liquid culture

This study investigated the potential functions of Pleurotus florida (an edible mushroom) in the biodegradation of gas oil at concentrations of 0 (control), 2.5, 5, and 10% (V: V) for 30 days. The gas oil increased dry weight and protein concentration in all treatments (by an average of 19.5 and 108%, respectively). Moreover, the pH, surface tension (ST), and interfacial tension (IFT) were reduced by the mushroom supplementation. The lowest surface tension (31.9 mN m⁻¹) and the highest biosurfactant production belonged to the 10% gas oil treatment (0.845 ± 0.03 mg mL⁻¹). The results demonstrated that the adsorption isotherm agreed well with the Langmuir isotherm. The maximum Langmuir adsorption capacity was calculated at 0.743 mg g⁻¹ wet biomass of P. florida. The fungal supplementation efficiently remedied the total petroleum hydrocarbons (TPHs) by an average of 55% after 30 days. Gas chromatography (GC) analysis revealed that P. florida effectively detoxified C₁₃–C₂₈ hydrocarbons, Pristane, and Phytane, implying its high mycoremediation function. The toxicity test showed that mycoremediation increased the germination by an average of 35.82% ± 8.89 after 30 days. Laccase activity increased significantly with increasing gas oil concentration in the treatments. The maximum laccase activity was obtained in the 10% gas oil treatment (142.25 ± 0.72 U L⁻¹). The presence of pollutants was also associated with induction in the tyrosinase activity when compared to the control. These results underline the high mycoremediation capacity of P. florida through the involvement of biosurfactants, laccase, and tyrosinase.     

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L. cv. Poinsett76) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L⁻¹ benzyladenine (BA), 20 mg L⁻¹ l-glutamine and 2 mg L⁻¹ phosphinothricin (PPT) or 100 mg L⁻¹ kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L⁻¹) containing MS media with 0.5 mg L⁻¹ gibberellic acid (GA₃) and 0.6 mg L⁻¹ indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T₀ and the progenies. T₁ segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.