Hexavalent chromate reduction by immobilized Streptomyces griseus

Hexavalent chromium, which is a mutagen and carcinogen, was efficiently reduced by Streptomyces griseus. This activity was associated with the cell. Cr⁶⁺ reduction by free as well as immobilized cells was studied: cells in PVA-alginate had the highest (100%) Cr⁶⁺ removal efficiency in 24 h with reduction rates similar to free cells. Immobilized cells completely reduced 25 mg Cr⁶⁺ l⁻¹ in 24 h. PVA-alginate immobilized cells could be reused four times to completely reduce 25 mg Cr⁶⁺ l⁻¹ in 24 h each time. Chromate in a simulated effluent containing Cu²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ was completely reduced by PVA-alginate immobilized cells within 9 h.     

Evaluation of methods for celery (<Emphasis Type="Italic">Apium Graveolens</Emphasis> L.) transformation using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the <Emphasis Type="Italic">bar</Emphasis> gene as selectable marker

Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery. Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for 2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and Skoog (MS) medium + 2,4-Dichlorophenoxyacetic acid (2,4-D) (2.3 μM) + kinetin (2.8 μM) + timentin (300 mg·l⁻¹). Calluses 4- to 6-wk-old were selected for glufosinate (GS) resistance by a two step method. First, calluses were transferred to C medium + GS 0.35, 0.5, 1, 2, 5, or 10 mg·l⁻¹; calluses formed only with 0, 0.35 and 0.5 mg·l⁻¹ GS. All growing calluses from 0 and 0.35 mg·l⁻¹ and a few from 0.5 mg·l⁻¹, were divided and placed back on C + GS 0.35–0.5 mg·l⁻¹ for another 5–6 wk. Second, tolerant clones were again divided and placed on C + GS 1–50 mg·l⁻¹. When cultivar XP85 was inoculated with both strains, using pGPTVBAR, 19 glufosinate resistant (GR) callus clones were selected, but shoots regenerated only for strain EHA105 inoculations. When both of the strains (each with pDHB321.1) were inoculated on cv. XP166, 3 and 12 GR calluses occurred for EHA105 and GV3101, respectively. Using CS, a total of 34 GR callus clones were selected, and shoots were regenerated from over 50% of them on Gamborg B5 medium + 6-(γ, γ-dimethylallylamino) purine 2ip (4.9 μM) + naphthaleneacetic acid (NAA; 1.6 μM) and rooted on MS in 5–6 mo total time. Conversely, using FB with inoculation by GV3101/pDHB321.1 on cv. XP166 yielded putative transgenic celery plants confirmed by polymerase chain reaction (PCR) in just 6 wk. Transformation of the bar gene into celery was confirmed by PCR for 5 and 6 CS and FB lines, respectively. Southern blot analyses indicated 1–2 copies in CS lines and 1 copy in FB lines. Herbicide assays on whole plants with 100 and 300 mg·l⁻¹ glufosinate indicated a range of low to high tolerance for lines derived by both methods. The bar gene was found to be Mendelian inherited in one self-fertile CS derived line.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Heavy metal resistant <Emphasis Type="Italic">Distigma proteus</Emphasis> (Euglenophyta) isolated from industrial effluents and its possible role in bioremediation of contaminated wastewaters

Summary The alga, Distigma proteus, isolated from industrial wastewater showed tolerance against Cd²⁺ (8.0 μg/ml), Cr⁶⁺ (12 μg/ml), Pb²⁺ (15 μg/ml) and Cu²⁺ (10 μg/ml). The metal ions slowed down the growth of the organism after 4–5 days of exposure. The reduction in cell population was 90% for Cu²⁺, 84% for Cd²⁺, 71% for Cr⁶⁺, and 63% for Pb²⁺ after 8 days of metal stress. The order of resistance to heavy metal, in terms of reduction in the cellular population, was Cu²⁺ > Cd²⁺ > Cr⁶⁺ > Pb²⁺. Chromium- and cadmium-processing capabilities of the alga were worked out for its potential use as a bioremediator of wastewater. The reduction in the amount of Cr⁶⁺ after 2, 4, 6 and 8 days of algal culture containing 5.0 μg Cr⁶⁺ ml⁻¹ of culture medium was 77, 85, 92 and 97%, respectively. Distigma could also remove 48% Cd²⁺after 2 days, 68% after 4 days, 80% after 6 days and 90% after 8 days from the medium. The heavy metal uptake ability of Distigma can be exploited for metal detoxification and environmental clean-up operations.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Application of the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in biotreatment of bagasse effluent

Biotreatment of bagasse effluent using Phanerochaete chrysosporium (white rot fungus) is investigated. This study confirmed that lignin is the major pollutant component in this effluent followed by different carbohydrates. The treatment conditions must be very proper, especially in terms of biomass culture to achieve a successful treatment. The best conditions of temperature, biomass concentration, pH and duration for biotreatment of this effluent were 35°C, 552 mg l⁻¹, 6 and 5 to 9 days, respectively. Under these conditions, a 9 days long treatment reduced by 98.7% the original biochemical oxygen demand (of 2,780 mg l⁻¹) and by 98.5% the dissolved chemical oxygen demand (initial 4,200 mg l⁻¹). Moreover, fungal treatment reduced total dissolved solids from 3,950 to 575 mg l⁻¹ and color from 560 mg l⁻¹ PtCo to 111 mg l⁻¹ PtCo.     

Plant regeneration via somatic embryogenesis of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> in temporary immersion system (TIS)

The present study describes a protocol for plant regeneration via somatic embryogenesis in temporary immersion system (TIS) for Camptotheca acuminata. Somatic embryos were induced by culturing hypocotyl segments from 14-day-old in vitro grown C. acuminata seedlings in TIS. Hypocotyl segments were placed in culture vessels modified with a mechanical device to support the fixation of explants. Cultures were maintained under a 16 h photoperiod with a light intensity of 60 μmol m⁻² s⁻¹ PPF at 25 ± 1°C. After 16 weeks of incubation embryogenic calli were formed above the edge of the mechanical device in the basal Murashige and Skoog (MS) medium containing 35 g l⁻¹ sucrose and without hormonal supplementation. For plantlet regeneration, somatic embryos at cotyledonary stage were cultured in three different concentrations of 6-benzylamino-purine (0.5, 1.0 and 1.5 mg l⁻¹ BAP) and in plant growth regulator (PGR) free medium. In general, 0.5 mg l⁻¹ BAP was found to be the most effective concentration for growth and development of Camptotheca embryos in TIS. Conversion of somatic embryos into plantlets was also successfully achieved on sterile substrates moistened with 0.5 mg l⁻¹ BAP. Plantlets derived from cotyledonary embryos were rooted in vitro with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) before transfer to ex vitro conditions.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Metal resistance and removal by two strains of the green alga, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> Beijerinck,isolated from Laguna de Bay,Philippines

Two strains of Chlorella vulgaris Beijerinck isolated from two different sites in Laguna de Bay, Philippines, were studied for their resistance and ability to remove four metal ions, i.e., Cu²⁺, Cr⁶⁺, Pb²⁺, and Cd²⁺ added separately in BG-11 growth medium. The growth of the two strains was severely inhibited at 2 mg.L⁻¹ of Cu²⁺, 5 mg.L⁻¹ of Cr⁶⁺, 8 mg.L⁻¹ of Pb²⁺, and 10 mg.L⁻¹ of Cd²⁺. However, the two strains exhibited different EC₅₀ values for the same metal ion. The WB strain had a significantly higher resistance (p < 0.01) for Cd²⁺ and Cr⁶⁺ compared with the SB strain, while the SB strain had significantly higher resistance (p < 0.01) for Cu²⁺ compared with the WB strain. On the other hand, the two strains behaved differently in their capacity to remove the metal ions in BG-11 medium containing 1.0 mg.L⁻¹ of the three metal ions, except for Cu²⁺, which was added at 0.1 mg.L⁻¹. The WB strain showed the highest removal of Cd²⁺ at 70.3% of total, followed by Pb²⁺ at 32%, while the SB strain exhibited the highest removal of Pb²⁺ at 48.7% followed by Cd²⁺ at 40.7% of the total. Both strains showed the least removal of Cr⁶⁺ at 28% and 20.8% of the total for the WB and SB strains respectively. The percentage removal for Cu²⁺ was 50.7% and 60.8% for the WB and SB strains respectively. After 12 days of incubation, both strains showed that a greater percentage of the metal ions removed were accumulated intracellularly than adsorbed at a ratio of at least 2:1. Both strains manifested the same cytological deformities, like a loss of pyrenoids at 10 mg.L⁻¹ in all four metal ions. Discoloration and disintegration of chloroplasts were observed at 1.0 mg.L⁻¹ in Cu²⁺ and 5 mg.L⁻¹ in Cr⁶⁺. The nonrelease of autospores from the mother cells was observed at 10 mg.L⁻¹ in Cu²⁺ and Cr⁶⁺. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Adventitious bud regeneration from leaf explants of <Emphasis Type="Italic">Platanus occidentalis</Emphasis> L. and genetic stability assessment

The aim of this work is to develop a method of plant regeneration from leaf explants of Platanus occidentalis L. successfully. Woody plant medium (HortScience 16:453–459, 1981) and Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium were used as induced and rooted basal medium, respectively. The effects of combinations of 6-BA, IBA, NAA and KT with different concentrations on adventitious bud regeneration from P. occidentalis leaf explants were compared. The results showed that the highest shoot regeneration frequency (90%) and maximum number (13.72 ± 0.44) of shoots per explant was recorded on WPM medium supplemented with 22.20 mmol l⁻¹ 6-BA and 0.49 mmol l⁻¹ IBA. A 40-day-old explants were much more productive for shoot formation than others in this study. The regenerated shoots were cultured on MS medium supplemented with 1.33 mmol l⁻¹ 6-BA, 0.16 mmol l⁻¹ NAA and 2% (w/v) adenine, after 2-week shoots were transferred to 1/2 MS medium supplemented with 0.49 mmol l⁻¹ IBA for rooting. Hardened plantlets via acclimatization were transferred to pots and transplanted to the soil finally. To ascertain whether tissue culture had effects on the genetic stability of plantlets regenerated, the genetic diversity was assessed using RAPD marker. A total of 96 bands ranging from 0.5 to 2.2 kb with an average of 6.4 bands per primer, were obtained using 15 primers. Amplified products exhibited few of polymorphic patterns across all the plants of P. occidentalis and the overall frequency of detection of somaclonal polymorphisms was lower than 0.0104%. Yuehua Sun, Yanling Zhao, and Xiaojuan Wang contributed equally to this work.     

Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

CO<Subscript>2</Subscript>-enriched microenvironment affects sucrose and macronutrients absorption and promotes autotrophy in the <Emphasis Type="Italic">in vitro</Emphasis> culture of kiwi (<Emphasis Type="Italic">Actinidia deliciosa</Emphasis> Chev. Liang and Ferguson)

In traditional in vitro culture, the low CO₂ concentration inside the vessels restricts photosynthesis and necessitates the addition of sucrose to the culture medium as the main energy source, thus bringing about changes in the absorption of mineral elements from the culture medium. In this study, we investigated macronutrient absorption and sugar consumption in Actinidia deliciosa Chevalier Liang and Ferguson cv. Hayward (kiwi), cultured on medium supplemented with varying amounts of sucrose (0, 10, and 20 g l⁻¹) under both heterotrophy and autotrophy, flushed with different concentrations of CO₂ (non-ventilation, 300, 600, and 2,000 μl l⁻¹). In ventilated systems with 20 g l⁻¹ of sucrose, sucrose absorption was less than under non-ventilation. The lowest rate of sucrose absorption was recorded when the explants were cultured on medium supplemented with 20 g l⁻¹ of sucrose and flushed with 600 μl l⁻¹ CO₂. Absorption of NO₃ ⁻, PO₄ ³⁻, and Mg²⁺ were high (maximum) at the end of the culture period (40 d) in explants flushed with 600 μl l⁻¹ CO₂ that have been cultured 20 d in the presence of sucrose and then transferred to a sucrose-free medium. These autotrophic conditions promoted maximum plant growth in terms of both fresh and dry mass as well as the length and number of shoots and leaves. The study shows that to maintain an optimum regime of mineral nutrition for prolonged culture of kiwi in vitro, an increased amount of these three ions should be supplemented in Murashige and Skoog’s medium.     

Synergistic effect of morphactin on cytokinin-induced production of isoflavonoids in cell cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC

Isoflavonoids, the functional molecules of Fabaceae, are under clinical trials against cancer, osteoporosis and cardiovascular diseases. In this study, the efficacy of different plant growth regulators was evaluated for optimizing the production of isoflavonoids in Pueraria tuberosa. The cultures were maintained in Murashige and Skoog’s medium containing 0.1 mg l⁻¹ 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 0.1 mg l⁻¹ kinetin. The addition of 5.0 mg l⁻¹ N⁶-(2-Isopentenyl) adenine (2iP) resulted in about ∼32-folds increase in production of isoflavonoids, while about ∼23-folds increase was recorded in the absence of kinetin in the maintenance medium. A maximum yield of isoflavonoids (∼80 mg l⁻¹; 82-folds increase) was obtained in cultures grown at 0.1 mg l⁻¹ morphactin and 5.0 mg l⁻¹ of 2iP. However, 2,4,5-T in combination with 2iP was ineffective for their production. Among different plant growth regulators tested, maximum yields of puerarin, genistin, daidzein and genistein were 17.4, 15.9, 69.0 and 0.04 mg l⁻¹, respectively. The study suggested that the presence of two cytokinins or 2iP with morphactin in the culture medium markedly enhanced the production of isoflavonoids in P. tuberosa.     

In vitro root culture of <Emphasis Type="Italic">Ocimum sanctum</Emphasis> L. and evaluation of its free radical scavenging activity

Young leaf explants of Ocimum sanctum L. incubated on solidified Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 1-naphthaleneacetic acid (NAA) and 0.2 mg l⁻¹ kinetin (Kn) developed rhizogenic callus. When these were subcultured onto MS medium supplemented with 1.5 mg l⁻¹ 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.5 mg l⁻¹ NAA, friable rhizogenic callus was observed. Upon transfer of this friable callus onto liquid MS medium containing 4 mg l⁻¹ NAA and 1.3 mg l⁻¹ 6-benzyladnine (BA) under continuous agitation at 90 rpm and 16 h photoperiod, roots with an optimum dry weight of 1,460 mg l⁻¹ were obtained. An ethyl acetate extract of these roots exhibited 1, 1–diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity.     

Stimulation of menthol production in <Emphasis Type="Italic">Mentha piperita</Emphasis> cell culture

Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid to initiate cell suspension culture. This culture was maintained in half-strength MS medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid at 15 d interval and used for further studies. Precursor feeding alone, i.e., menthone, at 35 μM concentration showed slightly improved productivity. γ-Cyclodextrin alone at 60 μM concentration and in combination with menthone feeding at 35 μM increased menthol yield up to 92 and 110 mg l⁻¹ in comparison to 77 mg l⁻¹ of control culture. Synergistic potentiation effect of menthone feeding at 35 μM and γ-cyclodextrin at 60 μM treatment followed by in situ adsorption with RP-8 also showed potential stimulation of menthol production in M. piperita cell culture. Fungal elicitor treatment showed enhanced production level up to 140.8 mg l⁻¹ in comparison to that of control. Further studies were carried out with the establishment of Agrobacterium tumefaciens (Ach5) gall-mediated calli, and consequently, cell suspension culture and results showed the significant enhancement of menthol yield up to 278 mg l⁻¹. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by <Emphasis Type="Italic">Pseudomonas nitroreducens</Emphasis> isolated from soil

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens isolated from petroleum-contaminated soil was investigated. The effects of carbon, nitrogen and carbon to nitrogen ratio on biosurfactant production were examined using mineral salts medium as the growth medium. The tenso-active properties (surface activity and critical micelle concentrations of the produced biosurfactant were also evaluated. The best carbon source, nitrogen source were glucose and sodium nitrate giving rhamnolipid yields of 5.28 and 4.38 g l⁻¹, respectively. The maximum rhamnolipid production of 5.46 g l⁻¹ was at C/N (glucose/sodium nitrate) of 22. The rhamnolipid biosurfactant reduced the surface tension of water from 72 to ~37 mN/m. It also has critical micelle concentration of ~28 mg l⁻¹. Thus, the results presented in our reports show that the produced rhamnolipid can find wide applications in various bioremediation activities such as enhanced oil recovery and petroleum degradation.