Micropropagation of <Emphasis Type="Italic">Glossonema varians</Emphasis> (Stocks) Benth. ex Hook.f.—a rare Asclepiadeae of Indian Thar Desert

In the present study, an in vitro regeneration protocol for Glossonema varians (Stocks) Benth. ex Hook.f. of family Asclepiadaceae was optimized. Cotyledonary nodes of in vitro cultured seedlings were used as explants for activation of axillary shoot buds. Axillary shoot buds were initially activated on 0.1 mg L^?1 6-benzylaminopurine (BAP) and then multiplied on 0.05 mg L^?1 BAP. Shoots were rooted in vitro on 1/4 strength Murashige and Skoog medium containing 0.1 mg L^?1 2-naphthoxyacetic acid and 100 mg L^?1 activated charcoal. The cultures were maintained in a 12 h photoperiod at 40–50 μmol m^?2 s^?1 spectral flux photon, 25–30?±?2°C, and 60% relative humidity (RH). Up to 80% of in vitro regenerated plantlets were acclimatized on soilrite in cotton-plugged culture tubes in the greenhouse. This protocol can be a useful method to mass propagate and conserve this rare plant to balance biodiversity in the desert ecosystem.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Evaluation of growth and phycobiliprotein composition of cyanobacteria isolates cultivated in different nitrogen sources

Phycobiliproteins, light-harvesting pigments found in cyanobacteria and in some eukaryotic algae, have numerous commercial applications in food, cosmetic, and pharmaceutical industries. Colorant production from cyanobacteria offers advantages over their production from higher plants, as cyanobacteria have fast growth rate and high photosynthetic efficiency and require less space. In this study, three cyanobacteria strains were studied for phycobiliprotein production and the influence of sodium nitrate, potassium nitrate and ammonium chloride on the growth and phycobiliprotein composition of the strains were evaluated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources; however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate μ?=?0.284?±?0.03 and μ?=?0.274?±?0.13 day^?1, chlorophyll a 16.2?±? 0.5 and 12.2?±? 0.2 mg L^?1, and phycobiliprotein contents 19.38?±?0.09 and 19.99?±?0.14% of dry weight, whereas, for Arthrospira platensis, the highest growth rate of μ?=?0.304?±?0.0 day^?1, chlorophyll a 19.1?±?0.5 mg L^?1, and phycobiliprotein content of 22.27?±?0.21% of dry weight were achieved with sodium nitrate. The phycocyanin from the lyophilized cyanobacterial biomass was extracted using calcium chloride and food grade purity (A₆₂₀/A₂₈₀ ratio >?0.7) was achieved. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A₆₂₀/A₂₈₀ ratio >?4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α- and β-subunits of the C-phycocyanin. The results showed that Phormidium sp. and Pseudoscillatoria sp. could be good candidates for phycocyanin production.     

Quercetin and silver nitrate modulate organogenesis in <Emphasis Type="Italic">Carissa carandas</Emphasis> (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO₃), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L^?1 6-benzylaminopurine; 0.5 mg L^?1 kinetin, and 0.75 mg L^?1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L^?1 BAP, 0.5 mg L^?1 kinetin, and 1.5 mg L^?1 AgNO₃ was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L^?1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.     

Optimization of continuous-flow solid-phase denitrification via coupling carriers in enhancing simultaneous removal of nitrogen and organics for agricultural runoff purification

Coupling of biodegradable corncob and plastic carrier was optimized in continuous-flow solid-phase denitrification systems for enhancing simultaneously removal of nitrogen and organics in agricultural runoff. In compared with preposition of plastic carriers and mixed distribution method, it was demonstrated that the preposition of corncobs simultaneously enhanced nitrate (6.64 ± 1.35 mg L^?1 day ^?1) and organics removal (6.33 ± 1.44 mg L^?1 day^?1) at a hydraulic retention time (HRT) of 6 h. The operation performance could be further enhanced with extension of HRT to 12 h. The dominant genera found in corncob were denitrifiers for nitrate reduction (Bosea, Simplicispira, Desulfovibrio, Klebsiella, etc.) and fermentative bacteria (Pleomorphomonas, Actinotalea, Opitutus, Cellulomonas, Bacteroides, etc.) responsible for corncob degrading to simple organics for other denitrifiers. However, much lower and different denitrifiers abundances (Bradyrhizobium, Acinetobacter, Bacillus, etc.) exhibited on plastic filler than those of corncob. It well explained that the biofilm on plastic carrier was mainly related with organics removal while the biofilm on corncobs inclined to effectively remove nitrate, and simultaneous removal of nitrogen and organics could be achieved in coupling carriers system with preposition of biodegradable corncob.     

Micropropagation, <Emphasis Type="Italic">in vitro</Emphasis> flowering,and tuberization in <Emphasis Type="Italic">Brachystelma glabrum</Emphasis> Hook.f., an endemic species

Brachystelma glabrum Hook.f. is an endemic plant species of Eastern Ghats, India. In this study, efficient protocols for in vitro micropropagation, flowering, and tuberization of this plant were developed. Sterilized shoot tip and nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) and additives for shoot induction and multiplication. Both shoot tip and nodal explants showed the best response (90 and 100%, respectively) on MS medium supplemented with thidiazuron (TDZ) at 1.0 mg L^?1. The microshoots multiplied best on MS + TDZ (1.0 mg L^?1) in combination with α-naphthaleneacetic acid (NAA) at 0.5 mg L^?1 and coconut water (CW) at 25%. The highest number of in vitro flowers (4.0 flowers per microshoot) was observed on MS medium supplemented with a combination of N6-benzyladenine (BA) and indole-3-butyric acid (IBA), each at 1.5 mg L^?1. In vitro-derived shoots produced aerial tubers on MS + TDZ (2.0 mg L^?1) + IBA (0.5 mg L^?1) and basal tubers on MS + TDZ at 2.0 mg L^?1. In vitro shoots were best rooted on half-strength (½) MS + NAA at 0.5 mg L^?1. The rooted plantlets were successfully acclimatized in pots with 70% survival after a hardening period of 1 mo. This protocol provides an effective method for the conservation of this endemic plant species.     

Biodegradation of the Pyrethroid Pesticide Esfenvalerate by Marine-Derived Fungi

Esfenvalerate biodegradation by marine-derived fungi is reported here. Esfenvalerate (S,S-fenvalerate) and its main metabolites [3-phenoxybenzaldehyde (PBAld), 3-phenoxybenzoic acid (PBAc), 3-phenoxybenzyl alcohol (PBAlc), and 2-(4-chlorophenyl)-3-methylbutyric acid (CLAc)] were quantitatively analyzed by a validated method in triplicate experiments. All the strains (Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. CBMAI 1675, Acremonium sp. CBMAI 1676, Westerdykella sp. CBMAI 1679, and Cladosporium sp. CBMAI 1678) were able to degrade esfenvalerate, however, with different efficiencies. Initially, 100 mg L^?1 esfenvalerate (Sumidan 150SC) was added to each culture in 3 % malt liquid medium. Residual esfenvalerate (64.8–95.2 mg L^?1) and the concentrations of PBAc (0.5–7.4 mg L^?1), ClAc (0.1–7.5 mg L^?1), and PBAlc (0.2 mg L^?1) were determined after 14 days. In experiments after 7, 14, 21, and 28 days of biodegradation with the three most efficient strains, increasing concentrations of the toxic compounds PBAc (2.7–16.6 mg L^?1, after 28 days) and CLAc (6.6–13.4 mg L^?1, after 28 days) were observed. A biodegradation pathway was proposed, based on HPLC-ToF results. The biodegradation pathway includes PBAld, PBAc, PBAlc, ClAc, 2-hydroxy-2-(3-phenoxyphenyl)acetonitrile, 3-(hydroxyphenoxy)benzoic acid, and methyl 3-phenoxy benzoate. Marine-derived fungi were able to biodegrade esfenvalerate in a commercial formulation and showed their potential for future bioremediation studies in contaminated soils and water bodies.     

Plantlet formation via somatic embryogenesis and LC ESI Q-TOF MS determination of secondary metabolites in <Emphasis Type="Italic">Butea monosperma</Emphasis> (Lam.) Kuntze

Medicinal properties of Butea monosperma (BM) and overexploitation of bark as a rich source of flavonoids for different biological activities, development of efficient method for high frequency somatic embryos and in vitro synthesis of bioactive secondary metabolites using plant tissue culture technology is important. Initially, callus was induced from leaf explants of BM on Murashige and Skoog (MS) medium containing 0.25 mg L^?1 2,4-d-dichlorophenoxyacetic acid (2,4-d) with 0.1 mg L^?1 kinetin (Kn) and ascorbic acid (AA). MS half strength macronutrients and full strength micronutrients containing 0.25 mg L^?1 2,4-d with 0.1 mg L^?1 Kn, and 0.5 mg L^?1 AA provided fragile callus with 84.0 ± 1.00 % optimal growth response. Shoot formation occurred via somatic embryogenesis through an intermediary callus phase. However, 2.1 mg L^?1 thidiazuron with 0.5 mg L^?1 AA provides high frequency (79.6 ± 2.02 %) of somatic embryogenesis within 5 weeks. Developed embryos when transferred to woody plant medium containing 0.5 mg L^?1 AA with 3.0 mg L^?1 Kn and 0.5 mg L^?1 α naphthalene acetic acid responded 44.0 ± 0.00 % embryo maturation, whereas 0.5 mg L^?1 Kn, 0.3 mg L^?1 indole-3-butyric acid, and 0.25 mg L^?1 AA induced rooting within 6 and 8 weeks, respectively. Liquid chromatography electro spray ionization quadrupole time of flight mass spectrometry (LC ESI Q-TOF MS) analysis of in vitro cultures showed similarity to those compounds identified in wild grown leaf samples known for osteogenic activity. Histological investigation through scanning electron microscopy demonstrates the developmental stages of somatic embryos, shoot bud formation, and induction of root primordial.     

Respirometric response and microbial succession of nitrifying sludge to <Emphasis Type="Italic">m</Emphasis>-cresol pulses in a sequencing batch reactor

A nitrifying consortium was kinetically, stoichiometrically and molecularly characterized via the in situ pulse respirometric method and pyrosequencing analysis before and after the addition of m-cresol (25 mg C L^?1) in a sequencing batch reactor (SBR). Five important kinetic and stoichiometric parameters were determined: the maximum oxygen uptake rate, the maximum nitrification rate, the oxidation yield, the biomass growth yield, and the substrate affinity constant. An inhibitory effect was observed in the nitrification process with a recovery of this by up to eight SBR cycles after m-cresol was added to the system. However, full recovery of the nitrification process was not observed, as the maximum oxygen uptake rate was 25% lower than that of the previous operation without m-cresol addition. Furthermore, the pyrosequencing analyses of the nitrifying consortium after the addition of only two pulses of 25 mg C L^?1 m-cresol showed an important microbial community change represented by a decrease in the nitrifying populations and an increase in the populations degrading phenolic compounds.     

Ribosome engineering and fermentation optimization leads to overproduction of tiancimycin A,a new enediyne natural product from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CB03234

Tiancimycin (TNM) A, a recently discovered enediyne natural product from Streptomyces sp. CB03234, showed rapid and complete killing of cancer cells and could be used as a payload in antibody drug conjugates. The low yield of TNM A in the wild-type strain promoted us to use ribosome engineering and fermentation optimization for its yield improvement. The Streptomyces sp. CB03234-R-16 mutant strain with a L422P mutation in RpoB, the RNA polymerase β-subunit, was obtained from the rifamycin-resistant screening. After fermentation optimization, the titers of TNM A in Streptomyces sp. CB03234-R-16 reached to 22.5 ± 3.1 mg L^?1 in shaking flasks, and 13 ± 1 mg L^?1 in 15 L fermentors, which were at least 40-fold higher than that in the wild-type strain (~ 0.3 mg L^?1). Quantitative real-time RT-PCR revealed markedly enhanced expression of key genes encoding TNM A biosynthetic enzymes and regulators in Streptomyces sp. CB03234-R-16. Our study should greatly facilitate the future efforts to develop TNM A into a clinical anticancer drug.     

Enhancement of electricity production in a mediatorless air–cathode microbial fuel cell using <Emphasis Type="Italic">Klebsiella</Emphasis> sp. IR21

A novel dissimilatory iron-reducing bacteria, Klebsiella sp. IR21, was isolated from the anode biofilm of an MFC reactor. Klebsiella sp. IR21 reduced 27.8 % of ferric iron to ferrous iron demonstrating that Klebsiella sp. IR21 has electron transfer ability. Additionally, Klebsiella sp. IR21 generated electricity forming a biofilm on the anode surface. When a pure culture of Klebsiella sp. IR21 was supplied into a single chamber, air–cathode MFC fed with a mixture of glucose and acetate (500 mg L^?1 COD), 40–60 mV of voltage (17–26 mA m^?2 of current density) was produced. Klebsiella sp. IR21 was also utilized as a biocatalyst to improve the electrical performance of a conventional MFC reactor. A single chamber, air–cathode MFC was fed with reject wastewater (10,000 mg L^?1 COD) from a H₂ fermentation reactor. The average voltage, current density, and power density were 142.9 ± 25.74 mV, 60.5 ± 11.61 mA m^?2, and 8.9 ± 3.65 mW m^?2, respectively, in the MFC without inoculation of Klebsiella sp. IR21. However, these electrical performances of the MFC were significantly increased to 204.7 ± 40.24 mV, 87.5 ± 17.20 mA m^?2, and 18.6 ± 7.23 mW m^?2, respectively, with inoculation of Klebsiella sp. IR21. The results indicate that Klebsiella sp. IR21 can be utilized as a biocatalyst for enhancement of electrical performance in MFC systems.     

Phycoremediation potential,physiological, and biochemical response of <Emphasis Type="Italic">Amphora subtropica</Emphasis> and <Emphasis Type="Italic">Dunaliella</Emphasis> sp. to nickel pollution

Metal pollution can produce many biological effects on aquatic environments. The marine diatom Amphora subtropica and the green alga Dunaliella sp. possess a high metal absorption capacity. Nickel (Ni) removal by living cells of A. subtropica and Dunaliella sp. was tested in cultures exposed to different Ni concentrations (100, 200, 300, and 500 mg L^?1). The amount of Ni removed by the microalgae increased with the time of exposure and the initial Ni concentration in the medium. The metal, which was mainly removed by bioadsorption to Dunaliella sp. cell surfaces (93.63% of total Ni (for 500 mg Ni L^?1) and by bioaccumulation (80.82% of total Ni (for 300 mg Ni L^?1) into Amphora subtropica cells, also inhibited growth. Exposure to Ni drastically reduced the carbohydrate and protein concentrations and increased total lipids from 6.3 to 43.1 pg cell^?1, phenolics 0.092 to 0.257 mg GAE g^?1 (Fw), and carotenoid content, from 0.08 to 0.59 mg g^?1 (Fw), in A. subtropica. In Dunaliella sp., total lipids increased from 26.1 to 65.3 pg cell^?1, phenolics from 0.084 to 0.289 mg GAE g^?1 (Fw), and carotenoid content from 0.41 to 0.97 mg g^?1 (Fw). These compounds had an important role in protecting the algae against ROS generated by Ni. In order to cope with Ni stress shown by the increase of TBARS level, enzymatic (SOD, CAT, and GPx) ROS scavenging mechanisms were induced.     

The effect of temperature on growth and lipid and fatty acid composition on marine microalgae used for biodiesel production

Cultivation temperature is one of the major factors affecting the growth and lipid accumulation of microalgae. In this study, the effects of temperature on the growth, lipid content, fatty acid composition and biodiesel properties of the marine microalgae Chaetoceros sp. FIKU035, Tetraselmis suecica FIKU032 and Nannochloropsis sp. FIKU036 were investigated. These species were cultured at different temperatures (25, 30, 35 and 40 °C). The results showed that the specific growth rate, biomass and lipid content of all microalgae decreased with increasing temperature. With regards to fatty acids, the presence of saturated fatty acids (SFAs) in T. suecica FIKU032 and Nannochloropsis sp. FIKU036 decreased with increasing temperature, in contrast with polyunsaturated fatty acids (PUFAs). Moreover, Chaetoceros sp. FIKU035 was the only species that could grow at 40 °C. The highest lipid productivity was observed in Chaetoceros sp. FIKU035 when cultivated at 25 °C (66.73 ± 1.34 mg L^?1 day^?1) and 30 °C (61.35 ± 2.89 mg L^?1 day^?1). Moreover, the biodiesel properties (cetane number, cold filter plugging point, kinematic viscosity and density) of the lipids obtained from this species were in accordance with biodiesel standards. This study indicated that Chaetoceros sp. FIKU035 can be considered as a suitable species for biodiesel production in outdoor cultivation.     

<Emphasis Type="Italic">In vitro</Emphasis> sucrose concentration influences microtuber production and diosgenin content in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir)

Dioscorea spp. is an important food crop in many countries and the source of the phytochemical diosgenin. Efficient microtuber production could provide source materials for farm-planting stock, for food markets, and for the production of high-diosgenin-producing cultivars. The first step in this study was optimizing the plant growth regulators for plantlet production, followed by a study of the effects of sucrose concentration on microtuber induction and diosgenin production. Significantly, more shoots (3.5) were produced at 4.65 μM (1 mg L^?1) kinetin (KIN), longer shoots (4.1 cm) were obtained at 2.46 μM (0.5 mg L^?1) indole-3-butyric acid (IBA), and root number (3.9) was significantly higher at 5.38 μM (1 mg L^?1) naphthalene acetic acid (NAA) than in other treatments. Increased sucrose concentrations in the optimized growth medium with 4.65 μM KIN and 5.38 μM NAA had significant effects on microtuber production (p < 0.01) and diosgenin content (p < 0.05). The most microtubers (6.2) were obtained with 100 g L^?1 sucrose, while those on 80 g L^?1 sucrose were the heaviest (0.7 g) and longest (7.4 mm). Microtubers formed in medium with 80 g L^?1 sucrose had significantly higher diosgenin content (3.64% [w/w]) than those in other sucrose treatments (< 2%) and was similar to that of field-grown parent tubers (3.79%). This result indicates an important role for sucrose in both microtuber growth and diosgenin production. Medium containing 4.65 μM KIN and 5.38 μM NAA is recommended for plantlet production, and medium containing 80 g L^?1 sucrose is recommended for microtuber and diosgenin production.     

Potential Outdoor Cultivation of Green Microalgae Based on Response to Changing Temperatures and by Combining with Air Temperature Occurrence

In this study, our working hypothesis was to examine whether temperature alters biomass and metabolite production by microalgae according to strain. We also addressed whether it is possible to choose a strain suitable for growing in each season of a given region. A factorial experiment revealed a significant interaction between chlorophylls a and b (Chl a and Chl b), carotenoid/Chl (a?+?b) ratio, biomass and total lipid productivity of six green microalgae (four Chlorella spp., Chlorella sorokiniana and Neochloris oleoabundans) after 15 days at four temperatures. At 39/35 °C, two Chlorella sp. strains (IPR7115 and IPR7117) showed higher total carotenoids/Chl (a?+?b) (0.578 and 0.830), respectively. N. oleoabundans had the highest Chl a (8210 μg L^?1) and Chl b (1909 μg L^?1) at 19/15 °C and highest maximum dry biomass (2900 mg L^?1), specific growth rate (0.538 day^?1) and total lipids (1003 mg L^?1) at 15/8 °C. We applied a method to infer the growth of these six green microalgae in outdoor ponds, as based on their response to changing temperatures and by combining with historical data on day/night air temperature occurrence for a given region. We conclude that the use of regionalized maps based on air temperature is a good strategy for predicting microalgal cultivation in outdoor ponds based on their features and tolerance to changing temperature.     

The establishment of cell suspension culture of sabah snake grass (<Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Burm.F.) Lindau)

Clinacanthus nutans (Burm.F.) Lindau is an herbaceous plant that has long been used for traditional medicinal purposes in Asia. It has recently gained popularity as an alternative treatment for cancer. The aim of this study was to establish cell suspension cultures of C. nutans and to identify targeted bioactive compounds in the cultures. Young leaf explants were cultured on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin to identify a suitable medium for callus induction and proliferation. Proliferated, friable calluses were cultured in different combinations of plant growth regulators (2,4-D, naphthaleneacetic acid [NAA], picloram, kinetin, and 6-benzylaminopurine) in liquid medium to establish cell suspension cultures. Three cell lines of suspension culture, callus, and intact plant parts were subjected to ethyl acetate extraction followed by thin layer chromatography for identification of selected bioactive compounds. Medium supplemented with 0.25 mg L^?1 2,4-D and 0.75 mg L^?1 kinetin was found to be optimal for callus induction, whereas supplementation with 0.50 mg L^?1 2,4-D was efficient for callus proliferation. Liquid medium supplemented with 0.25 mg L^?1 2,4-D and 0.50 mg L^?1 NAA produced the highest growth index (2.52). Quercetin, catechin, and luteolin were present together in the callus and cell suspension cultures of C. nutans, but all three compounds were detected separately in young leaves, mature leaves, and stems. This study is the first to report the establishment of cell suspension culture of C. nutans with both cell and callus cultures producing quercetin, catechin, and luteolin.     

Iron homeostasis in tropical <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars having contrasting grain iron concentration

Iron homeostasis was studied in two tropical indica rice cultivars viz. Sharbati (high Fe) and Lalat (low Fe) having contrasting grain Fe concentration. Plants were hydroponically grown with 5 concentrations of Fe (0.05, 2, 5, 15, 50 mg L^?1) till maturity. The effect of incremental Fe treatment on the plant was followed by analyzing accumulation of ferritin protein, activities of aconitase enzyme, enzymes of anti-oxidative defense and accumulation of hydrogen peroxide and ascorbic acid. Plant growth was adversely affected beyond 15 mg L^?1 of Fe supplementation and effects of Fe stress (both deficiency and excess) were more apparent on the high Fe containing cultivar Sharbati than the low Fe containing Lalat. Level of ferritin protein and aconitase activity increased up to 5 mg L^?1 of Fe concentration. Lalat continued to synthesize ferritin protein at much higher Fe level than Sharbati and the cultivar also had higher activities of peroxidase, superoxide dismutase and glutathione reductase. It was concluded that the tolerance of Lalat to Fe stress was because of its higher intrinsic ability to scavenge free radicals of oxidative stress for possessing higher activity of antioxidative enzymes. This, together with its capacity to sequester the excess Fe in ferritin protein over a wider range of Fe concentrations made it more tolerant to Fe stress.     

Application of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> (Chlorophyceae) as supplement and/or an alternative medium for the in vitro cultivation of <Emphasis Type="Italic">Schomburgkia crispa</Emphasis> (Orchidaceae)

Schomburgkia crispa Lindley (Orchidaceae) is an epiphytic species found in gallery forests and dry vegetation in the Brazilian Cerrado. It is typically unable to germinate or exhibits low germination because of dependency on mycorrhizal associations. In vitro cultivation techniques have helped circumvent difficulties involved in propagation from seeds. Alternative media and organic biostimulant substances that reduce costs and promote satisfactory in vitro growth are constantly sought. This study evaluated in vitro multiplication and rooting of S. crispa in a modified culture medium containing extract of the microalga Chlorella sorokiniana. We analyzed supplementation of WPM (Woody Plant Medium) with microalgae suspended in NPK medium, or as the supernatant resulting from the centrifugation of a culture in NPK medium. The extracts were added to WPM instead of distilled water. The compounds 6-benzylaminopurine (BAP) and indolebutyric acid (IBA) were used as reference in the in vitro multiplication and rooting of S. crispa, respectively. Both growth regulators were tested at 0, 2.5, and 5.0 mg L^?1. During in vitro multiplication of S. crispa, WPM supplemented with 5.0 mg L^?1 BAP favored the formation of more sprouts, whereas WPM containing 2.5 mg L^?1 IBA supplemented with microalgae extract stimulated in vitro rooting. Schomburgkia crispa explants cultivated in medium supplemented with microalgae suspension or the supernatant of C. sorokiniana showed growth similar to explants cultivated in WPM alone. Therefore, it is possible to use the microalga C. sorokiniana as a supplement and/or alternative to WPM for the in vitro cultivation of S. crispa.     

<Emphasis Type="Italic">In vitro</Emphasis> preservation and micropropagation of <Emphasis Type="Italic">Oreocharis mileense</Emphasis> (W.T. Wang) M. Möller &amp; A. Weber (Gesneriaceae) through shoot organogenesis

Oreocharis mileense (W.T. Wang) M. Möller & A. Weber is endemic to China and was considered to be extinct because it had not been seen in the wild since the first collection in 1906. In 2006, the species was rediscovered in Shilin County, Yunnan Province. Oreocharis mileense was considered critically endangered for its narrow geographic range and extremely small population. An efficient method to preserve plant germplasm by in vitro culturing of O. mileense has not been reported. In this study, an orthogonal array with three factors (6-benzyladenine, BA; α-naphthaleneacetic acid, NAA; and sucrose), at four levels was performed, and shoot induction as well as shoot proliferation were recorded. The results were analyzed to determine the most significant components and the optimum combination for micropropagation of O. mileense. The results showed that: (1) organogenesis was easily induced by different combinations of plant-growth regulators and sucrose; (2) NAA and sucrose had the most significant effect on shoot induction and shoot multiplication, and (3) the optimum induction and proliferation media were 0.5 mg L^?1 BA + 0.2 mg L^?1 NAA?+?30 g L^?1 sucrose and 1 mg L^?1 BA + 0.1 mg L^?1 NAA?+?30 g L^?1 sucrose, respectively.