Antibiotic MIC/MBC analysis of Bacillus-based commercial insecticides: use of bioreduction and DNA-based assays

Bacillus thuringiensis subsp israelensis (Bti) and subsp kurstaki (Btk); (2) vegetative cells derived from these BT products; and (3) Gram-positive and Gram-negative bacteria used as controls. The XTT-kinetic assay improved sensitivity and early reading of MIC breakpoints. The conventional colony count method for determining minimal bactericidal concentration (MBC) was used to validate a multi-sample dot-blot assay in which organisms in individual MIC assays are trapped free of residual antibiotic and their viability is estimated by in situ conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to insoluble formazan. Tolerance (MBC/MIC) for most antibiotics was low (≤4). Resistance to β-lactams was attributed to β-lactamase activity in both BT products and cultures derived from them. MIC and MBC breakpoints in spore-based assays were also approximated by changes in genome copy, using δ-endotoxin and β-lactamase genes as probes. The DNA assays are effective for monitoring and authenticating organisms in microbe-containing biotechnology products. Received 29 September 1998/ Accepted in revised form 12 February 1999     

Streptomyces daliensis sp. nov. from soil

A novel actinomycete strain YIM 31724^T was isolated from a soil sample collected from Dali, Yunnan Province, People’s Republic of China. The strain is characterized by white to yellow white aerial mycelia, spiral spore chains and smooth spore surface. The cell wall of strain YIM 31724^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell hydrolysates contained mainly glucose and small amounts of galactose and xylose. The menaquinones were MK-9(H₆) (31%) and MK-9(H₈) (69%). Phosphatidylethanolamine was the diagnostic phospholipid. The DNA G+C content of strain YIM 31724^T was 67.2 mol%. Phylogenetic analysis indicated that the strain belongs to the genus Streptomyces, with highest similarity to Streptomyces rimosus subsp. rimosus JCM 4667^T (rRNA gene sequence similarity value of 98.9%) and Streptomyces erumpens DSM 40941^T (rRNA gene sequence similarity value of 98.7%). Based on its phenotypic and genotypic characteristics, including low DNA–DNA hybridization results, strain is proposed as the type strain of a novel species, Streptomyces daliensis sp. nov.     

In Vitro and In Vivo Antioxidant Activity of <Emphasis Type="Italic">Bifidobacterium animalis</Emphasis> 01 Isolated from Centenarians

Several studies reported the antioxidant activity of bifidobacteria using assays in vitro. In present study, the in vitro and in vivo antioxidant activity of Bifidobacterium animalis 01 was investigated. Culture supernatant, intact cells, and intracellular cell-free extracts of B. animalis 01 were involved in this study. The antioxidant assays in vitro included lipid peroxidation assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, hydroxyl radical ( ^• OH) assay and superoxide anion ( \textO₂ ^- {\text{O}}_{2}^{ - } ) assay. The antioxidant assays in vivo were conducted using mice model. Activities of antioxidative enzymes, malondialdehyde (MDA) content in serums and livers of aging mice were evaluated. Monoamine oxidase (MAO) activity and lipofuscin level in brains of aging mice were also characterized. Results showed that culture supernatant, intact cells and intracellular cell-free extracts of B. animalis 01 could effectively scavenge free radicals, significantly enhance mice’s activities of antioxidative enzymes and reduce mice’s MDA content, lipofuscin level and MAO activity. Our results indicated that B. animalis 01 has the potential to be developed into a dietary antioxidant supplements.     

Reactive oxygen species production and antioxidative defense system in pea root tissues treated with lead ions: the whole roots level

The lead absorbed by the roots induce oxidative stress conditions through the Reactive oxygen species (ROS) production for the pea plants cultivated hydroponically for 96 h on a Hoagland medium with the addition of 0.1 and 0.5 mM of Pb(NO₃)₂. The alterations in \textO₂ ^{- ·} {\text{O}}_{2}^{ - \cdot } and H₂O₂ concentrations were monitored spectrophotometrically which show a rapid increase in \textO₂ ^{- ·} {\text{O}}_{2}^{ - \cdot } production during the initial 2 h, and in case of H₂O₂, during the eighth hour of cultivation. The level of ROS remained higher at all the time points for the roots of the plants cultivated with Pb²⁺ and it was proportional to metal concentration. The production of \textO₂ ^{- ·} {\text{O}}_{2}^{ - \cdot } and H₂O₂ was visualized by means of fluorescence microscope technique. They are produced in nonenzymatic membrane lipid peroxidation and its final product is Malondialdehyde, the level of which increased together with the level of H₂O₂. As stress intensity raised (duration of treatment and Pb²⁺ concentration), so did the activities of superoxide dismutases, catalase and ascorbate peroxidase antioxidative enzymes and of low-molecular antioxidants, particularly glutathione (GSH), homoglutathione (h-GSH) and cysteine substrate toward their synthesis. The root cells redox state (GSH/GSSG) dropped proportionally to lead stress intensity.     

Interfacial Tension of the Lipid Membrane Formed from Phosphatidylcholine–Decanoic Acid and Phosphatidylcholine–Decylamine Systems

Interfacial tension has been determined for phosphatidylcholine (PC)–decanoic acid (DA) and PC–decylamine (DE) membranes. PC (lecithin), DA and DE were used in the experiments; the interfacial tension values of the pure components are 1.62 × 10⁻³, −2.38 × 10⁻² and −3.88 × 10⁻² N/m (hypothetical values for DA and DE), respectively. The 1:1 complexes were formed during formation of PC–DA and PC–DE membranes. The following parameters describing the complexes were determined: the surface concentrations of the lipid membranes formed from these complexes, A₃ ^{- 1} A_{3}^{ - 1} ; the interfacial tensions of such membranes, γ ₃; and the stability constants of these complexes, K.     

Starch processing wastewater as a new medium for production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillus thuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL⁻¹ and higher entomotoxicity of 1,050 IU μL⁻¹ were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for safe SPW disposal and production of high potency and low cost bioinsecticide.     

A Novel Approach for Bioremediation of a Coastal Marine Wastewater Effluent Based on Artificial Microbial Mats

A novel alternative for wastewater effluent bioremediation was developed using constructed microbial mats on low-density polyester. This biotechnology showed high removal efficiencies for nitrogen and phosphorous in a short retention time (48 h): 94% for orthophosphate (7.78 g m³ d⁻¹), 79% for ammonium (11.30 g m⁻³ d⁻¹), 78% for nitrite (7.46 g m⁻³ d⁻¹), and 83% for nitrate (8.55 g m⁻³ d⁻¹). The microbial mats were dominated by Cyanobacteria genera such as Chroococcus sp., Lyngbya sp., and bacteria of the subclass Proteobacteria representative of the Eubacteria Domain. Nitzschia sp. was the dominant Eukaryote Domain. Various N and P substrates in the wastewater permit the growth of self-forming and self-sustaining bacterial, microalgal, and cyanobacterial communities on a polyester support. The result is the continuous, self-sufficient growth of microbial mats. This is an innovative, economical, and environmentally safe alternative for the treatment of wastewater effluents in coastal marine environments.     

Influence of {\text{NH - }}{{\text{S}}^\gamma } bonding interactions on the structure and dynamics of metallothioneins

Mammalian metallothioneins ( \textM₇^\textIIMTs {\text{M}}_7^{\text{IIMTs}} ) show a clustered arrangement of the metal ions and a nonregular protein structure. The solution structures of Cd₃-thiolate cluster containing β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Molecular dynamics simulations revealed a substantially increased number of \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd₃-thiolate cluster and the enfolding protein domain. Alterations in the \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biological function.     

Modification of the data-processing method for the turbidimetric bioassay of nisin

The data processing method of the turbidimetric bioassay of nisin was modified to facilitate its industrial application. The influence of the initial indicator concentration was minimized by a redefined specific dose of the bacteriocin as the quotient between the titer of the added bacteriocin and the initial population density of the indicator in the suspension. It was found that d _c = 0.125 μg ml⁻¹ was the critical dose of nisin that can cause a complete inhibition of the indicator, Pediococcus acidilactici UL5, with an initial OD of 0.135. To eliminate the interference of the cell debris, an equation, , exploiting d _c, was formulated to obtain the intrinsic survival proportion. The use of the specific dose of the bacteriocin and the intrinsic survival proportion as parameters of the dose/response curve greatly enhanced its repeatability and feasibility. A dual-dosage approach was developed to further simplify the conventional standard dose/response curve method.     

A Combined Patch-Clamp and Electrorotation Study of the Voltage- and Frequency-Dependent Membrane Capacitance Caused by Structurally Dissimilar Lipophilic Anions

Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA⁻), tetraphenylborate (TPB⁻) and [W₂(CO)₁₀(S₂CH)]⁻, the putative lipophilic anion and three new heterocyclic W(CO)₅ derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA⁻, TPB⁻ and increased and that of W(CO)₅ derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA⁻ and W(CO)₅ derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB⁻ and no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.     

Genetic effects of forest fragmentation in high-density Araucaria angustifolia populations in Southern Brazil

Araucaria angustifolia is an endangered tropical/subtropical coniferous of great interest for conservation due its economical, ecological, and social value. Only 3% of original Araucaria forests remain, which are generally confined to small forest fragments. Forest fragmentation can have serious consequences on genetic process in tree population, affecting long-term fitness and adaptability. To investigate the effects of forest fragmentation on genetic diversity and the structure of A. angustifolia populations, the genetic diversity of eight microsatellite loci was compared in four small fragmented populations (<22 ha), four tree groups (five to 11 trees) occurring in pastures and in three plots in a large continuous population. The clearest effect of fragmentation was the loss of rare alleles (p ≤ 0.05) in fragmented populations (19.4% to 47.2%) and intermediate frequency (0.05 < p ≤ 0.25) and rare alleles (p ≤ 0.05) in tree groups (19% to 86.1%) in comparison to continuous populations. Fragmented populations have significant higher fixation index ( [^(F)]_\textIS = 0.121 \widehat{F}_{\text{IS}} = 0.121 , P < 0.05) than continuous populations ( [^(F)]_\textIS = 0.083 \widehat{F}_{\text{IS}} = 0.083 , P < 0.05). High genetic differentiation was detected among tree groups ( [^(G)]_\textST^￠ = 0.258 \widehat{G}_{{{\text{ST}}}}^{\prime } = 0.258 , P < 0.01) and low among fragments ( [^(G)]_\textST^￠ = 0.031 \widehat{G}_{{{\text{ST}}}}^{\prime } = 0.031 , P < 0.05) and continuous populations ( [^(G)]_\textST^￠ = 0.026 \widehat{G}_{{{\text{ST}}}}^{\prime } = 0.026 , P < 0.05), showing a significant bottleneck effect in tree groups. Evidence that forest fragments have experienced a recent bottleneck was confirmed in at least two studied fragments. The implications of the results for conservation of the fragmented A. angustifolia populations are discussed.     

Model of Active Transport of Ions Through Diatom Cell Biomembrane

A model of the active transport of ions in the Cascinodiscus wailesii diatom cell is constructed taking into account the transport of H⁺, Na⁺, K⁺, Ca⁺², NO₃^-\mathrm{NO}_{3}^{-}, Cl⁻, and NH₄⁺\mathrm{NH}_{4}^{+} ions. This model allows calculating intracellular concentrations of basic ions and the biomembrane resting potential. A hierarchical algorithm “one ion—one transport system” is used in the model. The dependence of the resting potential on the extracellular concentration of potassium is plotted in terms of the model. The calculated values are in good agreement with the corresponding experimental data.     

Studies in imitative behavior: A generalization of the rashevsky model; Its mathematical properties

This paper is based on N. Rashevsky's theory of imitative behavior, the underlying idea being that performance of one reaction by a given individual produces an increased stimulation (or tendency) toward the same reaction in other individuals. For simplicity, consideration is limited to cases in which each individual may choose only between two (or two main categories of) reactions, denoted byA andB in the following. However, upon suggestion from Dr. Rashevsky and certainly in better agreement with actual facts, the strength of imitative interaction is assumed to vary from individual to individual. More precisely, if Ψ_i denotes the additional excitation caused by imitation in theith individual,PA_i the probability for performance of reactionA, andPB_i the probability for performance of reactionB by theith individual, we postulate that where the constants α _ik ^′ and β _ik ^′ (coefficients of imitative interaction) measure the amount of imitative influence exerted by thekth individual upon theith,N being the total number of individuals in the population. The term — α_i Ψ_i accounts for the spontaneous decay of excitation, and the quantities α _ik ^′ and α _ik ^′ are assumed to benon-negative. The expressions forPA_i andPB_i are obtained from H. D. Landahl's theory of conflicting stimuli; they depend non-linearly on the values Ψ_i. It is implicit in this formulation that the theory can only be applied if the frequency of contacts between individuals is not too small. Some further shortcomings and limitations of the model are outlined, and the discussion includes suggestions for reinterpretation and improvement of the theory. If all the quantities α _ik ^′ and α _ik ^′ have the same value, sayA, we return to the case treated by Rashevsky (and Landau, 1950); these authors, however, replace the sums in the equation above by integrals, which automatically restricts the validity of their results to very large values ofN. Their work may therefore be characterized by the assumption of uniform interaction in large populations. Our equations, on the other hand, are applicable even to very small groups, and therein lies one of their main advantages. In this paper the mathematical properties of the non-linear system of equations above are studied with particular reference to the existence and stability of steady states [dΨ_i/dt ≡ 0;, i = 1 , 2, . . . N]. A sufficient condition for the existence of only one stable steady state is derived. It may be formulated roughly by stating that all the coefficients of interaction should be sufficiently small. It that is not the case, there may exist a greater number of stationary states. In particular, two of them (called “extremal”) have the following properties: they arestable and such that the average number of individuals in the group performing one or the other reaction is the largest (or smallest) possible as compared with the other steady states. Hence the situation is qualitatively similar to that found by Rashevsky and Landau.Quantitatively, however, important differences may arise, depending on the nature of the matrix specifying the interaction. A stable state may be approached through damped oscillations, but this effect is important only if the damping is sufficiently small for the oscillations to become practically observable. Little information could be obtained on this point, due to mathematical difficulties. As mentioned above, the most interesting applications of this theory will be with respect to small populations or to populations partitioned into subgroups with varying amounts of imitative interaction within as well as between groups.     

Hydrochemical fluctuations and crustacean community composition in an ephemeral saline lake (Sua Pan,Makgadikgadi Botswana)

Fluctuating hydrochemistry, as a result of extreme hydrological regimes, imposes major physiological constraints on the biota of ephemeral saline lakes. While the inverse relationship between salinity and zooplankton species richness is well-known across salinity gradients, few studies have documented closely the response of zooplankton to seasonal changes in salinity. Weekly sampling during two flood seasons at Sua Pan, an intermittent saline lake in central Botswana demonstrated the importance of spatial and temporal salinity gradients for crustacean community composition, associated with a decline in species richness, from 11 to three species. Conductivity ranged between 320 and 125,800 μS cm⁻¹ during seasonal flooding; changing from dominance by and , Ca²⁺ and Mg²⁺, at the beginning of the floods, to NaCl dominated waters as the lake dried out and salinities increased. pH estimates generally ranged between 8.6 and 10, with maximum values recorded during initial flooding. Crustaceans comprised mainly Branchinella spinosa, Moina belli, Lovenula africana and Limnocythere tudoranceai, all of which occurred across a wide range of salinities, while halotolerant freshwater species (Metadiaptomus transvaalensis, Leptestheria striatochonca and the ostracods Plesiocypridopsis aldabrae, Cypridopsis newtoni and a newly identified Potamocypris species) disappeared above conductivities of 1,500 μS cm⁻¹. A unique crustacean composition in southern Africa was attributed to Sua Pans’ rare chemical composition among southern African saline lakes; flood waters on Sua Pan contained a higher proportion of Na⁺ and , and less K⁺, Mg²⁺ and than over 80% of records from salt pans elsewhere in southern African. The freshwater species of crustaceans in Sua Pan were similar to those found in other southern Africa lakes, and these similarities decreased in lakes with higher pH and proportions of Na, and less SO₄ and Mg in their chemical composition. The predominant saline tolerant species on Sua Pan, however, showed a greater similarity to those in saline lakes in southern and East Africa with higher proportions of and, particularly, Mg²⁺ in their chemical composition. Handling editor: J. M. Melack     

13C isotopic discrimination: a starting point for new insights in competition for nitrogen and water under contour hedgerow systems in tropical mountainous regions

Competition for nutrients and water between crops and associated hedgerows reduces overall performance of contour hedgerow systems and hampers its acceptance by rural communities in tropical mountainous regions. Therefore, it is imperative to better understand competition leading to a decline in crop response close to hedges. In the highlands of North East Thailand spatial variability in grain yield of maize (Zea mays L., cv. Suwan 1) was assessed for two contour hedgerow systems based on Brachiaria ruziziensis Germain et Evrard (Ruzi grass) barriers or Leucaena leucocephala (Lam) de Wit hedges without or with fertilizer (60 kg N ha⁻¹ and 14 kg P ha⁻¹). Available was analyzed across the slope. In addition, shoot N concentration and δ¹³C values in leaves were measured for maize plants in the center of the alley and in the row next to and at the upper side of barriers or hedges. Despite variable field conditions, δ¹³C values were significantly (p < 0.05) less depleted close to the barriers or hedges, except for 2 out of 16 plots, suggesting that water deficiency was not the main driver for spatial variability along the alleys. The negative correlation between ¹³C isotopic discrimination and available in the soil, with R ² ranging from 0.5 (p < 0.10) to 0.9 (p < 0.01), assigned a major role to N availability in the reduced crop response towards the barriers. The proposed framework of ¹³C isotopic discrimination, together with plant and soil N data, is a new approach and was shown to be suitable to determine N and water competition between hedgerows and crops grown in alleys under field conditions.     

Soil sampling for <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> spores in Queensland sugarcane fields

Three sugarcane fields in Bundaberg and four fields in each of the Burdekin, Tully and Innisfail (Queensland, Australia) were sampled for spores of Metarhizium anisopliae (Metchnikoff) (Deuteromycotina: Hyphomycetes). This entomopathogenic fungus is the active ingredient in the biocide “BioCane^TM”, which was developed for the management of the greyback canegrub Dermolepida albohirtum (Waterhosue) (Coleoptera: Scarabaeidae) and other scarabs in cane fields. Fields sampled were of different crop ages and all had a history of BioCane^TM treatment in Plant Cane in past years. Soil samples were taken in each field from four depths (0–10, 10–20, 20–30 and 30–40 cm below soil level) with the use of an auger. Spore levels were highest at the depths of 10–20 and 20–30 cm. Spore levels differed between locations with Innisfail and Tully recording the highest spore counts. Spores were also found in the inter-row space in plots sampled in Tully. Sampling statistics were determined for M. anisopliae spores at the four soil depths with 0.1 and 0.25 precision levels. Three sampling methods were compared (use of marker beads; use of 100 mm auger and 150 mm auger). Samples that relied on marker beads resulted in higher spore counts, however, an auger can still be used since BioCane^TM does not normally contain coloured markers. Results obtained demonstrate the ability of the pathogen to translocate in soil profile and across rows, most likely due to grub movements and other soil fauna. Sampling for M. anisopliae spores provides good monitoring of their levels in soil. The implications of this on grub management decisions are discussed.     

Soil algal communities inhabiting zinc and lead mine spoils

Algal communities inhabiting four calamine mine spoils differing in time since cessation of exploitation and loaded with high concentrations of zinc (20,284–61,599 μg g⁻¹ soil DW), lead (2,620–3,885 μg g⁻¹ DW) and cadmium (104–232 μg g⁻¹ DW) were studied. In dump soils of slightly alkaline pH (7.28–7.52) and low nutrient (, , ) concentrations, chlorophyll a content ranged from 0.41 to 2.27 μg g⁻¹ soil DW. In total, 23 algal species were recorded. Chlorophyta were the dominant taxonomic group (42–55% of all identified species) followed by Cyanobacteria (28–36%) and Heterokontophyta (13–21%). The highest species richness (18) was observed in the oldest dump (120 years old) with natural succession, while in younger dumps it was lower (11–15). Total algal abundance ranged between 5.5 and 19.1 × 10² ind. g⁻¹ soil DW, and values of Margalef’s diversity indices (1.59–2.25) were low. These results may suggest that both high concentrations of heavy metals and low nutrient content influenced the algal communities in all the dumps studied. The differences in algal microflora observed between tailing dumps may indicate that habitat quality improved with time and that algae isolated from Zn/Pb-loaded soils may be Zn/Pb-resistant ecotypes of ubiquitous species.     

Can biological nitrification inhibition (BNI) genes from perennial Leymus racemosus (Triticeae) combat nitrification in wheat farming?

Using a recombinant luminescent Nitrosomonas europaea assay to quantify biological nitrification inhibition (BNI), we found that a wild relative of wheat (Leymus racemosus (Lam.) Tzvelev) had a high BNI capacity and releases about 20 times more BNI compounds (about 30 ATU g⁻¹ root dry weight 24 h⁻¹) than Triticum aestivum L. (cultivated wheat). The root exudate from cultivated wheat has no inhibitory effect on nitrification when applied to soil; however, the root exudate from L. racemous suppressed formation and kept more than 90% of the soil’s inorganic-N in the -form for 60 days. The high-BNI capacity of L. racemosus is mostly associated with chromosome Lr#n. Two other chromosomes Lr#J, and Lr#I also have an influence on BNI production. Tolerance of L. racemosus to is controlled by chromosome 7Lr#1-1. Sustained release of BNI compounds occurred only in the presence of in the root environment. Given the level of BNI production expressed in DALr#n and assuming normal plant growth, we estimated that nearly 87,500,000 ATU of BNI activity ha⁻¹ day⁻¹ could be released in a field of vigorously growing wheat; this amounts to the equivalent of the inhibitory effect from the application of 52.5 g of the synthetic nitrification inhibitor nitrapyrin (one AT unit of BNI activity is equivalent to 0.6 μg of nitrapyrin). At this rate of BNI production it would take only 19 days for a BNI-enabled wheat crop to produce the inhibitory power of a standard commercial application of nitrapyrin, 1 kg ha⁻¹. The synthetic nitrification inhibitor, dicyandiamide, blocked specifically the AMO (ammonia monooxygenase) pathway, while the BNI from L. racemosus blocked the HAO (hydroxylamine oxidoreductase) pathway in Nitrosomonas. Here we report the first finding of high production of BNI in a wild relative of any cereal and its successful introduction and expression in cultivated wheat. These results demonstrate the potential for empowering the new generation of wheat cultivars with high-BNI capacity to control nitrification in wheat-production systems. Responsible Editor: Hans Lambers.     

Nitric oxide promotes in vitro organogenesis in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

The effects of nitric oxide (NO) on caulogenesis, shoot organogenesis and rhizogenesis from hypocotyl explants of Linum usitatissimum were investigated. Exogenously supplied NO donors, 5 μM sodium nitroprusside (SNP), 2 μM S-nitroso-N-acetylpenicillamine (SNAP) and 2 μM 3-morpholinosydnonimine (SIN-1), significantly promoted shoot differentiation from the hypocotyl explants of L. usitatissimum excised from its in vitro raised seedlings. Potassium ferrocyanide, a structural analogue of SNP, lacking NO group, did not promote shoot organogenesis. Likewise, products of NO, \textNO₂ ^- {\text{NO}}_{2}^{ - } and \textNO₃ ^- {\text{NO}}_{3}^{ - } supplied as 5 μM NaNO₂ and 5 μM NaNO₃ did not enhance shoot differentiation. Another source of NO, a mixture of sodium nitrite (SN) provided along with ascorbic acid (AsA), also caused significant promotion in the average number of shoots per responding explant. SNP also augmented the rhizogenic response of the microshoots in terms of percentage of responding explants, number of roots per responding explant and average root length. The NO scavengers, 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimideazoline-1-oxyl-3-oxide (cPTIO) or methylene blue (MB), provided along with SNP, SNAP, SIN-1 or SN + AsA, at concentrations equimolar to the optimum concentration of the donors, reversed the promotory influence, thereby, confirming the role of NO in promotion of in vitro morphogenesis. However, NO scavengers individually did not affect the observed morphogenic processes. Morphological and histological studies of hypocotyl segments cultured on BM or BM + SNP for 4, 8 and 12 days demonstrated that SNP enhanced shoot differentiation by inducing a higher number of shoot primordia, each of which develops into a single shoot.