Enhanced boron transport into the ear of wheat as a mechanism for boron efficiency

Genotypic variation in boron (B) efficiency in wheat (Triticum aestivum L.) is expressed as large differences in grain set and pollen fertility under low soil B, but the mechanisms responsible for such differences are unknown. This paper aims to determine whether differences in B transport and retranslocation can explain cultivar differences in B efficiency between B-efficient (Fang 60) and B-inefficient (SW41) wheat cultivars. Plants were grown with adequate ¹¹B (10 μM), until the premeiotic interphase stage in anther development, then transferred into ¹⁰B at 0.1 or 10 μM. After five days, ending at the young microspore stage, plants were returned to adequate ¹¹B. Plants were harvested at 0, 1 and 5 days after transferring into ¹⁰B, and at anthesis when fresh pollen was examined for viability. After 5 days in 0.1 μM B, pollen viability in SW41 was depressed by 47%, but pollen of Fang 60 was not affected. When B supply was low, the proportion of plant B partitioned into the ear of Fang 60 was almost twice as high as that in SW 41, enabling Fang 60 to maintain B concentration in the ear at 6.8 mg kg^?1 dry weight (DW), whereas it dropped to 3.8 mg kg^?1 DW in SW 41. Boron accumulation in the ear, when external supply was restricted, did not come from the ¹¹B previously taken up by the plant. The greater ¹⁰B accumulation in ears of Fang 60 compared to SW 41, with limited external B supply, indicated that B efficiency was associated with xylem transport of B. The greater increase of ¹⁰B:¹¹B ratio in the ear of Fang 60 compared to SW 41, over the 5 days of B interruption further indicated that greater B efficiency was associated with a stronger capability for long distance transport of B from the rooting medium into the ear via the xylem rather with than retranslocation of B from vegetative parts.     

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77?×?10⁵ protoplasts (Pp) per gram fresh weight with 94?% viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5?% cellulase Onozuka R10, and 1?% pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5?×?10⁵ Pp ml^?1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93?%. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62?%. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.     

Carotenoids in roots indicated the level of stress induced by mannitol and sodium azide treatment during the early stages of maize germination

Chemical mutagens, such as sodium azide, have attracted the interest of plant breeders. Azide creates DNA point mutations and affects plant growth and development, disturbs metabolic activity and inhibits protein and DNA replication, whereas mannitol is used to simulate drought stresses in tissue culture. To identify biochemical markers for stress tolerance, maize seeds were germinated under mannitol and sodium azide induced stress in controlled conditions for 7 days. Then levels of chlorophyll, carotenoids, phenolics and aldehydes produced were subsequently determined. Germination percentage was not affected by either mannitol or sodium azide and was always above 85%. However, total fresh weight decreased by 50% with the application of 153.4 mM mannitol and 0.26 mM azide in combination. This treatment significantly reduced plantlet growth from 0.94 g in the control to 0.53 g in the treated materials. Root weight reduced by 68.1%, cotyledons by 14.3%, stems by 65.0% and leaves by 70.0% in treated samples. The level of carotenoids in roots was the clearest biochemical indicator of stress produced by the mannitol and sodium azide treatment. Carotenoids increased from 0.01 µg g^??1 fresh weight in the control to 9.03 µg g^??1 fresh weight in the treated materials. A large-scale seed treatment with mannitol and sodium azide was carried out. 2296 seeds were placed in magenta containers with 153.4 mM mannitol and 0.26 mM NaN₃. At 7 days of germination, the heaviest seedlings (450) (450/2296?=?20%) were transferred to soil environment. Forty-two plants (42/450?=?9.3%) were off-type phenotypes at 45 days. Genetic variants may have been obtained following the novel procedure described here which combines chronic treatment with sodium azide and selection pressure with mannitol to simulate drought conditions.     

Assessment of the efficacy of amino acids and polyamines on regeneration of watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis> Thunb.) and analysis of genetic fidelity of regenerated plants by SCoT and RAPD markers

A simple and efficient regeneration protocol was developed for watermelon from cotyledonary node explants excised from 7-day-old in vitro grown seedlings. This study describes the effect of amino acids and polyamines (PAs) along with plant growth regulators (PGRs) on multiple shoot induction and rooting. The highest number of multiple shoots (46.43 shoots/explant) was obtained from cotyledonary node and they were also elongated (6.3 cm/shoot) on MS medium supplemented with 1 mg l^??1 N ⁶ –Benzyladenine (BA), 5 mg l^??1 leucine, and 10 mg l^??1 spermidine. The elongated shoots developed profuse roots (23.03 roots/shoot) in MS medium containing 1 mg l^??1 indole-3-butyric acid (IBA), 5 mg l^??1 isoleucine, and 10 mg l^??1 putrescine. All the rooted plantlets were successfully hardened and acclimatized in the greenhouse with a survival rate of 98%. The present study described an efficient method to obtain a 1.5-fold increase in the number of shoots, compared with the available regeneration protocols for watermelon. The plants developed in this study showed fivefold higher photosynthetic pigments compared to the control plants. The genetic fidelity of the regenerated plants was evaluated by SCoT and RAPD marker analyses, and banding patterns confirmed the true-to-type nature of in vitro regenerated plants.     

Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol

D-Ribitol, a five–carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27–30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid–Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol–KLH–Sepharose CL-6B resulted in pure ribitol–specific antibodies (~45–50 μg/mL). The affinity constant of ribitol antibodies was found to be 2.9?×?10⁷ M^?1 by non-competitive ELISA. Ribitol antibodies showed 100 % specificity towards ribitol, ~800 % cross–reactivity towards riboflavin, 10–15 % cross–reactivity with sorbitol, xylitol and mannitol, and 5–7 % cross–reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4 %) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.     

Enhanced shoot organogenesis in Bixa orellana L. in the presence of putrescine and silver nitrate

An efficient protocol for direct shoot organogenesis in Bixa orellana, known as achiote or annatto or Latkhan (India), has been developed. Using nodal shoot-tip explants, significant organogenetic responses, mean shoot number and shoot elongation were observed when these were incubated on Murashige and Skoog (MS) medium containing 6.66 ??M 6-benzyladenine (BA) and 4.9 ??M indole-3-butyric acid (IBA), and supplemented with either 200?C1,500 ??M putrescine or 40 ??M silver nitrate (AgNO₃). Putrescine at 800 and 1,000 ??M promoted the highest mean shoot length and mean shoot number per explant, respectively. Moreover, various concentrations of putrescine induced callus development. Incorporation of a polyamine biosynthesis inhibitor Difluro-Methyl Ornithine (DFMO) inhibited in vitro shoot multiplication and also altered the endogenous polyamine pool of B. orellana shoots. The field survival of in vitro-derived plants of putrescine and AgNO₃ treatments was 70%. This protocol can be used for improving the in vitro regeneration of B. orellana for transformation studies.     

Influence of Polyols on the Stability and Kinetic Parameters of Invertase from Candida utilis: Correlation with the Conformational Stability and Activity

Invertase (β-d-fructofuranoside fructohydrolase-E.C. 3.2.1.26) is a sucrose hydrolyzing enzyme found in microbial, plant and animal sources. Invertase from Candida utilis is a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. We investigated the mechanism of stabilization of invertase with polyols (glycerol, xylitol, and sorbitol). Activity, thermodynamic and kinetic measurements of invertase were performed as a function of polyol concentration and showed that polyols act as very effective stabilizing agents. The result from the solvent-invertase interaction shows preferential exclusion of the polyols from the protein domain leading to preferential hydration of protein. Apparent thermal denaturation temperature of the protein (T _m ) rose from 75 °C to a maximum of 85 °C in 30% glycerol. The stabilization has been attributed to the preferential hydration of the enzyme.     

Plasmolysis effects and osmotic potential of two phylogenetically distinct alpine strains of Klebsormidium (Streptophyta)

The osmotic potential and effects of plasmolysis were investigated in two different Klebsormidium strains from alpine habitats by incubation in 300–2,000 (3,000) mM sorbitol. Several members of this genus were previously found to tolerate desiccation in the vegetative state yet information was lacking on the osmotic potentials of these algae. The strains were morphologically determined as Klebsormidium crenulatum and Klebsormidium nitens. These species belong to distinct clades, as verified by phylogenetic analysis of the rbcL gene. K. crenulatum is part of to the K. crenulatum/mucosum (‘F’ clade) and K. nitens of the ‘E2’ clade. Plasmolysis occurred in K. crenulatum at 800 mM sorbitol (961 mOsmol kg^?1, Ψ?=??2.09 MPa) and in K. nitens at 600 mM sorbitol (720 mOsmol kg^?1, Ψ?=??1.67 MPa). These are extraordinarily high osmotic values (very negative osmotic potentials) compared with values reported for other green algae. In K. crenulatum, the maximum photosynthetic rate (Pmax) in the light-saturated range was 116 μmol O₂ h^?1 mg^?1 chl a. Incubation in 1,000 mM sorbitol decreased Pmax to 74.1% of the initial value, whereas 2,000 mM sorbitol (Ψ?=??5.87 MPa) lead to an almost complete loss of oxygen production. In K. nitens, Pmax was 91 μmol O₂ h^?1 mg^?1 chl a under control conditions and incubation in 800 mM sorbitol did not decrease Pmax, 2,000 mM sorbitol decreased Pmax only to about 62.6% of the initial value whereas 3,000 mM sorbitol stopped oxygen evolution. This indicated a broader amplitude for photosynthesis in the examined strain of K. nitens. Control samples and samples plasmolysed for 3 h in 800 mM sorbitol (K. nitens), 1,000 mM sorbitol (K. crenulatum), or 2,000 mM sorbitol were investigated by transmission electron microscopy after chemical or high-pressure freeze fixation. In cells undergoing plasmolysis the protoplasts were retracted from the cell wall, the cytoplasm appeared dense, vacuoles were small and fragmented, and the cytoplasm was filled with ribosomes. Thin cytoplasmic strands were connected to the cell wall; 2,000 mM sorbitol increased the effect. The content of soluble carbohydrates in these two strains was investigated by HPLC, as this is one known mechanism for cells to maintain high osmotic pressure of the cytosol. Both Klebsormidium species contained diverse soluble carbohydrates, including a dominant mixed peak of unidentified oligosaccharides, and more minor amounts of raffinose, sucrose, glucose, xylose, galactose, mannose, inositol, fructose, glycerol, mannitol, and sorbitol. The total content of soluble carbohydrates was approximately 1.2% of the dry weight, indicating that this is not a major factor contributing to the high osmotic potential in these strains of Klebsormidium.     

Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-Symport of numerous substrates, including myo-inositol, glycerol, and ribose

Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5–green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H⁺-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.     

Boron neutron capture synovectomy (BNCS) as a potential therapy for rheumatoid arthritis: boron biodistribution study in a model of antigen-induced arthritis in rabbits

Boron neutron capture synovectomy (BNCS) is explored for the treatment of rheumatoid arthritis (RA). The aim of the present study was to perform boron biodistribution studies in a model of antigen-induced arthritis (AIA) in female New Zealand rabbits, with the boron carriers boronophenylalanine (BPA) and sodium decahydrodecaborate (GB-10) to assess the potential feasibility of BNCS for RA. Rabbits in chronic phase of AIA were used for biodistribution studies employing the following protocols: intra-articular (ia) (a) BPA-f 0.14 M (0.7 mg ¹⁰B), (b) GB-10 (5 mg ¹⁰B), (c) GB-10 (50 mg ¹⁰B) and intravenous (iv), (d) BPA-f 0.14 M (15.5 mg ¹⁰B/kg), (e) GB-10 (50 mg ¹⁰B/kg), and (f) BPA-f (15.5 mg ¹⁰B/kg) + GB-10 (50 mg ¹⁰B/kg). At different post-administration times (13–85 min for ia and 3 h for iv), samples of blood, pathological synovium (target tissue), cartilage, tendon, muscle, and skin were taken for boron measurement by inductively coupled plasma mass spectrometry. The intra-articular administration protocols at <40 min post-administration both for BPA-f and GB-10, and intravenous administration protocols for GB-10 and [GB-10 + BPA-f] exhibited therapeutically useful boron concentrations (>20 ppm) in the pathological synovium. Dosimetric estimations suggest that BNCS would be able to achieve a therapeutically useful dose in pathological synovium without exceeding the radiotolerance of normal tissues in the treatment volume, employing boron carriers approved for use in humans. Radiobiological in vivo studies will be necessary to determine the actual therapeutic efficacy of BNCS to treat RA in an experimental model.     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

Phytotoxic doses of boron in contrasting soils depend on soil water content

Boron (B) affects plant growth in soil at B doses (mg added B kg^-1 soil) that appear in the range of natural background B concentrations. A study was set up to determine B bioavailability by testing B toxicity to plant as affected by soil properties and ageing after soil dosing. Nineteen soils (pH 4.4?C7.8) and 3 synthetic soils (sand-peat mixtures) were amended with 7 doses of H₃BO₃. Barley root elongation was determined immediately after B amendment and after 1 and 5 months ageing. Soil solution B concentrations increased linearly with added B concentrations with almost no detectable adsorption. In contrast, the ratio of aqua regia soluble B/soil solution B in unamended soils (no B added) was 10?C25 times higher than in B amended soils at similar aqua regia soluble B concentrations illustrating a much lower B availability in unamended soils. Soil solution B concentrations did not decrease by ageing. The toxic B doses or soil B concentrations that decreased barley root growth by 10% (EC10 values) varied about tenfold (respectively 3?C27 mg added B kg^-1 and 5?C52 mg B kg^-1) among soils. Corresponding thresholds in soil solution varied less than fourfold (16?C59 mg B l^-1). Soil ageing for 5 months did not significantly change EC10 and EC50 values, expressed either as total soil B or as soil solution B, unless in 1 soil. Variability in EC10 and EC50 values was explained by various soil properties (soil moisture content, background B, %clay, cation exchange capacity), but covariance of these properties with the soil moisture content suggest that B dilution is the critical factor explaining B toxicity. It is concluded that effects of B amendments do not decrease by ageing and that soil solution B or B doses corrected for soil moisture content may be used as an index for B toxicity across different soils.     

In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding

Digitalis purpurea L. (Scrophulariaceae; Foxglove) is a source of cardiotonic glycosides such as digitoxin and digoxin which are commercially applied in the treatment to strengthen cardiac diffusion and to regulate heart rhythm. This investigation deals with in vitro propagation and elicited production of cardiotonic glycosides digitoxin and digoxin in shoot cultures of D. purpurea L. In vitro germinated seedlings were used as a primary source of explants. Multiple shoot formation was achieved for three explant types (nodal, internodal, and leaf) cultured on Murashige and Skoog (MS) medium with several treatments of cytokinins (6-benzyladenine—BA; kinetin—Kin; and thidiazuron—TDZ) and auxins (indole-3-acetic acid—IAA; α-naphthaleneacetic acid—NAA; and 2,4-dichlorophenoxy acetic acid—2,4-D). Maximum multiple shoots (12.7?±?0.6) were produced from nodal explants on MS?+?7.5 μM BA. Shoots were rooted in vitro on MS containing 15 μM IAA. Rooted plantlets were successfully acclimatized. To further maintain the multiple shoot induction, mother tissue was cut into four equal parts and repeatedly sub-cultured on fresh shoot induction liquid medium after each harvest. On adaptation of this strategy, an average of 18 shoots per explant could be produced. This strategy was applied for the production of biomass and glycosides digitoxin and digoxin in shoot cultures on MS medium supplemented with 7.5 μM BA and several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). The treatment of KCl, mycelial mass of Helminthosporium sp., and progesterone were highly effective for the production of cardenolides. In the presence of progesterone (200 to 300 mg/l), digitoxin and digoxin accumulation was enhanced by 9.1- and 11.9-folds respectively.     

Susceptibility Test of Two Ca2+-ATPase Conformers to Denaturants and Polyols to Outline Their Structural Difference

To determine the effect of denaturants [guanidine hydrochloride (GdnHCl) and urea] and polyols [with various molecular masses (62.1–600)] on calcium binding at the two hypothesized conformers (A and B forms) of the chemically equivalent sarcoplasmic reticulum Ca²⁺-ATPase, which bind two calcium ions in different manners, we examined the effect of these reagents on the calcium dependence of ATP-supported phosphorylation of the ATPase molecules and of their calcium-activated, acetyl phosphatate hydrolytic activity. (1) GdnHCl (~0.05 M) and urea (~0.5 M) increased the apparent calcium affinity (K _0.5) of 2–6 μM of noncooperative binding [Hill coefficient (n _H) ~ 1] of the A form to 10–40 μM. (2) The employed polyols transformed the binding of the A form into cooperative binding (n _H ~ 2), accompanying the approach of its K _0.5 value to that (K _0.5 = 0.04–0.2 μM) of the cooperative binding (n _H ~ 2) of the B form; the transition concentration (0.025–2 M) of the polyols, above which such transformation occurs, was in inverse relation to their molecular mass. (3) The binding of the B form was resistant to these denaturants and polyols. Based on these data, a structural model of the two forms, calcium-binding domains of which are loosely and compactly folded, is presented.     

Nitrogen-15 signals of leaf-litter-soil continuum as a possible indicator of ecosystem nitrogen saturation by forest succession and N loads

Understanding forest carbon cycling responses to atmospheric N deposition is critical to evaluating ecosystem N dynamics. The natural abundance of ¹⁵N (??¹⁵N) has been suggested as an efficient and non-invasive tool to monitor N pools and fluxes. In this study, three successional forests in southern China were treated with four levels of N addition. In each treatment, we measured rates of soil N mineralization, nitrification, N₂O emission and inorganic N leaching as well as N concentration and ?? ¹⁵N of leaves, litters and soils. We found that foliar N concentration and ??¹⁵N were higher in the mature broadleaf forest than in the successional pine or mixed forests. Three-year continuous N addition did not change foliar N concentration, but significantly increased foliar ?? ¹⁵N (p < 0.05). Also, N addition decreased the ?? ¹⁵N of top soil in the N-poor pine and mixed forests and significantly increased that of organic and mineral soils in N-rich broadleaf forests (p < 0.05). In addition, the soil N₂O emission flux and inorganic N leaching rate increased with increasing N addition and were positively correlated with the ¹⁵N enrichment factor (?? _p/s) of forest ecosystems. Our study indicates that ?? ¹⁵N of leaf, litter and soil integrates various information on plant species, forest stand age, exogenous N input and soil N transformation and loss, which can be used to monitor N availability and N dynamics in forest ecosystems caused by increasing N deposition in the future.     

Kinsenoside and polysaccharide production by rhizome culture of <Emphasis Type="Italic">Anoectochilus roxburghii</Emphasis> in continuous immersion bioreactor systems

Anoectochilus roxburghii (Wall) Lindl. (Orchidaceae) is a precious raw material for medicine. However, the wild resource of A. roxburghii has been endangered, and artificial cultivation results in low yields. To provide rhizomes of A. roxburghii as alternative plant materials, the present study used continuous immersion bioreactor systems to investigate several factors affecting rhizome biomass and bioactive compound accumulation. The bioreactor with a net at the bottom of the sphere in the bioreactor was suitable for production of rhizomes. The rhizome biomass and kinsenoside and polysaccharide accumulation peaked at 30 days of the bioreactor culture. Thus, 30 days was the appropriate culture period. Maximum rhizome biomass and kinsenoside and polysaccharide accumulation were determined when a bioreactor was inoculated with 12.5 g L^??1 (fresh weight) of rhizomes, aerated at 500 mL min^??1, and maintained under 45 µmol m^??2 s^??1 light intensity. This process resulted in the production of 2980.5 mg L^??1 of kinsenoside and 5672.9 mg L^?1 of polysaccharides.     

Mammalian <Emphasis Type="Italic">CYP2E1</Emphasis> gene triggered changes of relative ions fluxes,CaM content and genes expression profiles in <Emphasis Type="Italic">Petunia hybrida</Emphasis> cells to enhance resistance to formaldehyde

The influence of light quality and cytokinin content in media on growth, development, photosynthetic pigments and secondary metabolite content of Myrtus communis L. was evaluated in an in vitro culture. Various treatments with light emitting diodes (LEDs): 100% blue (B), a mix of 70% red and 30% blue (RB) and 100% red were applied and compared with a traditional fluorescent lamp as control. Axillary shoots were incubated on Murashige and Skoog medium with 30 g dm^?3 sucrose, 0.5% BioAgar, 0.5 μM 1-naphthaleneacetic acid and different concentrations of 6-benzyladenine (BA): 1, 2.5 and 5 µM. Cultures were maintained for 6 weeks in 23/21?±?1 °C (day/night), 80% relative humidity and 16/8 h photoperiod; photosynthetic photon flux density (PPFD) was 35 µmol m^?2 s^?1 in all treatments. Light spectra and BA content in media affected biometrical and phytochemical M. communis properties. Red LEDs and 5 µM BA resulted in the highest multiplication rate. The highest shoots were obtained under red LEDs, but with the lowest concentration of cytokinin in media. Fresh weight was greatest on LEDs containing blue light in the spectrum (B and RB); moreover, 5 µM BA increased dry weight. Photosynthetic pigment levels were lower under LED light compared to control lamps. Phenolic acids and flavonoids were identified in M. communis leaf extracts. Myricetin was the major constituent with highest concentration under red LEDs and highest BA level.     

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

Optimal concentration of Beauveria bassiana vectored by bumble bees in relation to pest and bee mortality in greenhouse tomato and sweet pepper

Greenhouse cage trials were conducted to determine the optimal concentration of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) (BotaniGard 22WP^® formulation) as vectored by the bumble bee, Bombus impatiens (Cresson) (Hymenoptera: Apidae) pollinator for control of greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) on greenhouse tomato, tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae) and green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) on greenhouse sweet pepper. Three inoculum concentrations of B. bassiana: low, 9 × 10⁹; middle, 6.24 × 10¹⁰; and high, 2 × 10¹¹ conidia g^?1 of inoculum and two controls (one with bees and heat-inactivated inoculum, and the other which contained only the host plants and pest species) were tested in a completely randomized block design. Beauveria bassiana killed 18, 54 and 56% of the adult T. vaporariorum and 33, 70 and 67% of the adult L. lineolaris, respectively, at the low, middle and high concentrations; but no infection from B. bassiana occurred in each of the control treatments. Internal infection rates after surface sterilization of the pest insects were 11, 34 and 35% for adult T. vaporariorum, 29, 54 and 58% for adult L. lineolaris, 22, 34 and 30% for nymphal M. persicae and 17, 29 and 32% for nymphal T. vaporariorum, respectively, at the low, middle and high concentrations. Significantly more bumble bees died at the high concentration of B. bassiana (42–45%) than at the other concentrations (9–15%) and the controls (5–7%). Spores of B. bassiana were collected throughout the plant canopy with the greatest numbers sampled from the top third of the canopy [ca. 1,200 colony forming units (CFU) per cm^?2]. The middle concentration was selected as the optimal concentration because it provided the best pest control with the least impact on the bees.     

Enhancement of succinate production by metabolically engineered Escherichia coli with co-expression of nicotinic acid phosphoribosyltransferase and pyruvate carboxylase

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD⁺. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD⁺ ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l^?1 glucose and produced 14.08 g l^?1 succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased.