Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

Optimization of <Emphasis Type="Italic">Renealmia mexicana</Emphasis> (Klotzsch ex. Petersen) cultivation <Emphasis Type="Italic">in vitro</Emphasis>

Renealmia mexicana (Klotzsch ex. Petersen) is a tropical plant found in southern México with an ornamental value and a potential source of curcuminoids. Its distribution in Chiapas has decreased because of deforestation and low propagation and germination rate, so a protocol for in vitro propagation was developed. An orthogonal experimental design of L₉ (3⁴) in triplicate was used to investigate the effect of 6-benzyl adenine (BA), indole butyric acid (IBA), silver nitrate (AgNO₃), and sucrose on shoot, root, and leaf development of plantlets grown in vitro. Plantlets with well-developed shoots and roots were transferred to pots containing a mixture of peat moss and agrolite for hardening before transfer to soil. The Murashige and Skoog (Physiol. Plant. 15:473–497, 1962) mineral medium (MS) supplemented with 4.4 μM BA, 2.5 μM IBA, 11.7 μM AgNO_3y and 5.5% (w/v) sucrose gave most shoots, 8.9 μM BA, 2.5 μM IBA, 17.7 μM AgNO₃ and 5.5% (w/v) sucrose most roots, and 8.9 μM BA, 4.9 μM IBA, 11.7 μM AgNO₃ and 3.0% (w/v) sucrose most leaves, although other combinations were statistically equivalent in each case. Sucrose was the factor that most explained the variation in the promotion of shoots, roots, and leaves. The protocol developed resulted in up to 100% survival when plantlets were transferred to soil using AgNO₃, confirming that hardening of plantlets in vitro using hormonal stimulation was a suitable strategy to improve acclimatization.     

<Emphasis Type="Italic">In vitro</Emphasis> multiplication of heavy metals hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0–0.2 mg dm⁻³), gibberellic acid (GA₃; 0–1.0 mg dm⁻³) and riboflavin (0–3.0 mg dm⁻³). The maximum number of shoots was developed on medium containing 1.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA. GA₃ (0.5 mg dm⁻³) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm⁻³ BA + 0.5 mg dm⁻³ GA₃ + 1.0 mg dm⁻³ riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm⁻³ sucrose, 8 g dm⁻³ agar and 1.0 mg dm⁻³ indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

The peroxidase activity of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach chloroplasts

The cytochrome b ₆ f (Cyt b ₆ f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H₂O₂) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (K _m) values for H₂O₂ and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k _obs) was about 26 M⁻¹ s⁻¹ and the turnover number (K _cat) was about 60 min⁻¹ (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H₂O₂]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Characterization of <Emphasis Type="Italic">Francisella</Emphasis> sp., GM2212, the first <Emphasis Type="Italic">Francisella</Emphasis> isolate from marine fish,Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella sp., isolate GM2212^T, previously isolated from diseased farmed Atlantic cod Gadus morhua in Norway is characterized. The complete 16S rDNA, 16S–23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and a hypothetical lipoprotein (LpnB) is sequenced and compared with Francisella tularensis and Francisella philomiragia. All these sequences support a close relationship between GM2212^T and F. philomiragia. The bacterium grows at 10–25°C with an optimum at about 20°C, a temperature range clearly different from F. tularensis and F. philomiragia. GM2212^T is catalase-positive, indole positive, oxidase-negative, do not produce H₂S in Triple Sugar Iron agar, and does not hydrolyze gelatin, is resistant to erythromycin and susceptible to ceftazidime, the latter five characteristics separating it from F. philomiragia. Cysteine enhances growth. Acid is produced from d-glucose, maltose, sucrose (weak) but not from lactose or glycerol. GM2212^T grows on both MacConkey agar and in nutrient broth (6% NaCl). The bacterium is resistant to trimethoprim-sulfamethoxazole, penicillines, cefuroxime and erythromycin; but is susceptible to ceftazidime, tetracycline, gentamicin, ciprofloxacin. Based on the molecular and phenotypical characteristics, we suggest that this GM2212 isolate, may represent a new species of Francisella. Isolate GM2212^T (=CNCM I-3481^T = CNCM I-3511^T = DSM 18777^T).     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Chemoorganoheterotrophic Growth of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrosomonas eutropha</Emphasis>

The ammonia oxidizers Nitrosomonas europaea and Nitrosomonas eutropha are able to grow chemoorganotrophically under anoxic conditions with pyruvate, lactate, acetate, serine, succinate, α-ketoglutarate, or fructose as substrate and nitrite as terminal electron acceptor. The growth yield of both bacteria is about 3.5 mg protein (mmol pyruvate)⁻¹ and the maximum growth rates of N. europaea and N. eutropha are 0.094 d⁻¹ and 0.175 d⁻¹, respectively. In the presence of pyruvate and CO₂ about 80% of the incorporated carbon derives from pyruvate and about 20% from CO₂. Pyruvate is used as energy and only carbon source in the absence of CO₂ (chemoorganoheterotrophic growth). CO₂ stimulates the chemoorganotrophic growth of both ammonia oxidizers and the expression of ribulose bisphosphate carboxylase/oxygenase is down-regulated at increasing CO₂ concentration. Ammonium, although required as nitrogen source, is inhibitory for the chemoorganotrophic metabolism of N. europaea and N. eutropha. In the presence of ammonium pyruvate consumption and the expression of the genes aceE, ppc, gltA, odhA, and ppsA (energy conservation) as well as nirK, norB, and nsc (denitrification) are reduced.     

Foliar nutrition of <Emphasis Type="Italic">in vitro</Emphasis>-cultured <Emphasis Type="Italic">Prosopis chilensis</Emphasis> (Molina) Stuntz shoots

Summary Foliar nutrition has been conceived as a possible means of overcoming the recalcitrance of Prosopis chilensis (Molina) Stuntz explants to standard in vitro culture. The foliar uptake of cations (K from 20 gl⁻¹ KNO₃ and Ca from 50 gl⁻¹ CaCl₂), anions (NO₃ from 50 gl⁻¹ KNO₃ and PO₄ from 50 gl⁻¹ NaH₂PO₄), and glucose from a 100 mg l⁻¹ solution studied. All of the nutrients examined were absorbed. The efficacy of foliar nutrition in prolonging the vigor of micropropagated P. chilensis shoot tips was compared with nutrients supplied as a liquid to the base of the stem (liquid) or as an agar-solidified medium (agar). A foliar-feeding apparatus was constructed that employed pressurization of the medium reservoir to drive the medium into the culture vessel with a passive return by a siphoning effect. The medium used was Murashige and Skoog with 30 gl⁻¹ sucrose, 0.1 mgl⁻¹ benzylaminopurine, and 1 mgl⁻¹ indole-3-butyric acid. Over a 9-wk test period it was found that explants cultured by foliar nutrition performed significantly better than those grown on agar for shoot length, nodal production, and leaf retention; and better than liquid MS for node production. There was no significant difference among the three treatments in percentage survival, percentage rooting, or the mean number of roots.     

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between <Emphasis Type="Italic">Castanea sativa</Emphasis> and <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis>

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H₂O₂ and O₂ ^·− during the early stages of fungal contact. Three peaks of H₂O₂ production were detected, the first two coinciding with O₂ ^·− bursts. The first H₂O₂ production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H₂O₂ scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H₂O₂ production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H₂O₂ results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Direct shoot regeneration from immature inflorescence cultures of <Emphasis Type="Italic">Chlorophytum arundinaceum</Emphasis> and <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L⁻¹ BA, 150 mg L⁻¹ Ads, 0.1 mg L⁻¹ NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L⁻¹ IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.     

Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> strains: <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

Features of alloplasmic wheat-barley substitution and addition lines (<Emphasis Type="Italic">Hordeum marinum</Emphasis> subsp. <Emphasis Type="Italic">gussoneanum</Emphasis>)-<Emphasis Type="Italic">Triticum aestivum</Emphasis>

Two alloplasmic wheat-barley substitution lines were studied: a line replaced at three pairs of chromosomes 1H ^mar (1B), 5H ^mar (5D), and 7H ^mar (7D), and the disomic-substituted line 7H ^mar (7D). The lines were constructed on the basis of individual plants from BC₁F₈ and BC₂F₆ progeny of barley-wheat hybrids (H. marinum subsp. gussoneanum Hudson (= H. geniculatum All.) (2n = 28) × T. aestivum L.) (2n = 42) (Pyrotrix 28), respectively. Moreover, the alloplasmic wheat-barley ditelosomic addition line 7HL ^mar isolated among plants from the BC₁F₆ progeny of a barley-wheat amphiploid was studied, which in this work corresponds to BC₂F₁₀ and BC₂F₁₁ progeny. It was ascertained that when grown in the field, these alloplasmic lines manifest stable self-fertility. Plants of the given lines are characterized by low height, shortened ears, the fewer number of stems and ears, and of spikelets in the ear, by decreased grain productivity and weight of 1000 grains, in comparison with the common wheat cultivar Pyrotrix 28. The inhibition of trait expression in alloplasmic wheat-barley substitution and addition lines may be connected not only with the influence of wild barley chromosomes functioning in the genotypic environment of common wheat, but also with the effect of the barley cytoplasm. The alloplasmic line with substitution of chromosomes 1H ^mar (1B), 5H ^mar (5D), and 7H ^mar (7D) or the alloplasmic line 5HL ^mar with ditelosomic addition have, in comparison with the common wheat cultivar Pyrotrix 28, an increased grain protein content, which is explained by the effect of wild barley H. marinum subsp. gussoneanum chromosomes.