Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

Establishment and characterization of immortalized cell lines from rat parotid glands

Summary This study reports for the first time the establishment of immortalized cell lines from normal adult rat parotid glands. The freshly prepared cellular clumps obtained from parotid glands of isoproterenol-treated rats were incubated in 0.2% trypsin solution without EDTA. These clumps were transfected with plasmid vectors pSV ₃ ^neo and pSV ₅ ^neo by electroporation and calcium phosphate-Co-DNA-precipitation techniques. The untransfected and transfected cellular clumps were plated in precoated dishes containing modified MCDB-153 medium. Epithelial cells grew from the clumps that were attached. All epithelial cells from untransfected culture died within 6 to 8 wk. Two cell lines which were isolated from transfected cultures subsequently grew on regular tissue culture dishes. One of them, which was isolated from pSV ₅ ^neo transfected cultures, exhibited non-epithelial cell morphology, but at confluency, many cells mature to acinar-like cells containing numerous granules. The other cell line (2RS), which was isolated from pSV ₃ ^neo transfected culture, contained cells of non-epithelial and epithelial morphology. During the initial phase of the growth, MCDB-153 medium was essential; however, at a later time, RPMI medium was better than MCDB-153 or F12 medium for maintaining morphology and growth of these cells. The immortalized cells grew in RPMI with a doubling time of about 25 h, synthesize T-antigen,α-amylase mRNAs of 1176 and 702 bp, andα-amylase and were non-tumorigenic. These amylase-producing cells can be a useful model to study the mechanisms of regulation of growth and differentiation in these cells.     

Phenotypic Characteristics of Thiobacillus ferrooxidansStrains

Phenotypicpolymorphism of Thiobacillus ferrooxidansstrains isolated from various ecological niches was studied. The strains differed both in rates of growth and oxidation of Fe²⁺, S⁰, FeS₂, and sulfide minerals contained in concentrate. Each strain, irrespective of its original environment, required a period of adaptation to a new substrate. Strains TFN-d, TFBk, TFO, and TFL-2, isolated from ores and concentrates rich in oxidizable substrates, showed an equal adaptation rate (five culture transfers) but differed in their adaptation efficiency. Strain TFV-1, isolated from low-grade ore and showing the lowest rates of growth and oxidation of all the substrates, required five culture transfers to adapt to S⁰and FeS₂and seven culture transfers to adapt to the concentrate. It is concluded that the phenotypic properties of the strains correlate with their genotypic polymorphism and the environmental conditions under which their microevolution took place.     

Effects of environmental settings on MTBE removal for a mixed culture and its monoculture isolation

A mixed culture was utilized to evaluate methyl tert-butyl ether (MTBE) removal under various conditions and to isolate a MTBE-degrading pure culture. The results showed that high MTBE removal efficiencies can be reached even in the presence of other substrates. The biodegradation sequence of the target compounds by the mixed culture, in order of removal rate, was toluene, ethyl benzene, p-xylene, benzene, MTBE, ethyl ether, tert-amyl methyl ether, and ethyl tert-butyl ether. In addition, preincubation of the mixed cultures with benzene and toluene showed no negative effect on MTBE removal; on the contrary, it could even increase the degradation rate of MTBE. The kinetic behavior showed that the maximum specific growth rate and the saturation constant of the mixed culture degrading MTBE are 0.000778 h⁻¹ and 0.029 mg l⁻¹, respectively. However, a high MTBE concentration (60 mg l⁻¹) was slightly inhibiting to the growth of the mixed culture. The pure culture isolated from the enrichments in the bubble-air bioreactor showed better efficiency in MTBE removal than the mixed culture; whereas, tert-butyl alcohol was formed as a metabolic intermediate during the breakdown of MTBE.     

Isolation and characterization of arsenite-oxidizing bacteria from arsenic-contaminated soils in Thailand

Two hundred and eighty-eight arsenic-resistant bacteria were isolated by an enrichment culture method from a total of 69 arsenic-contaminated soil-samples collected from Dantchaeng district in Suphanburi province (47 samples), and from Ron Phiboon district in Nakhon Sri Thammarat province (22 samples), in Central and Southern Thailand, respectively. Twenty-four of the 288 isolated arsenic-resistant bacteria were found to be arsenite-oxidizing bacteria. On the basis of their morphological, cultural, physiological, biochemical and chemotaxonomic characteristics, and supported by phylogenetic analysis based upon their 16S rRNA gene sequences, they were divided into five groups, within the genera Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium and Sphingomonas, respectively. Within genera, phylogenetic analysis using the 16S rRNA gene sequences suggested that they were comprised of at least ten species, five isolates being closely related to known bacteria (Acinetobacter calcoaceticus NCCB 22016^T, Pseudomonas plecoglossicida FPC951^T, Ps. knackmussii B13^T, Sinorhizobium morelense Lc04^T, and Sphingomonas subterranea IFO16086^T). The other five proposed species are likely to be new species closely related to Flavobacterium johnsoniae, Sinorhizobium morelense, Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, but this awaits further characterization for confirmation of the taxonomic status. No overlap in isolated species or strains was observed between the two sites. The strain distribution and characterization are described.     

Production of mannitol by a novel strain of Candida magnoliae

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l^–1, it yielded 67 g mannitol l^–1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l^–1 after 200 h, corresponding to an 83% yield and a 1.03 g l^–1 h^–1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.     

Extracellular metalloendopeptidase of Streptomyces rimosus

Metalloendopeptidase was isolated from Streptomyces rimosus culture filtrates in a homogeneous form. It was determined to be a 15 kDa basic protein, most active around pH 7.5, and susceptible to inhibition by chelating agents, N-bromosuccinimide, thiorphan, and 10⁻⁴ M zinc. The enzyme was highly specific for phenylalanine at the N-side of endopeptide bonds. Determination of amino acid sequence of the enzyme’s NH₂-part allowed the recognition of its structure homology with isolated and predicted metallopeptidases from several Streptomyces species. The data contribute to the definition of M7 family of metalloendopeptidases in streptomycetes.     

Isolation of a gram-positive bacterium effective in suppression of brown blotch disease of cultivated mushrooms,Pleurotus ostreatus andAgaricus bisporus, caused byPseudomonas tolaasii

A Gram-positive bacterium was isolated from a rottingPleurotus ostreatus fruiting body that markedly reduced the level of extracellular toxins (i.e., tolaasins) produced byPseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The isolated bacterium is saprophytic but not parasitic nor pathogenic toP. ostreatus. A low ratio, ca. 10⁻³ cells of the isolated bacterium for oneP. tolaasii cells, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease inP. ostreatus andAgaricus bisporus. The suppression of the disease development, however requires the initial cell density equivalent to ca. 10⁻¹ cells of the isolated bacterium for one cells of the pathogen. The effects is ascribed to the inactivation of tolaasin by the live, suppressive bacterial cells, and not to metabolites secreted from the organism into culture media. Examination by conventional bacteriological tests and with testing kits, i.e., MicroStation^TMSystem Release 3.5 (Biolog Inc., Hayward, CA), ATB Expression (bioMerieux Inc. Japan) and VITEK (bioMerieux Inc. Japan), failed to assign the organism to any defined bacterial genus. The suppressive bacterium may be useful in future for the development of biocontrol system and/or the construction of genetically modified edible fungi resistant to the disease caused byP. tolaasii.     

Induction of axenic culture of Arthrospira (Spirulina) platensis based on antibiotic sensitivity of contaminating bacteria

Arthrospira platensis SAG 21.99 and the isolated bacteria (Halomonas spp., Staphylococcus sp., etc.) from the culture of A. platensis SAG 21.99 were treated with five antibiotics to determine the minimal lethal concentrations. The combination of a washing step and a consecutive treatment with antibiotics, imipenem (100 μg ml⁻¹), neomycin (100 μg ml⁻¹) and cycloheximide (20 μg ml⁻¹), treatment step was highly effective in eliminating bacteria. An axenic culture of A. platensis SAG 21.99 could be induced within 3 days using this method. This technique is a simple and rapid method for obtaining axenic cultures of filamentous cyanobacteria.     

Glutamine synthetase specific activity and protein concentration in symbiotic Anabaena associated with Azolla caroliniana

Glutamine synthetase (GS) is the primary NH₄ ⁺ assimilating enzyme of cyanobacteria. The specific activities and cellular protein concentration of GS in symbiotic cyanobacteria associated with the water fern Azolla caroliniana were determined and compared to free-living cultures of Nostoc sp. strain 7801, a strain originally isolated from symbiotic association with the bryophyte Anthoceros punctatus. Both the in vitro specific activity and concentration of GS in symbiotic cyanobacteria separated from A. caroliniana were approximately 3-fold lower than the free-living Nostoc sp. strain 7801 culture. These results imply depressed synthesis of GS by the symbiont associated with A. caroliniana.     

Anti-<Emphasis Type="Italic">Helicobacter pylori</Emphasis> substances from endophytic fungal cultures

The human pathogenic bacterium Helicobacter pylori has been ascertained to be an aetiological agent for chronic active gastritis and a significant determinant in peptic and duodenal ulcer diseases. Endophytic metabolites are being recognized as a versatile arsenal of antimicrobial agents, since some endophytes have been shown to possess superior biosynthetic capabilities owing to their presumable gene recombination with the host, while residing and reproducing inside the healthy plant tissues. A total of 32 endophytic fungi isolated from the medicinal herb Cynodon dactylon(Poaceae) were grown in in vitroculture, and the ethyl acetate extracts of the cultures were examined in vitro for the anti-H. pylori activity. As a result, a total of 16 endophyte culture extracts were identified as having potent anti-H. pyloriactivities. Subsequently, a detailed bioassay-guided fractionation of the extract of the most active endophyte (strain number: CY725) identified as Aspergillussp., was performed to afford eventually four anti-H. pylori secondary metabolites. The four isolated compounds were identified through a combination of spectral and chemical methods (IR, MS, ¹H- and ¹³C-NMR) to be helvolic acid, monomethylsulochrin, ergosterol and 3β-hydroxy-5α,8α-epidioxy- ergosta-6,22-diene with corresponding MICs of 8.0, 10.0, 20.0 and 30.0 μg/ml, respectively. The MIC of ampicillin co-assayed as a reference drug against H. pylori was 2.0 μg/ml. Furthermore, preliminary examination of the antimicrobial spectrum of helvolic acid, the most active anti-H. pylori metabolite characterized from the endophyte culture, showed that it was inhibitory to the growth of Sarcina lutea, Staphylococcus aureusand Candida albicans with MICs of 15.0, 20.0 and 30.0 μg/ml, respectively.     

Protoplasts from <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed cell line of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. regenerated to hairy roots

Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×10⁶ per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2 mgl⁻¹ (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl⁻¹ (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl⁻¹ casein hydrolyzate at a plating density of 1.0×10⁵ per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.     

Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua

Scaled-up hairy root culture of Artemisia annua L. was established in three-liter Erlenmeyer flask. Both artemisinin and stigmasterol that derive from the common precursors of isopentenyl diphosphate and farnesyl pyrophosphate were isolated from hairy roots. The production rate of artemisinin isolated by column chromatography from hairy root cultures was 0.54% (mg.gDW⁻¹). Stigmasterol was identified by mass spectrometry and nuclear magnetic resonance analysis. The production of stigmasterol isolated by column chromatography from hairy root cultures was 108.3% (mg.gDW⁻¹). In hairy root cultures, the production rate of stigmasterol was estimated to be 201 times greater than that of artemisinin. Our results suggest that investigation of secondary metabolites may provide a new insight to study artemisinin production in hairy root cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

Evaluation of the phytic acid effect on the labeling of blood elements with technetium-99m and on the survival of a strain of Escherichia coli treated with stannous fluoride

The labeling of red blood cells with technetium-99m(^99mTc) depends on a reducing agent and stannous ions, as chloride or fluoride, are widely utilized. This labeling may also be altered by drugs. Moreover, some authors have reported that the survival of Escherichia coli (E. coli) cultures decreases in presence of stannous ions. Phytic acid is present in the daily diet and we evaluated its influence on: (i) the labeling of blood elements with ^99mTc and (ii) on the survival of an E. coli strain treated with stannous fluoride. Heparinized whole blood was withdrawn from Wistar rats and it was incubated with stannous chloride and with ^99mTc, as sodium pertechnetate, centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P and BC were also precipitaded with trichloroacetic acid, centrifuged and soluble (SF) and insoluble fractions (IF) isolated. E. coli culture was treated with stannous fluoride in presence of phytic acid. As phytic acid altered the fixation of ^99mTc on BC, on IF-P and on IF-BC and, moreover, it abolished the lethal effect of stannous fluoride on the E. coli culture, we can suggest that, probably, phytic acid would have chelating properties to the stannous ions.     

Effects of organic carbon sources on cell growth and eicosapentaenoic acid content of <Emphasis Type="BoldItalic">Nannochloropsis</Emphasis> sp.

A strain of Nannochloropsis isolated originally from the East China Sea and obtained from Institute of Hydrobiology, Chinese Academy of Sciences was shown to utilize glucose or ethanol for mixotrophic and heterotrophic growth. The highest cell density, 550 mg L^{− 1} dry weight after culture for 8 days, was obtained during mixotrophic culture with 30 mM glucose. The organic carbon sources had no effect on the net photosynthetic rate, but enhanced the respiratory rate. The addition of an organic carbon source led to an increase in the cell lipid content and a decrease in their eicosapentaenoic acid (EPA) content. The EPA yield was 21.9 mg L^{− 1} using photoautotrophic culture, and 23.4 mg L^{− 1} and 23.0 mg L^{− 1}, respectively, in mixotrophic cultivation with glucose or ethanol as the carbon source.