Efficient secretion of human lysozyme from the yeast, <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis, was achieved by using more stable vectors in the order of S11 replication origin-containing episomal vector < full-length K. lactis plasmid pKD1-containing vector < centromeric vector < chromosome-integrated vectors. Cells containing a PGK (phosphoglycerate kinase) promoter-driven integration vector grown in non-selective rich medium achieved the highest level of secretion, 100 g lysozyme secretion ml ^–1 culture: this level was 10-fold higher than that achieved by episomal vectors. An additional copy of the protein disulfide isomerase gene further facilitated the secretion.     

Effect of aluminium on yield and plant chemical concentrations of some temperate legumes

The aluminium (Al) tolerance of 34 temperate legume species (143 genotypes, including 57 from Trifolium repens) was determined in 60 experiments over a 3 year period in a low ionic strength (2.7 × 10^-3 M) solution culture. For each genotype, the relationship between solution Al³⁺ activity (M) and relative yield was determined and the Al³⁺ activity associated with a 50% reduction in yield (Al_RY50) calculated. In addition, plant chemical concentrations were determined in at least one genotype from most species. For white clover, Al_RY50 over all genotypes had an approximately normal distribution with mean of 1.31 M for the tops and 1.51 M for the roots, and a standard deviation of about 0.4. This suggested that Al tolerance had a polygenic inheritance. For the other species tested, Al_RY50 ranged from 0.15 to 4.53 M in the tops and from 0.21 to 4.89 M in the roots. In the tops and roots, 37% and 26% respectively of the genotypes had an Al_RY50 less than 1 M, including all species tested in the genera Melilotus and Medicago. Only 8% or 23% of the genotypes, based on the tops and roots respectively, had an Al_RY50 greater than 2, including all genotypes in the species Lotus pedunculatus. Except for Lotus, there were no consistent differences between genera in plant chemical concentrations. In Lotus, concentrations of Ca, Zn, Mn and Cu in the tops and of all elements except B in the roots were lower than that of the other species. The Al_RY50 of the species was not related to plant chemical concentrations in the absence of Al. Depending on the plant element, increasing solution Al concentrations had no significant effect on plant chemical concentrations for 56–94% of the species. When a significant effect did occur, increasing Al in solution generally decreased S and K concentrations and increased Mn, Zn, Cu Fe, B and Al concentrations in the tops and roots and decreased Ca concentrations in the tops. Plant P concentrations decreased in the tops but increased in the roots. Increasing Al in solution increase plant Al at the average rate of 44 g g^-1 M ^-1 (range 20–87) in the tops and 333 g M ^-1 (range 162–616) in the roots.     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Organochlorine and organophosphorus pesticide concentrations in water,sediment, and selected organisms in Lake Naivasha (Kenya)

Water, sediment, red swamp crayfish (Procambarus clarkii) and black bass (Micropterus salmoides) from Lake Naivasha were analyzed for selected organochlorine and organophosphorus pesticide residues. The mean p,p'-DDT, o,p'-DDT and p,p'-DDE residue levels recorded in black bass (28.3 (± 30.0), 34.2 (±54.0) and 16.1 (±16.1) g kg^–1, respectively) and crayfish (4.6 (±5.1), 3.2 (±2.8), and 1.4 (±1.1) g kg^–1, respectively), were higher than previously recorded. This indicated recent usage of technical DDT in the lake's catchment. Levels of p,p'-DDT, higher than those of p,p'-DDE further emphasized this. Mean lindane, dieldrin, -endosulfan and aldrin concentrations in black bass were 100.5, 34.6, 21.6 and 16.7 g kg^–1, respectively. The same residues were detected at lower concentrations in crayfish at 2.0, 2.0, 2.0 and 1.9 g kg^–1, respectively. The higher fat content (3.7 ± 2.7% SD) in black bass (compared to 0.6 ± 0.3% in crayfish) accounted for the significantly higher residue concentrations in black bass. Organophosphate pesticides were the most commonly used pesticides in the lake's catchment, but none was detected in any of the samples. The results indicate that there is need for further work to identify sources and fate of pesticide contaminants, as well as to improve monitoring of pesticide use throughout the catchment.     

Micropropagation of Tamarix gallica from nodal explants of mature trees

Tamarix gallica L. was micropropagated from four-to six-node explants taken from mature trees. Shoot proliferation was induced on Linsmaier and Skoog medium containing 30 g l^-1 sucrose, 7 g l^-1 agar, 200 mg l^-1 reduced glutathione (basal medium) and supplemented with 3.3 M benzyladenine. Adding 0.5 or 1.0 M indole-3-butyric acid (IBA) to the basal medium increased lateral shoot formation and ease of rooting. Microcuttings repeatedly subcultured on 1.0 M IBA produced well-developed roots, a high number of axillary shoots and could be acclimatized in the greenhouse.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B₁ (FB₁) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB₁ proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

A novel antifungal from an Actinomadurae with preferential activity against the mycelial phase ofCandida albicans

Summary Sch 40873, a novel antifungal compound isolated from the fermentation broth of anActinomadura spp. was discovered in an assay designed to detect compounds with preferential activity against the invasive mycelial form ofCandida albicans. The geometric mean MIC of Sch 40873 against sevenCandida spp. in Sabouraud dextrose broth (yeast phase) was 58 g/ml and in Eagles minimum essential medium (mycelial phase) was <0.03 g/ml. Sch 40873 demonstrated slight in vivo topical activity in a hamster vaginal model.     

Ecotoxicity of lead to the zebra musselDreissena Polymorpha,Pallas

The effect of lead on the filtration rate of the zebra musselDreissena polymorpha was investigated, together with the accumulation of Pb in the soft tissues of the mussels. The NOEC-filtration was 116 g.l^–1 (0,56 mol.l^–1) and the EC₅₀-filtration was 370 g.l^–1 (1.79 mol.l^–1). The NOEC-accumulation was the concentration found in the control water (1.4g.l^–1). These experiments show that the EC₅₀-filtration for Pb is similar to that for Cd, higher than that for Cu and lower than that for Zn. The water quality criteria for lead allow 25 g Pb.l^–1 in surface water. This will not cause short-term effects. Long-term effects may, however, occur, since an accumulation of Pb as low as 16 g.l^–1 was recorded in this study.     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

Concentrations of fumonisin B1 in feeds associated with animal health problems

Ninety-eight samples of feeds associated with 44 cases of equine leukoencephalomalacia (ELEM) and 83 samples of feed associated with 42 cases of a porcine pulmonary edema syndrome (PPE) were analyzed for fumonisin B₁ (FB₁). For comparison purposes, 51 feed samples not associated with PPE or ELEM were also analyzed. Feed associated with ELEM contained FB₁ ranging from less than 1 g/g to 126 g/g with 75% of the cases having at least 1 sample above 10 g/g. Feeds associated with PPE ranged from less than 1 g/g to 330 g/g with 71% of the cases having at least 1 sample greater than 10 g/g. Quantitation was by high performance liquid chromatography (HPLC)/fluorescence using the fluorescamine derivative with confirmation by thin layer chromatography (TLC) and/or gas chromatography/mass spectroscopy (GC/MS).     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium sp. from cultures of Datura innoxia (Mill.)

We have discovered a bacterial contaminant in some cell cultures of Datura innoxia (Mill.). The bacterium was tentatively identified as a species of Hyphomicrobium on the basis of its morphology and life cycle, and was isolated and grown in pure culture on a defined medium. The contaminant was not macroscopically observable in plant cell cultures. It caused neither a reduction of plant cell growth nor a noticeable increase in culture turbidity. Furthermore, it was not readily detectable by many standard assays for culture contamination: it would not grow alone in plant culture medium or yeast extract potato dextrose medium, and grew only very slowly on nutrient agar or beef-peptone medium. Repeated treatments with a combination of streptomycin (100 g/ml) and carbenicillin (100 g/ml) eliminated the contaminant from D. innoxia cell cultures without harming the plant cells.     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

Micropropagation of garlic through in vitro bulblet formation

We developed a novel micropropagation method for garlic (Allium sativum L.) by the combination of initial shoot-tip culture, shoot multiplication and in vitro bulblet formation. Garlic shoot-tips were cultured on LS medium containing 1 M indole-3-acetic acid (IAA) and 1 M 6-benzyladenine (BA) to regenerate proliferative shoots. These shoot-tips produced multiple shoots when transferred to modified LS medium containing 5 M 1-naphthaleneacetic acid (NAA) and 10 M BA, and cultured at 20°C under 12-h light conditions. Higher ratios of KNO₃/NH₄Cl in the media promoted multiple shoot formation, together with suppressing vitrification of these shoots. The proliferated shoots of early maturing cultivars produced bulblets by culture on LS growth regulator-free medium at 25°C under 16-h light. On the other hand, the late maturing cultivar, Howaito-roppen, formed bulblets after a low temperature treatment of the proliferated shoots for 6 months followed by culture on LS medium containing 6 to 12% sucrose for two months. The dormancy of the bulblets of cv. Howaito-roppen was broken by successive treatments at a high (35°C), a middle (20°C), and then a low (5°C) temperature.Abbreviations IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine - LS Linsmaier and Skoog macro- and microelements     

Determination of mannitol in ectomycorrhizal fungi and ectomycorrhizas by enzymatic micro-assays

Two sensitive methods for the enzymatic determination of mannitol are described which were applied to fungal and mycorrhizal extracts. Both methods are based on the oxidation of mannitol by mannitol dehydrogenase from Agaricus hortensis and the fluorometric determination of the NADPH produced in this reaction. The detection limits are 125 pmol for the direct fluorometric assay and 100 fmol, when enzymatic cycling of NADPH is included. The levels of mannitol detected were 123 pmol/g dry wt (mycelia from Cenococcum geophilum, cultivated on malt medium), below 0.3 or about 2.4 pmol/g dry wt (mycelia from Amanita muscaria, dependent on carbon source in the cultivation medium), and between 1.9 and 5.2 pmol/g dry wt in mycorrhizal short roots of Picea abies/Amanita muscaria.