Fluctuations of Uridine Incorporation in the Shoot Apex ofChenopodium rubrum L. during Photoperiodic Induction

Uridine incorporation into the shoot apex of the short-day plantChenopodium rubrum was investigated during a 16 h period of darkness and the following transfer to light. Uridine incorporation during this single inductive cycle was compared to incorporation under non-inductive conditions of continuous light. After transfer of the plants from light to darkness RNA synthesis was reduced to about half after the first two hours. This occurred not only when the plants were precultivated in continuous light but also after an interruption of the dark period by light for 31/2 h. The low level of uridine incorporation was maintained for the whole duration of the dark period. Incorporation regained its initial level after exposure of the plants to light irrespective of the duration of the preceding dark period. After this immediate rise of uridine incorporation in plants transferred from darkness to light a slight temporary decrease was observed in light. In darkness the decrease of incorporation into the nucleoli was still more marked than the reduction of overall incorporation. After the termination of the dark period incorporation into the nucleolus rose slowly and extranucleolar incorporation was relatively enhanced during the first 10 h of light in induced plants. The fluctuations of RNA synthesis observed in the shoot apex during photoperiodic treatment may be regarded as a necessary condition for the transition from the vegetative to the reproductive state.     

Killing Rates for Caspofungin Against Candida albicans After Brief and Continuous Caspofungin Exposure in the Presence and Absence of Serum

It was previously demonstrated that brief (≤1 h) exposures to echinocandins are as effective to kill Candida albicans cells as continuous 24-h exposure. However, killing rates after continuous and short (1 h) echinocandin exposures to C. albicans have not yet been evaluated in RPMI-1640 with and without 50 % serum. We evaluated four echinocandin susceptible C. albicans bloodstream isolates, ATCC 10231 type strain and an echinocandin-resistant isolate (DPL20, FKS F645P). Caspofungin MICs, time-kill and postantifungal effect (PAFE) tests were performed in RPMI-1640 with and without 50 % serum. Killing rates (k values) in time-kill and PAFE experiments were determined for each strain and concentration. In time-kill experiments, colony count decreases were isolate- and concentration-dependent at 0.25, 1, 4, 8, 16 and 32 mg/L in RPMI-1640, but concentration-independent at 1, 4, 8, 16 and 32 mg/L in 50 % serum. One-hour caspofungin exposure at 4, 16 and 32 mg/L resulted in CFU decreases comparable with the results obtained in time-kill experiments in RPMI-1640, but 50 % serum at 4, 16 and 32 mg/L allowed growth of all isolates (k values were negative) (P < 0.05–0.001). PAFE in 50 % serum decreased markedly at 4, 16 and 32 mg/L. Killing rates remained high and concentration-independent in 50 % serum in case of continuous but not in case of brief caspofungin exposure. As only a short growth inhibition without killing was observed in 50 % serum, clinical relevance of caspofungin PAFE in vivo is questionable.     

In vitro induction of cell division in the epidermis of stem segments ofTorenia fournieri Lind.

Cell division in the epidermis of stem segments ofT. fournieri stopped immediately when the epidermis was separated from subjacent tissues after having been in contact with these tissues for some time. Thus, the effects of the inductive signals emanating from these tissues did not persist. However, cell division in isolated epidermis cultured alone could be induced by adding asparagine, alanine or glutamine to the medium. Asparagine, at 5 mM, had the greatest stimulatory effect. Growth substances had a synergistic effect on this induction by amino compounds. However, these cell divisions, unlike those in epidermis cultured together with subepidermal tissues, did not lead to organogenesis. The amino compounds which partially replaced the inductive action of subepidermal layers on the epidermis can be considered as one of the endogenous factors coming from the first-named layers in intact explants.     

Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’ production from Bacopa monnieri (L.) Wettst.

Stable lines of hairy roots were established from leaf explants of Bacopa monnieri using different strains (A4, R1000, SA79, MTCC 532 and MTCC 2364) of Agrobacterium rhizogenes. The efficiency of hairy roots induction of these strains varied significantly and the maximum transformation frequency (75 %) was observed in case of strain SA79 using leaf explants followed by internode (55 %) in the presence of acetosyringone. Different parameters such as cell density of Agrobacterium suspension, co-cultivation period and infection time influenced the root induction frequency. Maximum frequency of root induction was obtained with bacterial density of 0.6 OD₆₀₀, 2 days of co-cultivation period and 10 min of infection time. Integration of T-DNA in the genome of hairy roots was confirmed by PCR amplification of rolB gene. Elimination of Agrobacterium from the established root cultures was ascertained by amplifying the DNA fragment specific to 16S rDNA and virD gene. All lines of hairy roots except strain A4 induced showed higher growth rate and accumulated higher levels of ‘bacoside A’ than the untransformed roots. Maximum biomass accumulation (6.8 g l^?1) and ‘bacoside A’ content (10.02 mg g^?1 DW) were recorded in case of the hairy root line induced by strain MTCC 2364.     

A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum

Hairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L^?1) (as elicitor) and sucrose (20, 30, 40 and 50 g L^?1) on the growth of hairy roots were evaluated. The results showed that, 30 g L^?1 sucrose and 100 mg L^?1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.     

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

Chlorella pyrenoidosa 7-11-05 in batch and in homogeneous continuous culture under autotrophic conditions

The cell content of substances (proteins, nucleic acids and chlorophylls) which play a significant role in growth processes in algae was used to characterize the physiological state of a continuous culture ofChlorella pyrenoidosa. The ratio of the various components of the cell content did not alter significantly with changes in the dilution rate. An increase in the mean cell volume was accompanied by a proportional increase in the amount of the various components. Their respective dry weight concentrations rose with the dilution rate and after reaching a maximum either fell or remained constant. The specific rates of synthesis of the given substances tended to rise, i.e. maximum activity of the culture was not attained. Deoxyribonucleic acid synthesis appears to be the endogenous factor limiting growth of the culture. The proportion of the individual nucleic acid fractions was compared with protein synthesis.     

Enhancement of Festuca rubra L. germination and seedling growth by seed treatment with pathogenic Agrobacterium rhizogenes

The study concerned effects of two methods of red fescue seeds treating with wild type Agrobacterium rhizogenes strains (15834 and LBA 1334): soaking and matriconditioning with Micro-Cel E solid carrier. The effects were assessed from germination and seedlings growth rates. The bacterial indole-3-acetic acid (IAA) production and ACC deaminase activity were also determined. Both strains are able to produce the IAA and showed ACC deaminase activity in various amounts. The strains accelerated seeds germination, seedling emergence and development. The beneficial effect on those processes was visible when seeds were soaked for 1 h in bacterial suspension and, especially, when the bacteria were present during Micro-Cel E conditioning. The main effect observed upon inoculation of seeds during priming was increased growth of lateral roots and more complex architecture of the branching root system. Treating seeds with A. rhizogenes simultaneously with Micro-Cel E priming as a carrier is a promising method of enhancing grass germination and seedlings growth, particularly by improving the root system development.     

Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU)

Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l^?1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.     

Changes in nucleic acid synthesis in cotyledons and apical buds of chenopodium rubrum l. associated with photoperiodic induction

The nucleic acid (NA) fractions were analyzed in cotyledons and apical buds ofChenopodium rubrum plants by means of acrylamide electrophoresis at the end of the dark period of a different number of photoperiodic cycles or after transfer of the plants to light for 4 h subsequent to the termination of the dark period. The plants were labelled with³²P three hours prior to sampling. The uptake of³²P into the cotyledons was higher in light than in darkness in all cases, however, it was not in correlation with³²P incorporation into the NA fractions. After one dark period lasting 8 or 16 h NA synthesis in light did not increase in comparison with darkness. After two or more photoperiodic cycles NA synthesis was higher in light than in darkness irrespective of whether the dark period lasted 8 or 16 h. NA synthesis was distinctly highest after two inductive cycles lasting 16 h. In buds NA synthesis was slightly shifted in favour of ribosomal RNA as compared with cotyledons. In the cotyledons the increase in light was mainly duo to a raise of rRNA synthesis whereas in the buds synthesis of sRNA and DNA increased, as well.     

Phytoremediation of soil co-contaminated with heavy metals and deca-BDE by co-planting of Sedum alfredii with tall fescue associated with Bacillus cereus JP12

Aim

The objective of this study was to develop a remediation strategy for soil co-contaminated with decabromodiphenyl ether (BDE-209) and heavy metals (Cd, Pb and Zn) using co-plantation of the hyperaccumulator plant (Sedum alfredii) with tall fescue (Festuca arundinaceae) associated with a BDE degrader (Bacillus cereus strain JP12).

Methods

A 120-day remediation experiment was conducted under greenhouse conditions. S. alfredii and tall fescue were grown in monoculture and intercropped in artificially contaminated soil. Plant biomass, concentration of polybrominated diphenyl ethers, density of soil bacteria, soil enzyme activity, and the physiological profile of the soil microbial community were determined.

Results and discussion

Inoculation with JP12 significantly increased BDE-209 dissipation in soil. Phytoextraction of metals was also enhanced by JP12 inoculation due to the improved plant growth. Planting of tall fescue significantly enhanced BDE-209 dissipation as compared to that in the bare soil because of the increased soil microbial activity. Tall fescue showed higher Pb phytoextraction efficiency than S. alfredii, but Pb was principally retained in the roots of tall fescue. BDE-209 dissipation and metal phytoextraction were highest when co-planting S. alfredii with tall fescue inoculated with strain JP12. Pyrosequencing analysis revealed that the inoculated JP12 could functionally adapt to the introduced soil, against competition with indigenous microorganisms in soil.

Conclusions

Co-planting of S. alfredii with tall fescue combined with BDE-degrading bacterial strain JP12 is promising for remediation of soil co-contaminated with BDE-209 and metals.     

Production of plant growth substances by rhizosphere mycoflora of broad bean and cotton

The culture filtrates of the rhizosphere fungi of broad bean (Vicia faba L.) and cotton (Gossypium barbadense L.) were analysed for the presence of plant growth substances of auxin and gibberellins nature. Bioassay test and chromatographic analysis indicated that these fungi, each synthesized different auxins in their culture medium. These auxins were indole compounds. Similarly the rhizosphere fungi produced in their culture medium some gibberellins and gibberellin-like substances.     

The benefits of foliar inoculation with <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> in soybean are explained by an auxin signaling model

Azospirillum sp. is one of the most studied genera of plant growth-promoting rhizobacteria (PGPR). The ability of Azospirillum sp. to promote plant growth has been associated with its ability to produce several phytohormones, such as auxins, gibberellins and cytokinins, but mainly indole-3-acetic acid (IAA). It has been propoosed that the production of IAA explains the positive effects of co-inoculation with Azospirillum sp. on the rhizobia-legume symbiosis. In this study, we constructed an IAA-deficient mutant of A. brasilense Az39 (ipdC ^? ) by using a restriction-free cloning method. We inoculated soybean seeds with 1·10⁶ cfu·seed^?1 of Bradyrhizobium japonicum E109 and co-inoculating leaves at the V3 stage with 1·10⁸ cfu.plant^?1 of A. brasilense Az39 wt or ipdC ^? or inoculated leaves with 20 μg.plant^?1 synthetic IAA. The results confirmed soybean growth promotion as there was increased total plant and root length, aerial and root dry weight, number of nodules on the primary root, and an increase in the symbiosis established with B. japonicum E109. Nodule weight also increased after foliar co-inoculation with the IAA- producer A. brasilense Az39. The exogenous application of IAA decreased aerial and root length, as well as the number of nodules on primary roots in comparison with the Az39 wt strain. These results allow us to propose a biological model of response to foliar co-inoculation of soybean with IAA-producing rhizobacteria. This model clearly shows that both the presence of microorganism as part of the colonization process and the production of IAA in situ are co-responsible, via plant signaling molecules, for the positive effects on plant growth and symbiosis establishment.     

Inherent limitations to the problem of reducing the lysine microbiological assay time

A kinetic approach is proposed to shorten the microbiological assay time for the determination of unboundl-lysine. The present lysine bacterial assay takes from 16 to 24 h usingPediococcus cerevisiae P-60 ATCC 8042 (formerlyLeuconostoc mesenteroides P-60 ATCC 8042) and uses a medium in which lysine is the limiting substrate. Measurements of the final cell concentration are linearly correlated with the initial concentration of lysine,S*, to provide an indirect estimate ofS*. We propose to understand the limitations inherent to the reduction of the assay time to 4 h by focusing in our analysis on the bacterial late lag or early growth transient phases, rather than the stationary phase of growth. Generally, the Monod equation is expected to describe a hyperbolically increasing correlation between the bacterial specific growth rate at about 2–4 h and the initial lysine concentration. A hyperbolic correlation is obtained by 3 h, but the lysine region of interest falls in the saturated portion of the curve. Discriminations between different initial lysine levels are therefore difficult with this nearly flat curve. On the other hand, when the initial inoculum level is lowered, so that substrate inhibition becomes effective, a correlation with a large negative slope is obtained by 4 h. Limitations to using absorbance measurements for the rapid assay turn up in a lack of reproducibility and, hence, a large variance associated with the measurements. Alternative microbial measuring techniques, such as impedance methods, need to be examined in order to reduce that large variance.     

Effects of high osmotic potential of a medium on mitotic cycle in roots ofVicia faba L

The present paper deals with the effects of osmotic potential of a medium on cell reproduction and elongation of the roots ofVicia faba L. andVicia sativa L. As the osmoticum polyethylene glycol (PEG 4000) in various concentrations ranging from 5 % to 25 % (i.e. fromca.-0.11 up to -1.27 MPa) has been used. The results show that at higher concentrations than 7.5 %, the growth of roots is slowed down and at the concentration of 25 % PEG this decrease in growth rate is as much as 6 fold compared with the control. The mitotic cycle is prolonged, however, only 1.86 times. Thus, the inhibition of root growth is caused mainly by the inhibition of cell elongation. Concerning the effect of high osmotic potential on mitotic cycle it was found that the roots after immersion into 25% PEG are able to overcome this osmotic stress and after 6 h to renew the mitotic activity. The S phase of the cycle is the most sensitive to this factor and even after mitotic activity was renewed it showed a slower rate in comparison with the control.     

Development of embryoids in carrot root callus culture (Daucus carota L.)

Embryogenesis occurred in carrot root callus (Daucus carota L.) cultivated on simple synthetic medium containing IAA and 2,4-D. Embryoid development continued also during successive years when the tissue was cultivated on the same nutrient medium without those substances. Sometimes production of plants with atypical leaves was also observed. In those plants development of adventive embryoids occurred repeatedly. Result of this work confirmed reports about the organogenic potentiality of this species and about its sensitivity to some chemical substances.     

Purification and characterization of antimicrobial substances from Bacillus licheniformis BFP011

Bacillus licheniformis BFP011 isolated from papaya (Thailand) could produce extracellular antimicrobial substances which were active against some important phytopathogens, pathogenics and spoilage microorganisms such as Colletotrichum capsici, Escherichia coli O157: H7 and Salmonella typhi ATCC 5784. The antimicrobial substances of this bacterium showed resistance to pronase enzyme and high temperature at 100 and 121°C for 15 min. They were purified by TLC on silica gel plates F254 using the different solvent mixtures. The best solvent mixture was revealed as n-butanol: ethanol: acetic acid: water (30: 60: 5: 30, v/v). The spots F4, F5 and F6 from TLC were able to inhibit growth of S. typhi ATCC 5784 assayed in vitro by the disc diffusion method. The characterization of the active fractions F4, F5 and F6 from TLC and reversedphase HPLC indicated that the antimicrobial substances of B. licheniformis BFP011 contain peptides and unsaturated fatty acids.     

Fatty acids profile and temperature in the cultured marine diatom Odontella aurita

The diatom Odontella aurita has now been industrially cultured and commercialized as a dietary supplement rich in omega-3 fatty acids for several years. In this study, we investigated the effect of three temperatures (8, 16, and 24 °C) on the growth and fatty acid composition of cells harvested during the exponential and stationary growth phases. These temperatures were selected on the basis of photosynthesis responses previously obtained at different temperatures using a modulated fluorometer. Our results confirm that both growth and lipid composition were sensitive to culture temperature. Growth was reduced when O. aurita was cultured at low temperature (8 °C) compared to when it was cultured at high temperatures (16 and 24 °C), but the proportion of polyunsaturated fatty acids (PUFAs, 20:5 n-3 and 22:6 n-3) increased while the level of saturated fatty acids (SFAs, 14:0 and 16:0) decreased in the cells harvested during both the exponential and stationary growth phases. On the other hand, the cells grown at 24 °C displayed a marked decrease in PUFA and an increase in SFA levels. Harvesting time is also a critical parameter in achieving optimum n-3 PUFA productivity during batch cultivation. Indeed, changes in fatty acid composition with growth phase seem to be dependent on the culture temperature, with the most marked effects being observed at 24 °C. PUFA levels (i.e., levels of 20:5 n-3 and 22:6 n-3) increased during the stationary growth phase, while the proportion of SFAs and monounsaturated fatty acids (MUFAs) fell with time. As this species is currently grown in outdoor ponds with seasonal temperature variations (minimal and maximal average temperatures in winter and summer, from 3 to 9 °C and from 13 to 26 °C, respectively), this factor can be expected to have a strong influence on the fatty acid content and composition of the algal biomass harvested and commercialized.     

Chufa (cyperus esculentus,cyperaceae): A weedy cultivar or a cultivated weed?

Cyperus esculentus(Cyperaceae) is a very variable species. From a large group of wild, mainly weedy plants, a cultivated group often named chufa, can be segregated by tuber color, reluctance to flower, and short rhizomes. Cultivar status within Cyperus esculentus is proposed for this useful plant.